University of Groningen Experimental studies on signal transduction ...

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Experimental studies on signal transduction pathways in rheumatoid arthritisBijl-Westra, Johanna

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https://research.rug.nl/en/publications/f75c705c-6545-41d2-adda-a0299a4fede2

Experimental studies on signal transduction pathways

in rheumatoid arthritis

Hannie Westra

Studies presented in this thesis were financially supported by Dutch Arthritis Association and Johnson and Johnson Pharmaceutical Research and Development, Raritan, New Jersey, USA The printing of this thesis was financially supported by University of Groningen, Dutch Arthritis Association, Groningen University Institute for Drug Exploration (Guide), Pharma-Bio Research (Zuidlaren), Tebu-Bio (Heerhugowaard) Copyright 2005 Johanna Westra ISBN-number: 90-367-22669-1 Printed by Drukkerij van Denderen, Groningen Cover illustration: Microscopic images of rheumatoid synovial tissue, stained for phospho-p38 MAPK (Santa Cruz antibodies). Upper: positive staining of synovial lining layer cells (brown); Lower: positive staining of synovial endothelial cells

RIJKSUNIVERSITEIT GRONINGEN

Experimental studies on signal transduction pathways in rheumatoid arthritis.

Proefschrift

ter verkrijging van het doctoraat in de Medische Wetenschappen

aan de Rijksuniversiteit Groningen op gezag van de

Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op

maandag 13 juni 2005 om 14.45 uur

door

Johanna Bijl-Westra

geboren op 10 december 1960

te Leeuwarden

Promotores: Prof. dr. P.C. Limburg Prof. dr. M.H. van Rijswijk Copromotor: Dr. M.A. van Leeuwen Beoordelingscommissie: Prof. dr. C.G.M. Kallenberg Prof. dr. P.P. Tak Prof. dr. E. Vellenga

Voor Heit en Mem

CONTENTS page

Chapter 1 General introduction and aim of the thesis 9

Chapter 2 Signal transduction pathways in rheumatoid arthritis with emphasis 13

on the p38 mitogen-activated protein kinase (MAPK) pathway.

Chapter 3 Effects of RWJ 67657, a p38 mitogen activated protein 29

kinase (MAPK) inhibitor, on the production of inflammatory

mediators by rheumatoid synovial fibroblasts.

Chapter 4 Strong inhibition of TNF-α production and inhibition 45

of IL-8 and COX-2 mRNA expression in monocyte-derived

macrophages by RWJ 67657, a p38 mitogen activated protein

kinase (MAPK) inhibitor.

Chapter 5 Monocytes and monocyte-derived macrophages differ 59

in regulation of signal transduction pathways.

Chapter 6 Chemokine production and E-selectin expression in 73

activated endothelial cells are inhibited by p38 MAPK

(mitogen-activated protein kinase) inhibitor RWJ 67657.

Chapter 7 Differential effects of NF-κB and p38 MAP kinase 89

inhibitors and combinations thereof on TNFα and IL-1β

induced pro-inflammatory status of endothelial cells in vitro.

Chapter 8 Differential influence of p38 mitogen-activated protein 107

kinase (MAPK) inhibition on acute phase synthesis in human

hepatoma cell lines and human liver slices.

Chapter 9 Summary, general conclusions and future perspectives. 125

Chapter 10 Nederlandse samenvatting 131

Dankwoord 138

List of abbreviations 140

List of publications 142

1

General introduction and aim of the thesis

Johanna Westra

Chapter 1

10

GENERAL INTRODUCTION AND AIM OF THE THESIS Rheumatoid arthritis is a chronic inflammatory disease, located in the synovial joints 1. This inflammatory process leads to degradation of cartilage and bone in the joints with consequent disability and reduced quality of life. Drugs that are intended to inhibit both the inflammatory and destructive processes in RA are the so-called DMARDS (disease modifying anti-rheumatic drugs). Even the gold standard of these drugs, methotrexate (MTX), has limited efficacy and its use may be hampered by side-effects 2. The breakthrough in treatment of RA in the last decade has been the development of TNF-blockers, thereby recognizing the key role of TNF-α in the inflammatory process 3. Although treatment efficacy of RA has dramatically improved with TNF-blockers, still not all patients do respond to this treatment. Moreover these drugs are expensive and severe side effects have been reported. A novel approach to treat inflammatory diseases might be the inhibition of intracellular signal transduction pathways. In short, signal transduction is the process by which a cell converts an extracellular signal into a response. A key role is played by proteins called kinases, which act as regulators of cell function by catalyzing (facilitating) the addition of a negatively charged phosphate group to proteins, resulting in the activation of the catalytic potential of the protein involved. One of the major signal transduction pathways in inflammation is the p38 mitogen-activated protein kinase (MAPK) pathway 4. Specific inhibitors for this pathway have been developed but until now only two of them have entered phase II clinical trials for RA 5;6. It still remains to be elucidated which pathways are important in inflammatory disease and especially in RA and what maybe the clinical importance of signal transduction inhibitors. The aim of the thesis was to investigate the potential of inhibition of signal transduction pathways in treatment of rheumatoid arthritis (RA) The study was performed in vitro using cells, which in some way are relevant players in the inflammatory process in RA. The main focus was on inhibition of the p38 MAPK pathway, but in addition the involvement of the NF-κB and the JAK/STAT pathways was investigated. In chapter 2 a general review is given on signal transduction pathways, which play a role in inflammatory processes in general and in RA more specifically. In chapter 3 the effects of p38 MAPK inhibition on inflammatory mediator production by rheumatoid synovial fibroblasts is investigated. These cells display aggressive invasive behaviour and are to a large extent responsible for the destructive process in the synovial joints in RA. In chapter 4 the effects of p38 MAPK inhibition on the products of monocyte-derived macrophages from healthy controls and RA patients was evaluated. In inflammation macrophages are responsible for the production of the major pro-inflammatory cytokines TNF-α and IL-1β, the key cytokines in RA. In chapter 5 the differences in reactivity towards p38 MAPK inhibition between monocytes and monocyte-derived macrophages were evaluated. In this study the use

Introduction and aim of the thesis

11

of a new technique, the kinase array is introduced. This technique allows the simultaneous determination of the activity of a large number of kinases in a cell lysate. In chapter 6 the effects of p38 MAPK inhibition on activated endothelial cells was investigated. These cells line the blood vessels and play an important role in recruitment of inflammatory cells into the inflamed synovium in RA. In chapter 7 the previous study was expanded by using both an NF-κB inhibitor as well as the p38 MAPK inhibitor in investigating the effects on endothelial cells. In chapter 8 the effects of p38 MAPK inhibition on the acute phase response (APR) was evaluated. Although the APR predominantly is regulated by the JAK/STAT pathway there is cross-talk between the pathways. In the case of p38 MAPK treatment in RA patients this could mean that the production of acute phase proteins may be influenced both directly as well as indirectly by the treatment. REFERENCES 1 Choy EH, Panayi GS. Cytokine pathways and joint inflammation in rheumatoid arthritis.

N.Engl.J.Med. 2001; 344: 907-16. 2 Whittle SL, Hughes RA. Folate supplementation and methotrexate treatment in rheumatoid

arthritis: a review. Rheumatology.(Oxford) 2004; 43: 267-71. 3 Feldmann M, Maini RN. Anti-TNF alpha therapy of rheumatoid arthritis: what have we learned?

Annu.Rev.Immunol. 2001; 19: 163-96. 4 Kumar S, Boehm J, Lee JC. p38 MAP kinases: key signalling molecules as therapeutic targets

for inflammatory diseases. Nat.Rev.Drug Discov. 2003; 2: 717-26. 5 Haddad JJ. VX-745. Vertex Pharmaceuticals. Curr.Opin.Investig.Drugs 2001; 2: 1070-6. 6 Nikas SN, Drosos AA. SCIO-469 Scios Inc. Curr.Opin.Investig.Drugs 2004; 5: 1205-12.

2

Signal transduction pathways in rheumatoid arthritis with

emphasis on the p38 mitogen-activated protein kinase

(MAPK) pathway

Johanna Westra1

Pieter C Limburg1,2

From the Departments of 1 Rheumatology and 2 Pathology and Laboratory

Medicine, University Medical Center Groningen, The Netherlands

Chapter 2

14

CONTENTS

1. Rheumatoid arthritis 1.1 Pathogenesis 1.2 Therapy

2. Signal transduction pathways in RA 2.1 MAPK pathways

2.1.1 ERK 1/2 2.1.2 JNK 1,2,3 2.1.3 p38 MAPKα, β, γ, and δ 2.1.4 ERK5 / BMK1

2.2 NF-κB pathway 2.3 JAK-STAT pathway

3. p38 MAPK pathway 3.1 Identification 3.2 Downstream effects of p38

3.2.1 Gene expression 3.2.2 Post transcriptional regulation of mRNA stability

3.3 Inactivation by phosphatases 4 p38 MAPK inhibitors

4.1 development 4.2 p38 MAPK inhibitors in animal models for arthritis 4.3 p38 MAPK inhibitors in human studies

5 Conclusions

Signal transduction pathways in RA

15

1. RHEUMATOID ARTHRITIS Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, primarily located in the synovial joints, leading to destruction of the cartilage and bone as a result of the chronic disease activity 1. RA affects 0.5 - 1% of the population in the industrialized world is two to three times more frequent in women than men and can lead to disability and reduced quality of life. 1.1. Pathogenesis The cause of RA is still unknown, but both genetic and environmental factors appear to play a role. The association with certain human leukocyte antigen (HLA) DR4 subtypes and RA has been recognized for a long time 2, and now it is found that rather specific amino acid sequences, the so-called shared epitope (SE) confer the highest risk for developing RA 3. RA is characterized by the presence of autoantibodies, especially rheumatoid factors and antibodies to citrullinated proteins (anti-CCP) in the majority of the patients. Recently in a study in blood bank donors it was demonstrated that approximately half of patients with RA had specific serologic abnormalities (rheumatoid factor and anti-CCP antibodies) several years before the onset of symptoms 4. The earliest events in RA might involve activation of the innate immune system, which triggers a T-cell response possibly directed towards citrullinated proteins 5. Infiltrating T-cells in the synovial membrane may, by cell-cell contact, and activation by different cytokines, such as TNF-α, IFN-γ and IL-17, activate monocytes, macrophages and synovial fibroblasts. These cells than produce pro-inflammatory cytokines, mainly TNF-α, IL-1 and IL-6 6. As the disease progresses multiple cytokine networks enter a state of persistent activation, triggering the production of matrix metalloproteinases, ultimately resulting in irreversible damage of cartilage and bone 7. 1.2. Therapy DMARDS (disease modifying anti-rheumatic drugs) are drugs that are intended to inhibit both the inflammatory and destructive processes in RA. Of these DMARDS methotrexate (MTX) is the most commonly used and is regarded as the gold standard of DMARD therapy 8. At doses used in the treatment of RA, MTX is likely to act via a number of intracellular pathways. Upon transport into cells, MTX is converted to polyglutamated forms, which promote intracellular retention. This results eventually in induced release of adenosine, which has an anti-inflammatory effect on neutrophils and mononuclear cells 8;9. MTX modulates cytokine responses at a number of levels and may promote apoptosis of activated lymphocytes 10. Since five years a new DMARD has become available, Leflunomide, which is an active metabolite that inhibits dihydro-orotate-dehydrogenase, an enzyme involved in de novo pyrimidine synthesis 11. Inhibition of this enzyme affects various signal-transduction mechanisms, the generation of cytokines, and cell proliferation and migration. However, these DMARDS still have limited efficacy and may lead to toxicity problems. The pro-inflammatory role of cytokines and the involvement of different cell types led to the development of therapeutics to selectively target cytokines. The key role of TNF-α in the pathogenesis of RA was discovered both in experimental animal models and in RA patients by Feldmann, Maini and others 12. There are now

Chapter 2

16

three drugs clinically available for treatment that block the activity of TNF-α: Infliximab (chimaeric monoclonal antibody to human TNF), Adalumimab (human monoclonal antibody to TNF) and Etanercept (soluble TNF receptor construct) and one to block IL-1 activity: Anakinra (IL-1 receptor antagonist). The efficacy and possible toxic effects of these new drugs have been reviewed by Olsen and Stein 13. Although TNF blockade has been a major breakthrough in the therapy of RA in the last ten years, there are some drawbacks. About half of the patients in clinical trials do not achieve adequate clinical responses expressed as ACR50 responses, remission is rare and these drugs also have side effects, for instance increased risk of tuberculosis13. New drugs to inhibit the production and activity of inflammatory cytokines may be found in inhibitors of intracellular signal transduction pathways 14. These pathways are involved in the primary production of these cytokines, as well as in the responses generated by these cytokines. TNF-α and IL-1 induce a variety of signal transduction cascades that lead to the activation of transcription factors and next to the transcription and translation of genes, coding for inflammatory mediators. The cascades that are important in RA and the potential inhibitors will be discussed below. 2. SIGNAL TRANSDUCTION PATHWAYS IN RA Intracellular signal transduction pathways are intracellular mechanisms by which cells can react to extracellular stimuli such as stress and inflammatory cytokines. In short, intracellular signal transduction is the process by which a cell converts an extracellular signal into a response. A key role thereby is for proteins called kinases, which act as key regulators of cell function by catalyzing (facilitating) the addition of a negatively charged phosphate group to proteins. These signalling cascades ultimately lead to induction of gene transcription and translation into specific proteins. These inflammatory proteins, including cytokines, matrix metalloproteinases, and cyclo-oxygenase (COX-2) are involved in the pathogenesis of RA. The main intracellular signal transduction pathways implicated in RA include the mitogen-activated protein kinase (MAPK) pathways, nuclear factor-kappa B (NF-κB) and the Janus kinase (JAK-STAT) pathway. 2.1. Mitogen-activated protein kinase (MAPK) pathways (figure 1) There are four well-characterized families of MAPKs, acting by phosphorylation of specific serine (Ser), threonine (Thr) and tyrosine (Tyr) residues of target substrates thereby controlling important cellular functions, such as gene expression, mitosis, movement, metabolism and apoptosis. The MAPK isoforms themselves are phosphorylated by dual- specificity serine-threonine MAPK-kinases (MAPKK or MEK) which in turn are phosphorylated by upstream MAPK-kinase-kinases (MAPKKKs or MEKKs). The MAPK family include the extracellular signal-regulated kinases ERK1 and ERK2 (also known as the p42/p44 MAPK pathway), the c-jun NH2-terminal kinases JNK 1, JNK 2 and JNK 3, the four p38 enzymes, p38α, p38β, p38γ and p38δ, and the ERK5 or big MAP kinase 1 (BMK1) 15. All MAPKs share the amino-acid sequence Thr-Xxx-Tyr in which X differs: X is glutamic acid (Glu), proline (Pro) and glycine (Gly) for the ERK, JNK and p38 MAPK respectively 16.


17

Figure 1. Mitogen-activated protein kinase (MAPK) pathways.

2.1.1. The ERK 1/2 signaling pathway is a major determinant in the control of cell growth, cell differentiation and cell survival. Growth factors, cytokines, viral infection, and carcinogens are the main activators of the proto-oncogene Ras, a small GTP-binding protein, which is the common upstream molecule of the MEKKs Raf (Raf1, A-Raf or B-Raf) 17. Activated Rafs phosphorylate MEK1/2, which in turn activate ERK1 and ERK2. ERKs can directly phosphorylate a set of transcription factors including Ets-1, c-Jun and c-myc, or activate RSK (ribosomal S6 kinase), which leads to the activation of the transcription factor CREB (cyclic AMP-responsive element-binding protein). MEK1 and MEK2 activity has been detected in a significant number of primary human tumor cells. Inhibitors of the ERK pathway (Ras-, Raf- and Src-inhibitors) are entering clinical trials as potential anti-cancer agents. PD98059 and U0126 are non-ATP competive MEK 1/2 inhibitors, which block phosphorylation and activation of ERK1 and 2 by MEK 18.

2.1.2. JNKs were discovered to bind and phosphorylate the DNA-binding protein c-Jun (a component of the AP-1 (activator protein) transcription complex) and to increase its transcriptional activity. Regulation of the JNK pathway is extremely complex and is influenced by many kinases. In general, stress or cytokines can initiate a series of events in which GTP-binding proteins such as Cdc42, Rac or Ras can activate protein kinases such as GCK (germinal centre kinase), which in turn can activate ASK (apoptosis stimulating kinase), TAK (TGFβ-activated kinase) and the MEKKs 1,2 and 3 19. Then MKK4 and MKK7 are activated, which are the direct activators of the JNK MAPKs. Targets of this pathway include c-Jun, ATF2 (activating transcription factor

Chapter 2

18

2) and ELK-1. JNK is one of the primary MAPKs required for expression of matrix metalloproteinases and joint destruction in models of inflammatory arthritis 20. The best known JNK2 inhibitor is SP600125, while another JNK pathway inhibitor CEP1347, has been reported to inhibit members of the MLK (mixed lineage kinase) family, which are upstream activators of the JNK pathway 18. 2.1.3. p38 MAPKs are, like the JNKs, stress-activated protein kinases, that mediate responses to cellular stress factors, such as UV light, osmotic shock and cytokines. The main activation route for p38 MAPK is through phosphorylation by MMK3 / MKK6 and possibly by MKK4, which in turn are activated by the MAPKKKs (figure 2). Members of the MAPKKK superfamily include MEKK 1-4, MLK, Tpl2 (tumor progression locus-2), TAK-1 and ASK-1 21. The MAPKKKs themselves are activated by small GTP-binding proteins, including Rac and Cdc42, partly involving p21-activated kinases (PAKs). In inflammation the most important route for activation is via TNF-α and IL-1 by ligation of their respective membrane receptors and recruitment of intracellular adaptor molecules. Activated p38 MAPKs can phosphorylate downstream kinases (MAPKAPK-2, MSK-1, PRAK and MNK) and transcription factors such as ATF-2, CHOP (C/EBP homologous protein) and ELK-1. Lipopolysaccharide (LPS) signal transduction is also one of the activation routes for

Figure 2. p38 mitogen-activated protein kinase (MAPK) pathway.


19

p38 MAPK. LPS is a common constituent of Gram-negative bacterial outer membranes and is the principal initiator of septic shock. Signalling of LPS involves a receptor complex with Toll-like receptor 4 (TLR-4), CD14 and the adaptor molecules MyDD88 and IRAK (interleukin-receptor associated kinase). The route then resembles the IL-1 route: via TRAF6 and TAK1 p38 MAPK can be activated as well as the JNK and the NF-κB pathways 22. The p38 MAPK signal transduction pathway will be discussed in detail below.

2.1.4. The fourth and least studied MAP kinase pathway, BMK1 or ERK5, is activated in response to growth factors and stress. This pathway has not only been implicated in cell survival, proliferation and differentiation, but also in pathologic processes such as carcinogenesis, cardiac hypertrophy and atherosclerosis 23. MEK5 is the upstream kinase of BMK1, and can be inhibited by inhibitors of MEK 1/2 such as PD98059 and U0126. The MEK5 kinases identified thus far are MEKK2 and MEKK3, which are also known to regulate p38 MAPK and JNK activity through the activation of MKK3/6 and MKK4/7 respectively 23. 2.2. NF-κB pathway (figure 3) The transcription factor Nuclear Factor κ-B (NF-κB) is a key factor in the transcription of many inflammatory genes. NF-κB is a complex group of heterodimeric and homodimeric transcription factors, consisting of five members: c-Rel, RelA (p65 or NF-κB3), RelB, NF-κB1 (p50/p105) and NF-κB2 (p52/p100) 24. Normally these dimers bind to the specific inhibitors of NF-κB, known as IκB proteins. Upstream kinases, including members of the MAPKKK family and NF-κB- activating-kinase (NAK) can induce phosphorylation and degradation of IκB, thereby liberating the NF-κB dimers, which translocate to the nucleus and regulate gene transcription 24;25. The modification of the IκB proteins depends on the IKK (IκB kinase) complex, which is composed of three subunits: the catalytic subunits IKK-α(1) and IKK-β(2), and the regulatory subunit IKK-γ (also known as NEMO). These subunits have specific roles in the regulation of NF-κB activity. The activation of NF-κB has been implicated in the pathogenesis of RA. Translocation of nuclear p50 and p65 was demonstrated in RA synovial lining cells and in mononuclear cells of the sublining 26. It has been demonstrated that NF-κB suppression is beneficial in many models of inflammatory disease 27. Moreover, for many therapeutic agents it has been shown that at least some of their effects are due to NF-κB blockade. Efforts to block this pathway have led to the development of small-molecule inhibitors of various kinases and regulatory proteins and also to research in gene therapy. In arthritis IKK-β seems an attractive target for therapy, because it regulates cytokine production in many cell types including synovial fibroblasts 28;29. Although it is obvious that NF-κB plays a key role in inflammatory diseases, there are major safety concerns about inhibition of this transcription factor, because of the major role that NF-κB plays in host defence, homeostasis, cell survival and response to stress.

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Figure 3. NF-κB pathway. 2.3. JAK/STAT pathway (figure 4) The Janus kinase (JAK) family and the signal transducer and activator of transcription (STAT) transcription factors play an important role in cytokine signal transduction. Type I cytokine receptors (for colony stimulating factors, and several interleukins) and type II cytokine receptors (for interferons) lack intrinsic kinase activity and rely on Jak proteins to initiate signalling 30. In the case of IL-6 an association with the IL-6 receptor and gp130 subunits takes place that activates JAKs. This is followed by the phosphorylation of the tyrosine-based docking sites on the receptor and recruitment of STATs. They form homo-hetero-dimers and translocate to the nucleus, where they bind target sequences 31. Dimerization of the IL-6-type cytokine receptors does not only lead to activation of the JAK/STAT signalling pathway, but also of the MAPK cascade. Negative regulation of IL-6 signalling via the JAK/STAT pathway may occur in different ways 30. Suppressor of cytokine signalling (SOCS) proteins are induced in response to IL-6 binding and can bind directly to the JAKs. SH2-domain-containing tyrosine phosphatase-1 (SHP-1) either can dephosphorylate JAKs or activated receptor subunits. Protein inhibitors of activated STATs (PIAS) family members inactivate STAT dimers.


21

Figure 4. JAK/STAT pathway.

THE p38 MAPK SIGNAL TRANSDUCTION PATHWAY 3.1. Identification The human p38α MAPK were first identified as the molecular target of pyridinylimidazole class of compounds, that were known to block TNF-α and IL-1 release from lipopolysaccharide (LPS)- stimulated human monocytes 32. Originally these proteins were designated cytokine-suppressive anti-inflammatory drug-binding proteins (CSBP) 33. Until now, five isoforms of p38 MAPK have been identified: p38β1 and p38β2 have more than 70% identity to p38α, whereas p38γ and p38δ have approximately 60% identity to p38α. Functional differences between the isoforms are related to their differential expression, activation, and substrate specificity 34. p38α, p38β and p38δ are widely produced in various tissues, while p38γ is expressed primarily in skeletal muscle. Inflammatory cells synthesize predominantly p38α and p38δ protein, but endothelial cells also produce p38β 35;36. ERK, JNK and p38 MAPK activation were almost exclusively found in synovial tissue from RA, but not osteoarthritis patients. p38 MAPK activation was observed in the synovial lining layer and in synovial endothelial cells 37. 3.2. Downstream effects of p38 MAPK activation 3.2.1. Gene expression One of the main downstream effects of the p38 MAPK pathway is regulation of gene expression, which can be at the transcriptional level, but also at the translational level,

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leading to protein synthesis. p38 MAPK has been implicated in the activation of various transcription factors including: ATF-2, MEF-2C, CHOP, and NF-κB 38-40. Other transcription factors are phosphorylated by downstream protein kinases that are themselves activated by phosphorylated p38 MAPK, such as MAPKAPK-2 (MAPK activated protein kinase). Furthermore MAPKAPK-2 deficient mice show diminished production of IL-6 and TNF-α 41. These effects are thought to be mediated by a mechanism involving mRNA turnover and protein translation. The p38 MAPK/MAPKAPK-2 pathway is crucial to inflammatory cytokine production. 3.2.2. Post transcriptional regulation of mRNA stability As will be discussed later, several p38 MAPK inhibitors have been developed which were shown to block the production of inflammatory cytokines. This inhibition seems to be a combined effect at the level of transcription and translation. Inducible cytokines usually have short lived mRNAs and contain an AU-rich element (ARE) in their 3’untranslated region responsible for their high turn-over rate 42. These AREs contain repeats of the motif AUUUA, and were discovered as instability elements. In the case of inflammatory response mRNAs, the instability is countered by signalling in the p38 MAPK pathway 43. Under normal conditions, these AU-rich elements are occupied by AU-binding proteins, thereby blocking translation or transcription. It has been demonstrated that upon stimulation these AU-binding proteins are phosphorylated in a p38 MAPK-dependent manner, resulting in their release, and allowing translation of these mRNAs 43;44. Inhibitors of p38 MAPK can target these events directly or via MAPKAPK-2. It has been demonstrated that both translation and stability of TNF-α mRNA are regulated by the p38MAPK pathway 45, whereas IL-6 and IL-8 mRNA stability is regulated by p38 MAPK, but the extent of inhibition of protein production varies with cell type 46;47. Activation of p38 MAPK has also been shown to enhance the mRNA stability of collagenase-1 (MMP-1) and stromelysin-1 (MMP-3 ) 48. Finally, Lasa et al demonstrated that the gluco-corticoid dexamethasone destabilizes COX-2 mRNA by inhibiting p38 MAPK. This effect was induced by expression of MAPK-phosphatase-1 (MKP-1) 49. 3.3. Inactivation by phosphatases For control of the MAPK signal transduction pathways dephosphorylation is necessary. Over the last decade a family of endogenous negative regulators of MAPK, the MAPK phosphatases (MKPs), also known as DUSPs (dual-specificity phosphatases) have been described 50. The MKPs dephosphorylate the tyrosine and threonine motifs of MAPK to deactivate MAPK-dependent signalling. At least 10 mammalian MKPs have been cloned and characterized, which have different subcellular distribution, substrate specificity, and expression patterns. MKP-1 was the first isolated MKP, and is a 39.5 kD protein that is preferentially localized in the cell nucleus. It is capable of dephosphorylating all MAPK families, although a preference for dephosphorylating p38 MAPK and JNK has been described 51. MKP-1 is an early response gene that is induced by the same stimuli that activate the MAPKs, such as cytokines, osmotic shock and UV radiation 52;53. Recently the expression of MKP-1 in


23

rheumatoid synovial fibroblasts was demonstrated as well as a role for glucocorticoid dependent upregulation of MKP-152. 4. p38 MAPK INHIBITORS (figure 5) 4.1. Development Already in 1988 the inhibition of IL-1 production by the anti-inflammatory compound SK&F 86002 was reported 54, but it took until 1994 to discover that the molecular target of the pyridinyl imidazole class of compounds indeed proved to be p38 MAPK, originally known as CSBP 32. Since the original report of the efficacy of these compounds, they have become the most widely studied inhibitors of this kinase. The compounds have been used as framework for further synthetic work and have been utilized to elucidate the role of p38 MAPK in the immune system. Crystallographic and kinetic experiments have shown that the pyridinyl imidazole family of p38 MAPK inhibitors bind at the ATP binding site of p38 MAPK and compete with ATP for binding to active, phosphorylated p38 MAPK 55. When p38 MAPK is in the unactivated form, ATP is non-competitive with many p38 MAPK inhibitors. After the structure-activity relationship was established SB 203580 and other 2, 4, 5- triaryl imidazoles were prepared as pharmacological tools to regulate cytokine synthesis. A large number of preclinical studies have reported that specific and selective p38 MAPK inhibitors block the production of inflammatory cytokines in vitro and in vivo. Furthermore, the p38 MAPK pathway is involved in the induction of several other inflammatory molecules such as cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS).

Figure 5. p38 MAPK inhibitors.

The p38 MAPK inhibitor used in our studies, RWJ 67657, was developed by Johnson and Johnson Pharmaceutical Research and Development and was first described in 1999 56. RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol) has been shown to be effective in inhibiting the release of TNF-α from LPS-treated human peripheral blood mononuclear cells with an IC50 of 3 nM. In comparison to the literature standard SB 203580 this new compound proved to be approximately 10-fold more potent in all p38 MAPK dependent systems tested.

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Moreover the compound inhibited the enzymatic activity of p38α and β, but not of γ and δ, in vitro and had no significant activity against a variety of other enzymes. 4.2. p38 MAPK inhibitors in animal models for arthritis Several p38 MAPK inhibitors have been evaluated in animal arthritis models. Already in 1996 the pharmacological profile of SB 203580 (GlaxoSmithKline) was investigated in adjuvant-induced arthritis in Lewis rats 57, whereas in 1998 the pharmacologically effects of SB 220025 (GlaxoSmithKline) were investigated in an angiogenesis and chronic disease model 58. RPR-200765A (Aventis) reduced incidence and progression in the rat streptococcal cell wall (SCW) arthritis model at doses given orally 59, while prevention of the onset and progression of collagen-induced arthritis were reported for FR167653 (Fujisawa Pharmaceuticals) 60. Recently SB 242235 (GlaxoSmithKline) was evaluated in a new model of arthritis, pristane-induced arthritis, and demonstrated to significantly reduce all arthritis scores 61. 4.3. p38 MAPK inhibitors in human studies RWJ 67657 is one of the p38 MAPK inhibitors who until now have been used in human studies. The effects on clinical and cytokine response to endotoxaemia were studied in healthy human volunteers and reported by Fijen et al 62. Single oral doses of RWJ 67657 dose-dependently decreased symptoms and elevated cytokine levels, induced after administration of endotoxin. Furthermore single-dose pharmacokinetics and pharmacodynamics of RWJ 67657 were investigated in healthy male subjects 63. RWJ 67657 was rapidly absorbed (mean tmax = 0.6-2.5 h) and there were no significant adverse effects associated with single doses of this drug. This study demonstrates that RWJ 67657 has acceptable safety and pharmacokinetics to warrant further investigation in a repeat-dose setting. Other compounds that have been investigated in rheumatoid arthritis patients are VX-74564 and SCIO-469, which is now in phase II clinical trial 65. 5. CONCLUSIONS The discovery of p38 MAPK inhibitors have dramatically increased the understanding of signal transduction pathways involved in inflammation. It is widely expected that p38 MAPK inhibitors will have efficacy in arthritis and other inflammatory diseases. However, clinical trials have been stopped due to safety issues. One of the reasons for these undesirable effects might be the cross-reactivity against other kinases. Solutions for these problems might lie in the development of non-ATP competitive inhibitors or inhibitors, which target other molecules in the p38 MAPK pathway. REFERENCES 1 Choy EH, Panayi GS. Cytokine pathways and joint inflammation in rheumatoid arthritis.

N.Engl.J.Med. 2001; 344: 907-16. 2 Stastny P. Association of the B-cell alloantigen DRw4 with rheumatoid arthritis. N.Engl.J.Med.

1978; 298: 869-71. 3 McDonagh JE, Dunn A, Ollier WE, Walker DJ. Compound heterozygosity for the shared

epitope and the risk and severity of rheumatoid arthritis in extended pedigrees. Br.J.Rheumatol. 1997; 36: 322-7.

4 Nielen MM. Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. Arthritis and rheumatism 2004; 50: 380-6.


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5 Firestein GS, Zvaifler NJ. How important are T cells in chronic rheumatoid synovitis?: II. T cell-independent mechanisms from beginning to end. Arthritis Rheum. 2002; 46: 298-308.

6 Bombara MP, Webb DL, Conrad P et al. Cell contact between T cells and synovial fibroblasts causes induction of adhesion molecules and cytokines. J.Leukoc.Biol. 1993; 54: 399-406.

7 Dayer JM, Graham R, Russell G, Krane SM. Collagenase production by rheumatoid synovial cells: stimulation by a human lymphocyte factor. Science 1977; 195: 181-3.

8 Bathon JM, Martin RW, Fleischmann RM et al. A comparison of etanercept and methotrexate in patients with early rheumatoid arthritis. N.Engl.J.Med. 2000; 343: 1586-93.

9 Whittle SL, Hughes RA. Folate supplementation and methotrexate treatment in rheumatoid arthritis: a review. Rheumatology.(Oxford) 2004; 43: 267-71.

10 Genestier L, Paillot R, Quemeneur L, Izeradjene K, Revillard JP. Mechanisms of action of methotrexate. Immunopharmacology 2000; 47: 247-57.

11 Li EK, Tam LS, Tomlinson B. Leflunomide in the treatment of rheumatoid arthritis. Clin.Ther. 2004; 26: 447-59.

12 Feldmann M, Maini RN. Anti-TNF alpha therapy of rheumatoid arthritis: what have we learned? Annu.Rev.Immunol. 2001; 19: 163-96.

13 Olsen NJ, Stein CM. New drugs for rheumatoid arthritis. N.Engl.J.Med. 2004; 350: 2167-79. 14 Smolen JS, Steiner G. Therapeutic strategies for rheumatoid arthritis. Nat.Rev.Drug Discov.

2003; 2: 473-88. 15 Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK,

and p38 protein kinases. Science 2002; 298: 1911-2. 16 Herlaar E, Brown Z. p38 MAPK signalling cascades in inflammatory disease. Mol.Med.Today

1999; 5: 439-47. 17 Chang F, Steelman LS, Lee JT et al. Signal transduction mediated by the Ras/Raf/MEK/ERK

pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention. Leukemia 2003; 17: 1263-93.

18 English JM, Cobb MH. Pharmacological inhibitors of MAPK pathways. Trends Pharmacol.Sci. 2002; 23: 40-5.

19 Barr RK, Bogoyevitch MA. The c-Jun N-terminal protein kinase family of mitogen-activated protein kinases (JNK MAPKs). Int.J.Biochem.Cell Biol. 2001; 33: 1047-63.

20 Han Z, Boyle DL, Chang L et al. c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J.Clin.Invest 2001; 108: 73-81.

21 Lee JC, Kumar S, Griswold DE, Underwood DC, Votta BJ, Adams JL. Inhibition of p38 MAP kinase as a therapeutic strategy. Immunopharmacology 2000; 47: 185-201.

22 Diks SH, Richel DJ, Peppelenbosch MP. LPS signal transduction: the picture is becoming more complex. Curr.Top.Med.Chem. 2004; 4: 1115-26.

23 Hayashi M, Lee JD. Role of the BMK1/ERK5 signaling pathway: lessons from knockout mice. J.Mol.Med. 2004; 82: 800-8.

24 Karin M, Yamamoto Y, Wang QM. The IKK NF-kappa B system: a treasure trove for drug development. Nat.Rev.Drug Discov. 2004; 3: 17-26.

25 Sweeney SE, Firestein GS. Signal transduction in rheumatoid arthritis. Curr.Opin.Rheumatol. 2004; 16: 231-7.

26 Handel ML, McMorrow LB, Gravallese EM. Nuclear factor-kappa B in rheumatoid synovium. Localization of p50 and p65. Arthritis Rheum. 1995; 38: 1762-70.

27 McIntyre KW, Shuster DJ, Gillooly KM et al. A highly selective inhibitor of I kappa B kinase, BMS-345541, blocks both joint inflammation and destruction in collagen-induced arthritis in mice. Arthritis Rheum. 2003; 48: 2652-9.

28 Andreakos E, Smith C, Kiriakidis S et al. Heterogeneous requirement of IkappaB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: implications for therapy. Arthritis Rheum. 2003; 48: 1901-12.

29 Tak PP, Gerlag DM, Aupperle KR et al. Inhibitor of nuclear factor kappaB kinase beta is a key regulator of synovial inflammation. Arthritis Rheum. 2001; 44: 1897-907.

30 Ortmann RA, Cheng T, Visconti R, Frucht DM, O'Shea JJ. Janus kinases and signal transducers and activators of transcription: their roles in cytokine signaling, development and immunoregulation. Arthritis Res. 2000; 2: 16-32.

Chapter 2

26

31 Heinrich PC, Behrmann I, Haan S, Hermanns HM, Muller-Newen G, Schaper F. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem.J. 2003; 374: 1-20.

32 Lee JC, Laydon JT, McDonnell PC et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 1994; 372: 739-46.

33 Obata T, Brown GE, Yaffe MB. MAP kinase pathways activated by stress: the p38 MAPK pathway. Crit Care Med. 2000; 28: N67-N77.

34 Kumar S, McDonnell PC, Gum RJ, Hand AT, Lee JC, Young PR. Novel homologues of CSBP/p38 MAP kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles. Biochem.Biophys.Res.Commun. 1997; 235: 533-8.

35 Hale KK, Trollinger D, Rihanek M, Manthey CL. Differential expression and activation of p38 mitogen-activated protein kinase alpha, beta, gamma, and delta in inflammatory cell lineages. J.Immunol. 1999; 162: 4246-52.

36 Herlaar E, Brown Z. p38 MAPK signalling cascades in inflammatory disease. Mol.Med.Today 1999; 5: 439-47.

37 Schett G, Tohidast-Akrad M, Smolen JS et al. Activation, differential localization, and regulation of the stress- activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis. Arthritis Rheum. 2000; 43: 2501-12.

38 Han J, Jiang Y, Li Z, Kravchenko VV, Ulevitch RJ. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature 1997; 386: 296-9.

39 Zhu T, Lobie PE. Janus kinase 2-dependent activation of p38 mitogen-activated protein kinase by growth hormone. Resultant transcriptional activation of ATF-2 and CHOP, cytoskeletal re-organization and mitogenesis. J.Biol.Chem. 2000; 275: 2103-14.

40 Wesselborg S, Bauer MK, Vogt M, Schmitz ML, Schulze-Osthoff K. Activation of transcription factor NF-kappaB and p38 mitogen-activated protein kinase is mediated by distinct and separate stress effector pathways. J.Biol.Chem. 1997; 272: 12422-9.

41 Kotlyarov A, Neininger A, Schubert C et al. MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nat.Cell Biol. 1999; 1: 94-7.

42 Caput D, Beutler B, Hartog K, Thayer R, Brown-Shimer S, Cerami A. Identification of a common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflammatory mediators. Proc.Natl.Acad.Sci.U.S.A 1986; 83: 1670-4.

43 Frevel MA, Bakheet T, Silva AM, Hissong JG, Khabar KS, Williams BR. p38 Mitogen-activated protein kinase-dependent and -independent signaling of mRNA stability of AU-rich element-containing transcripts. Mol.Cell Biol. 2003; 23: 425-36.

44 Dean JL, Sully G, Clark AR, Saklatvala J. The involvement of AU-rich element-binding proteins in p38 mitogen-activated protein kinase pathway-mediated mRNA stabilisation. Cell Signal. 2004; 16: 1113-21.

45 Brook M, Sully G, Clark AR, Saklatvala J. Regulation of tumour necrosis factor alpha mRNA stability by the mitogen-activated protein kinase p38 signalling cascade. FEBS Lett. 2000; 483: 57-61.

46 Miyazawa K, Mori A, Miyata H, Akahane M, Ajisawa Y, Okudaira H. Regulation of interleukin-1beta-induced interleukin-6 gene expression in human fibroblast-like synoviocytes by p38 mitogen-activated protein kinase. J.Biol.Chem. 1998; 273: 24832-8.

47 Ridley SH, Sarsfield SJ, Lee JC et al. Actions of IL-1 are selectively controlled by p38 mitogen-activated protein kinase: regulation of prostaglandin H synthase-2, metalloproteinases, and IL-6 at different levels. J.Immunol. 1997; 158: 3165-73.

48 Reunanen N, Li SP, Ahonen M, Foschi M, Han J, Kahari VM. Activation of p38 alpha MAPK enhances collagenase-1 (matrix metalloproteinase (MMP)-1) and stromelysin-1 (MMP-3) expression by mRNA stabilization. J.Biol.Chem. 2002; 277: 32360-8.

49 Lasa M, Abraham SM, Boucheron C, Saklatvala J, Clark AR. Dexamethasone causes sustained expression of mitogen-activated protein kinase (MAPK) phosphatase 1 and phosphatase-mediated inhibition of MAPK p38. Mol.Cell Biol. 2002; 22: 7802-11.

50 Theodosiou A, Ashworth A. MAP kinase phosphatases. Genome Biol. 2002; 3: REVIEWS3009.


27

51 Franklin CC, Kraft AS. Conditional expression of the mitogen-activated protein kinase (MAPK) phosphatase MKP-1 preferentially inhibits p38 MAPK and stress-activated protein kinase in U937 cells. J.Biol.Chem. 1997; 272: 16917-23.

52 Toh ML, Yang Y, Leech M, Santos L, Morand EF. Expression of mitogen-activated protein kinase phosphatase 1, a negative regulator of the mitogen-activated protein kinases, in rheumatoid arthritis: up-regulation by interleukin-1beta and glucocorticoids. Arthritis Rheum. 2004; 50: 3118-28.

53 Xu Q, Konta T, Nakayama K et al. Cellular defense against H2O2-induced apoptosis via MAP kinase-MKP-1 pathway. Free Radic.Biol.Med. 2004; 36: 985-93.

54 Lee JC, Griswold DE, Votta B, Hanna N. Inhibition of monocyte IL-1 production by the anti-inflammatory compound, SK&F 86002. Int.J.Immunopharmacol. 1988; 10: 835-43.

55 Young PR, McLaughlin MM, Kumar S et al. Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site. J.Biol.Chem. 1997; 272: 12116-21.

56 Wadsworth SA, Cavender DE, Beers SA et al. RWJ 67657, a potent, orally active inhibitor of p38 mitogen-activated protein kinase. J.Pharmacol.Exp.Ther. 1999; 291: 680-7.

57 Badger AM, Bradbeer JN, Votta B, Lee JC, Adams JL, Griswold DE. Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function. J.Pharmacol.Exp.Ther. 1996; 279: 1453-61.

58 Jackson JR, Bolognese B, Hillegass L et al. Pharmacological effects of SB 220025, a selective inhibitor of P38 mitogen-activated protein kinase, in angiogenesis and chronic inflammatory disease models. J.Pharmacol.Exp.Ther. 1998; 284: 687-92.

59 Mclay LM, Halley F, Souness JE et al. The discovery of RPR 200765A, a p38 MAP kinase inhibitor displaying a good oral anti-arthritic efficacy. Bioorg.Med.Chem. 2001; 9: 537-54.

60 Nishikawa M, Myoui A, Tomita T, Takahi K, Nampei A, Yoshikawa H. Prevention of the onset and progression of collagen-induced arthritis in rats by the potent p38 mitogen-activated protein kinase inhibitor FR167653. Arthritis Rheum. 2003; 48: 2670-81.

61 Patten C, Bush K, Rioja I et al. Characterization of pristane-induced arthritis, a murine model of chronic disease: response to antirheumatic agents, expression of joint cytokines, and immunopathology. Arthritis Rheum. 2004; 50: 3334-45.

62 Fijen JW, Zijlstra JG, de Boer P et al. Suppression of the clinical and cytokine response to endotoxin by RWJ-67657, a p38 mitogen-activated protein-kinase inhibitor, in healthy human volunteers. Clin.Exp.Immunol. 2001; 124: 16-20.

63 Parasrampuria DA, de Boer P, Desai-Krieger D, Chow AT, Jones CR. Single-dose pharmacokinetics and pharmacodynamics of RWJ 67657, a specific p38 mitogen-activated protein kinase inhibitor: a first-in-human study. J.Clin.Pharmacol. 2003; 43: 406-13.

64 Haddad JJ. VX-745. Vertex Pharmaceuticals. Curr.Opin.Investig.Drugs 2001; 2: 1070-6. 65 Nikas SN, Drosos AA. SCIO-469 Scios Inc. Curr.Opin.Investig.Drugs 2004; 5: 1205-12.

3

Effects of RWJ 67657, a p38 mitogen activated protein

kinase (MAPK) inhibitor, on the production of

inflammatory mediators by rheumatoid synovial

fibroblasts

Johanna Westra1

Pieter C Limburg1,2

Peter de Boer3

Martin H van Rijswijk1

From the Departments of 1 Rheumatology, 2 Pathology and Laboratory Medicine,

University Medical Center Groningen, The Netherlands, and 3 Pharmaceutical

Research and Development, Johnson and Johnson, Saunderton, United Kingdom

Annals of the Rheumatic Diseases 2004; 63 (11): 1453 – 1459

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ABSTRACT Objective. To investigate the effect of the p38 mitogen activated protein kinase (MAPK) inhibitor RWJ 67657 on inflammatory mediator production by rheumatoid synovial fibroblasts (RSF). Methods. RSF were pretreated with RWJ 67657 and stimulated with TNF-α and/or IL-1β. Protein levels and mRNA expression of MMP-1, MMP-3, TIMP-1, IL-6 and IL-8 were determined, as was mRNA expression of COX-2 and ADAMTS-4. Results. MMP-3 production was significantly inhibited at 1 µM RWJ 67657, MMP-1 production at 10 µM, whereas TIMP-1 production was not inhibited. Significant inhibition of IL-6 and IL-8 protein production was already seen at 0.1 µM of RWJ 67657. mRNA expression profiles were in concordance with protein production. Significant inhibition of COX-2 mRNA expression already occurred at 0.01 µM. Conclusion. RWJ 67657 inhibits major proinflammatory mediator production in stimulated RSF at pharmacological relevant concentrations. These findings could have important relevance for treatment of rheumatoid arthritis. Key words. p38 MAPK inhibitor, synovial fibroblast, matrix metalloproteinase, cytokine, rheumatoid arthritis

p38 MAPK inhibition in RSF

31

INTRODUCTION The pathogenesis of rheumatoid arthritis (RA) involves complex interrelations between T cells, macrophages, fibroblasts and other immune cells. Growing evidence suggests that activated rheumatoid synovial fibroblasts (RSF) play a major role in both initiating and driving RA 1. Specially, RSF in the lining layer display numerous features of cellular activation that ultimately result in an aggressive, invasive behaviour. These cells can attach to the articular cartilage and invade the extra cellular matrix. Furthermore, RSF are important producers of inflammatory mediators such as cytokines and matrix-metalloproteinases (MMP). Many of these mediators are regulated by mitogen activated protein kinase (MAPK) pathways and downstream transcription factors 2. At least 3 subgroups of MAPK have been identified. These are the extra cellular signal regulated kinases (ERK), the c-Jun N-terminal or stress activated protein kinases (JNK/SAPK) and the p38 MAPK 3. In general ERK are activated by growth factors and hormones, whereas both JNK and p38 MAPK are activated by environmental stress and inflammatory cytokines 4. The involvement of p38 MAPK in the production of inflammatory mediators by fibroblasts has been reported in recent years. The role of p38 MAPK in relation to interleukin-6 (IL-6) and interleukin-8 (IL-8) production has been established in RSF 5. Also involvement of p38 MAPK in MMP-production was demonstrated in dermal fibroblasts 6 and gingival fibroblasts 7. An other matrix-degrading enzyme, aggrecanase-1 or ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin-1 motif), induced by cytokines in RSF, is important in cartilage degradation in RA 8. However its signal transduction pathways are not known at the moment. Prostaglandins have also been described as being under the influence of p38 MAPK 9. This has been confirmed in a study in which it was reported that glucocorticoids destabilize cyclo-oxygenase-2 (COX-2) mRNA by inhibiting the p38 MAPK route10. Interest in protein kinases as drug targets has increased in the recent years; in particular, p38 MAPK inhibitors have been developed 11-13, because p38 plays an important role as a major signal transducer responding to cellular stress stimuli such as cytokines. Because the production of interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) is influenced by p38 MAPK, p38 MAPK inhibitors are expected to inhibit not only the production of these principal pro-inflammatory cytokines, but also their subsequent actions, leading to interruption of the vicious cycle that often occurs in inflammatory diseases. The use of p38 MAPK inhibitors therefore could provide an important advantage in therapy. The p38 MAPK inhibitor RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol) has been shown to inhibit the release of TNF-α from lipopolysaccharide-treated human peripheral blood mononuclear cells with an IC50 of 3 nM, as well as inhibiting the release of TNF-α from peripheral blood mononuclear cells treated with the super antigen staphylococcal enterotoxin B14. The compound was approximately 10-fold more potent than the reference standard p38 MAPK inhibitor SB 203580 in all p38 dependent in vitro systems tested. RWJ 67657 specifically inhibited the enzymatic activity of recombinant p38 α and β, but not of γ and δ in vitro, and had no significant activity against a variety of other kinases 14. Furthermore it was reported that this compound suppressed clinical and cytokine

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32

responses to endotoxin in healthy human volunteers 15. Recently a study was published on pharmacokinetics and pharmacodynamics of RWJ 67657 in humans 16. This study demonstrated acceptable safety and pharmacokinetic characteristics, warranting further investigation in a repeat-dose setting. In the present study we investigated the effects of RWJ 67657 on the release of pro-inflammatory mediators produced by RSF, after stimulation with IL-1β or TNF-α or both. Collagenase-1 (MMP-1), stromelysin-1 (MMP-3) and the tissue inhibitor of matrix-metalloproteinases (TIMP-1) were studied at both on protein- and on mRNA expression level. The same was done for IL-6 and IL-8. In addition we looked at the effects on mRNA expression levels of aggrecanase-1 (ADAMTS-4) and COX-2. MATERIALS AND METHODS Reagents RWJ 67657 was provided by Johnson and Johnson (R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey, USA). Recombinant human IL-1β and recombinant human TNF-α were purchased from R&D Systems (Minneapolis, Minnesota, USA). Foetal calf serum (FCS) and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from Biowhittaker (Verviers, Belgium). Anti-CD14 antibodies were from IQP (Groningen, The Netherlands) and anti-fibroblast antibodies (clone 5B5) were from Dako (Glostrup, Denmark). All reagents for RNA isolation and reverse transcriptase reaction were obtained from Invitrogen, Life Technologies (Gaithersburg, Maryland, USA). Reagents for real-time RT-PCR were obtained from Applied Biosystems (Foster City, California, USA). Specific antibodies to p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 were purchased from Cell Signalling Technologies (Beverly, Massachusetts, USA). Isolation and culture of rheumatoid synovial fibroblasts (RSF) Synovial fibroblasts were isolated from synovium of 8 RA patients, who underwent total joint replacement. Synovium was minced and digested with 1 mg/ml collagenase (type 1A, Sigma, Zwijndrecht, The Netherlands) in DMEM (with L-glutamin and gentamycin) for two hours at 37ºC. The cell suspension was filtered through a cell strainer (70 µm) (Beckton Dickinson, Franklin Lakes, New Jersey, USA) and washed with phosphate buffered saline. Cells were cultured in a 5% CO2/37ºC incubator in DMEM with 10% FCS, and non-adherent cells were discarded after overnight incubation. At passage 3 the cell population consisted of CD14 neg/5B5 positive cells (fibroblast-like synoviocytes) and these cells were used for experiments until passage 8. For all experiments the cells were plated in 6-well or 48-well plates and serum starved for 24 hours in DMEM +1% FCS to synchronise cells in a non-activating and non-proliferating phase. Next they were pretreated with increasing concentrations (0.001 µM - 10 µM) of RWJ 67657 (stock solution 10 mM in DMSO) for 1 hour before stimulation with 1 ng/ml IL-1β and/or TNF-α.


33

Determination of MMP-1, MMP-3, TIMP-1, IL-6 and IL-8 levels in cell culture supernatants Confluent synovial fibroblasts (n=5) were plated in 48-well plates (10000 cells /ml per well) and treated as above. After 48 hours stimulation, supernatants were harvested and concentrations of MMP-1, MMP-3, TIMP-1, IL-6 17, and IL-8 18 were determined using enzyme linked immunosorbent assays (ELISAs) developed in our laboratory. The MMP-3 ELISA has been described previously 19. Briefly, 96-well plates (Greiner M129A) were precoated with F(ab)2 fragments of goat-anti-mouse IgG-Fc (Jackson, West Grove, Pennsylvania, USA) in 0.1 M carbonate buffer (pH=9.6) for at least 48 hours. Plates were subsequently coated with monoclonal antibody anti-MMP-3 (clone 10D6, R&D Systems) for 1 hour at 37°C. After sample incubation, bound MMP-3 was detected with rabbit-anti-human MMP-3 (Ab 810, Chemicon, Temecula, California, USA), and F(ab)2-goat-anti-rabbit IgG labelled with peroxidase (Zymed, San Francisco, California, USA). The colour-reaction was achieved with tetramethylbenzidin (TMB) (Roth, Karlsruhe, Germany). For the MMP-1 ELISA we used monoclonal anti-MMP-1 (clone 36665.111) and biotinylated goat-anti-human MMP-1 (both from R&D Systems). The TIMP-1 antibodies (R&D Systems) in the ELISA were monoclonal anti-TIMP-1 (clone 63515.111) and biotinylated goat-anti-human TIMP-1. The detection of the biotinylated antibodies was performed with streptavidin-poly-HRP (CLB, Amsterdam, The Netherlands) and TMB colour-reaction. RNA isolation Synovial fibroblasts (n=6) were plated in 6-well plates (0.5 x 106 cells/ well / 4 ml) and treated as mentioned before. After six or 24 hours of stimulation total RNA was isolated from the cells with TRIzol reagent according to the manufacturers instructions (Life Technologies). After DNase treatment (DNA-free, Ambion, Austin, Texas, USA) cDNA was synthesized from 2.0 µg of total RNA using M-MLV Reverse Transcriptase and oligo (dT)24. Real-time RT- PCR For quantitative detection of mRNA expression a fluorescence based real-time RT-PCR was performed, which allows relative quantification of steady-state mRNA. The amount of emitted fluorescence is proportional to the amount of PCR product and enables the monitoring of the PCR reaction 20. For the measurement of MMP-1, MMP-3, TIMP-1, ADAMTS-4, IL-6, IL-8, COX-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 1µl of cDNA in duplicate was used for amplification by the real-time quantitative PCR system (ABI Prism 7900HT Sequence Detection System, Applied Biosystems) with specific Taqman primers/probes. The amount of target, normalized to an endogenous reference and relative to a calibrator, is given by: 2-∆∆CT in which CT is the threshold cycle. The results are expressed as fold induction relative to untreated samples. Western blotting to detect phosphorylation of p38 MAPK and MAPKAPK-2 Phosphorylation of p38 MAPK was analysed by western blotting. Synovial fibroblasts were plated in 6-well plates (0.5 x 106 cells/ well/4 ml) and treated as mentioned

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above. After stimulation with TNF-α and/or IL-1β up till 60 minutes, cell extracts were prepared by lysing the cells with 1x SDS sample buffer (containing 2% SDS, 10% glycerol, 50 mM dithiothreitol, 62.5 mM Tris-HCl (pH=6.8) and 0.01% brome-phenol blue). Cells were scraped off the wells and the lysates were subsequently sonicated for 5-10 seconds and boiled for five minutes. After centrifugation the samples were loaded onto a 10% SDS-PAGE gel and resolved by running at 200 V and 15 Watt constant. Semidry-blotting onto nitrocellulose membrane was followed by immunoblotting with specific antibodies to p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2. Enhanced chemi-luminescence (ECL) detection was done according to the manufacturers guidelines (Lumi-Lightplus, Roche Diagnostics, Mannheim, Germany). Effects of RWJ 67657 on phosphorylation of p38 MAPK and its downstream substrate MAPKAPK-2 were measured after incubation of the cells with the p38 MAPK inhibitor and stimulation with TNF-α and/or IL-1β for 30 minutes. STATISTICS One-way ANOVA with Dunnett’s post test or Bonferroni’s Multiple Comparison Test was employed using GraphPad Prism version 3.00 for Windows, GraphPad Software (San Diego, CA, USA). RESULTS Effect of RWJ-67657 on protein production by rheumatoid synovial fibroblasts Figure 1 shows the results of the production of MMP-1, MMP-3, TIMP-1, IL-6 and IL-8 by RSF (n=5) after stimulation with TNF-α and/or IL-1β, and also after pre-treatment with RWJ 67657 is depicted. Production of MMP-3 after stimulation with IL-1β without RWJ 67657 led to higher production compared to stimulation with TNF-α (15.0 fold), and the same was true to a lesser extent for MMP-1 (1.2 fold). TIMP-1 production was not induced when stimulated with either cytokine. Stimulation with both cytokines had a synergistic effect on MMP-3 production: 22.7 fold compared to TNF-α alone and 1.6 fold compared to IL-1β alone. Pre-incubation with RWJ 67657 resulted in a significant dose-dependent decrease in MMP-3 production when cells were stimulated with IL-1β alone or together with TNF-α. Only a high concentration (10 µM) of the p38 MAPK inhibitor had an effect on MMP-1 production, while for TIMP-1 there was no effect of p38 MAPK treatment. Stimulation with IL-1β led to higher productions of IL-6 and IL-8 than stimulation with TNF-α: 24.0 fold for IL-6 and 14.3 fold for IL-8. A dose-dependent decrease in IL-6 and IL-8 production was seen after pre-treatment with RWJ 67657. We calculated the average percentage inhibition caused by treatment with RWJ 67657 compared to stimulated cells is shown. More than 50% inhibition of MMP-3 and IL-8 production could be achieved at 1 µM, more than 50% inhibition of MMP-1 at 10 µM and more than 50% inhibition of IL-6 production at 0.1 µM. Control experiments were done by adding 0.1% DMSO (at a concentration of 10 µM RWJ 67657) to stimulated RSF (n=3). No significant inhibition of protein production could be detected as a result of the DMSO (data not shown).


35

Figure 1. Protein production of MMP-1, MMP-3, TIMP-1, IL-6 and IL-8 by rheumatoid synovial fibroblasts (n=5). Cells were stimulated with TNF-α and/or IL-1β for 48 hours and pre-treated with a concentration range of RWJ 67657 (t=-1h). Protein production was measured in supernatants by ELISA and expressed in ng/ml. Legends: unst = unstimulated, 0-10 = concentration RWJ 67657 added. Bars show mean and SEM. (* p< 0.05, ** p<0.001, Dunnett’s post test, tested against the stimulated control). IL, interleukin; MMP, matrix-metalloproteinase; TIMP-1, tissue inhibitor of matrix-metalloproteinases; TNF, tumor necrosis factor Effect of RWJ 67657 on mRNA expression A time-course study after stimulation with TNF-α and/or IL-1β was carried out to determine the time required for optimal mRNA expression. MMP-1 and MMP-3 mRNA expression was maximal after 24 hours, while IL-6, IL-8 and COX-2 mRNA had already reached maximum expression after six hours (data not shown).

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Figure 2. mRNA expression of MMP-1, MMP-3, TIMP-1 and ADAMTS-4 of rheumatoid synovial fibroblasts (n=3). Cells were stimulated with TNF-α and/or IL-1β for 6 and 24 hours and pre-treated with a concentration range of RWJ 67657. mRNA expression was determined with real-time RT-PCR (reverse transcriptase polymerase chain reaction) and results were expressed as -fold induction compared to unstimulated cells (fold induction=1). White bars represent values after TNF-α stimulation (scale on left vertical axis); grey bars represent values after IL-1β stimulation and black bars after IL-1β+TNF-α stimulation (scale on right vertical axis). Bars show means and SEM (* p<0.05, ** p<0.001, Bonferroni Multiple Comparison Test, tested against the stimulated control). ADAMTS, a disintegrin and metalloproteinase and metalloproteinase with thrombospondin-1 motif; MMP, matrix-metalloproteinase; TIMP-1, tissue inhibitor of matrix-metalloproteinases.


37

Figure 2 shows the mean levels (expressed as -fold induction compared with untreated cells =1) for MMP-1, MMP-3, TIMP-1 and ADAMTS-4 mRNA expression of three different RSF after six and 24-hour stimulation and after pre-treatment with RWJ 67657. As with protein production, mRNA expression of MMP-3 was much greater after stimulation with IL-1β than after stimulation with TNF-α: 40.1 fold after six hours of stimulation, and up to 149.4 fold after 24 hours. The equivalent values for MMP-1 mRNA expression were 3.4 fold after six hours, and 2.1 after 24 hours, although the absolute expression increased with time. TIMP-1 mRNA expression hardly increased after stimulation (maximum two- to threefold compared with unstimulated cells). ADAMTS-4 mRNA expression could be measured in synovial fibroblasts and increased with time. Again the expression after IL-1β stimulation was greater than after TNF-α stimulation (4.7-fold after six hours, 2.3-fold after 24 hours). Inhibition by RWJ 67657 could be detected for MMP-1 mRNA, MMP-3 mRNA and ADAMTS-4 mRNA at both time points and after treatment with all stimuli. However, significant inhibition was seen for MMP-1 mRNA expression after six hours of IL-1β stimulation at 1µM RWJ 67657 and more. Significant inhibition of MMP-3 mRNA expression also occurred after six hours of IL-1β stimulation at 0.1µM and more, and with both stimuli at 1 µM and more. TIMP-1 mRNA expression increased after 24 hours of stimulation with increasing concentrations of RWJ 67657. This effect was significant at 10 µM. ADAMTS-4 mRNA expression was significantly inhibited after six hours stimulation with both cytokines at 1 µM RWJ 67657 and more. Figure 3 shows mRNA levels, expressed as -fold induction compared with unstimulated cells (-fold induction =1) for IL-6, IL-8 and COX-2 after stimulation for six hours and after treatment of three different RSF with different concentrations of RWJ 67657. Again there was a difference in expression after stimulation with TNF-α or IL-1β. For IL-6, IL-8 and COX-2 respectively, stimulation with IL-1β gave 38.0-fold, 21.1-fold, and 18.3-fold higher expression than after stimulation with TNF-α. A significant inhibition of IL-6 mRNA expression was already seen at 0.01 µM RWJ 67657 when RSF were stimulated with IL-1β alone or together with TNFα. Inhibition of IL-8 mRNA expression was not significant, possibly because of a large interindividual response in IL-8 expression. COX-2 mRNA expression was significantly inhibited at 0.01µM RWJ 67657, when the cells were stimulated with IL-1β or IL-1β+TNF-α combination. To determine whether RWJ 67657 also affected cells that were already stimulated, 0.01 µM and 1 µM of p38 MAPK inhibitor was added before and one hour after TNF-α + IL-1β stimulation of two RSF cultures, and IL-6 and COX-2 mRNA expression was analysed. One hour after stimulation, phosphorylation of p38 MAPK had already reached maximum. When RWJ 67657 was added one hour before stimulation, the decrease in mRNA expression with RWJ 67657 concentrations of 0.01 µM and 1 µM was 59.8% and 97.9% respectively, for COX-2, and 38.4% and 71.5% for IL-6. When RWJ 67657 was added one hour after stimulation these values were 57.1% and 95.4% for COX-2 and 45.2 % and 81.0% for IL-6. This shows that the p38 MAPK inhibitor also inhibits inflammatory mediator production in previously activated rheumatoid synovial cells. Control experiments were carried out by adding 0.1% DMSO to stimulated RSF (RWJ 67657concentration 10 µM). Significant reduction by 0.1%

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Figure 3. mRNA expression of IL-6, IL-8 and COX-2 of rheumatoid synovial fibroblasts (n=3). Cells were stimulated with TNF-α and/or IL-1β for six hours and pretreated with a concentration range of RWJ 67657. mRNA expression was determined with real-time RT-PCR (reverse transcriptase polymerase chain reaction) and results were expressed as fold induction compared to unstimulated cells (-fold induction=1). White bars represent values after TNF-α stimulation (scale on left vertical axis); grey bars represent values after IL-1β stimulation and black bars after IL-1β+TNF-α stimulation (scale on right vertical axis). Bars show means and SEM (* p<0.05, ** p<0.001, Bonferroni Multiple Comparison Test, tested against the stimulated control). COX, cyclo-oxygenase; IL, interleukin; TNF, tumor necrosis factor. DMSO was seen only after IL-1β-induced IL-6 mRNA expression and TNF-α + IL-1β induced COX-2 mRNA expression. However, in both cases a significant reduction in mRNA expression was already found with 0.01 µM RWJ 67657 at non-inhibiting DMSO concentrations. Effect of RWJ 67657 on phosphorylation First we investigated the phosphorylation rate of p38 MAPK after stimulation with TNF-α and/or IL-1β in rheumatoid synovial fibroblasts. As shown in figure 4A, phosphorylation occurred rapidly and started after five minutes, reaching its maximum at 15 to 30 minutes for both stimuli. As expected, no inhibition of phosphorylation of p38 MAPK by RWJ 67657 was found (figure 4B). For MAPKAPK-2, which is a


39

Figure 4. (A) Representative presentation of phosphorylation of p38 MAPK in rheumatoid synovial fibroblasts after stimulation with TNF-α and/or IL-1β at different time points. Phosphorylation was measured by Western blot using specific antibodies. (B) Effect of RWJ 67657 on phosphorylation of the direct substrate of p38 MAPK, MAPKAPK-2, measured after 30 minutes of stimulation. IL, interleukin; MAPK, mitogen activated protein kinase; TNF, tumor necrosis factor.

direct downstream substrate of p38 MAPK, a strong inhibition of phosphorylation was demonstrated at concentrations down to 0.1 µM RWJ 67657. DISCUSSION In this study we showed significant inhibition by the p38 MAPK inhibitor RWJ 67657 of proinflammatory mediator and protease production in rheumatoid synovial fibroblasts. When inhibition was seen at the protein level, there was also inhibition at the level of mRNA expression, which means that this inhibition is at least at the level of RNA transcription. TNF-α and IL-1 are considered the most important cytokines in the process of inflammation in rheumatoid arthritis. Studies in experimental models have shown that TNF-α is indeed a pivotal cytokine in acute joint swelling, whereas IL-1β is the dominant cartilage destroying cytokine 21. Therefore we used both cytokines for activation of synovial fibroblasts to investigate the effects of a p38 MAPK inhibitor. Stimulation of RSF with IL-1β or TNF-α had different effects. Production of MMP-3 was greater after stimulation with IL-1β than with TNF-α, although there was a synergistic effect. Significant inhibition of induced production was seen when the cells were pretreated with 1 µM of the p38 MAPK inhibitor. MMP-1 protein production could be induced after stimulation (five- to sevenfold), but relevant inhibition was seen

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only at a concentration of 10 µM of RWJ 67657, which is too high for use in humans. The study by Parasrampuria et al 16 showed that a single oral dosage of 0.25 to 30 mg/kg resulted in plasma concentrations of RWJ 67657 between 0.01 µM and 6 µM. No up- or down regulation was seen for TIMP-1 production after stimulation and treatment with RWJ. TIMP-1 is constitutively expressed, and the expression is not influenced by TNF-α or IL-1β, and consequently not by p38 MAPK inhibition either. With quantitative real-time RT-PCR we measured mRNA levels of MMP-1, MMP-3, TIMP-1 and ADAMTS-4. IL-1β induced higher levels of mRNA expression for MMP-1, MMP-3 and ADAMTS-4 than TNF-α. Moreover, we showed inhibition of mRNA expression for these genes by the p38 MAPK inhibitor. Others have found that activation of p38 MAPK in human skin fibroblasts enhances MMP-1 and MMP-3 expression by mRNA stabilization 22. This is in agreement with our findings, indicating that inhibiting the p38 MAPK signal transduction route in RSF decreased expression of MMP-3 mRNA and to a lesser extent of MMP-1 mRNA. Work from our group has established the importance of MMP-3 as indicator for radiological progression in early RA, especially of joint space narrowing, which represents cartilage degradation 23. As others have shown that both aggrecanases and matrix-metalloproteinases degrade cartilage in human joints 24;25, the inhibition by RWJ 67657 could be of importance in the treatment of RA. The expression of TIMP-1 mRNA was only affected after 24 hours stimulation with increasing concentrations of RWJ 67657, which could have a protective effect by neutralising MMPs. As GAPDH levels were constant, possible adverse effects of RWJ 67657 did not induce this phenomenon. IL-6 and IL-8 are important cytokines in inflammation and both are present at high concentrations in synovial fluids of RA patients17. Strong induction of both cytokines in RSF could be demonstrated after IL-1β stimulation particularly. RWJ 67657 significantly inhibited this induced IL-6 and IL-8 production at 0.01 µM and 0.1 µM. Suzuki et al. 5 reported the decrease of IL-6 and IL-8 protein production after treatment with SB 203580, but no effect on mRNA expression, measured by traditional RT-PCR at a concentration of 30 µM. With quantitative real-time RT-PCR we detected inhibition of both IL-6 and IL-8 mRNA expression. Therefore this study showed that IL-6 and IL-8 are inhibited by RWJ 67657 both at the protein- and mRNA level. In 1998 Guan et al reported that the induction of COX-2 and production of PGE2 were directly linked to activation of MEKK1 and consequently to activation of p38 MAPK 26. Recently it was reported that COX-2 mRNA stability is under regulation of p38 MAPK10;27. In our study we demonstrated upregulation of COX-2 mRNA after stimulation with IL-1β or TNFα, and very strong inhibition by RWJ 675657 especially after IL-1β-induced expression. Effects of p38 MAPK inhibition are partly mediated through its downstream kinase MAPKAPK-2 and may involve phosphorylation of hsp2728. Our results showed that after 30 minutes p38 MAPK was already maximally phosphorylated and that MAPKAPK-2 phosphorylation was blocked at a concentration of 0.1 µM. Addition of RWJ 67657 to stimulated cells did not affect the inhibitory capacities of the compound, so inhibition of the p38 MAPK signal transduction route in activated cells is possible. p38 MAPK inhibitors have very potent effects on TNF-α production by LPS stimulated monocytes at low concentration. For


41

RWJ 67657 this was established at 3 nM 14. For monocyte-derived-macrophages 50% inhibition was reached at a concentration of 30 nM 29. Our study here clearly showed that IL-1β is a stronger inducer of expression of inflammatory mediators by synovial fibroblasts than TNF-α and may as such be the major cartilage destructive cytokine. p38 MAPK inhibitors such as RWJ 67657 inhibit both IL-1β and TNF-α, as well as their responses induced by these cytokines. This dual activity of p38 MAPK inhibitors may be of major importance in the treatment of RA and other inflammatory conditions. p38 MAPK inhibitor have effects on different cell types, which could enhance the therapeutic effects, but also increase the risk of side effects. In the past, clinical trials with other p38 MAPK inhibitors have been stopped due to safety issues. One of the reasons for undesirable effects might be the cross-reactivity against other kinases, which was not the case for RWJ 67657. Furthermore we excluded induction of apoptosis in RSF following incubation with RWJ 67657 by staining cells with Annexin V and propidium iodide as described previously 30 (data not shown). However, RWJ 67657 has been shown to have acceptable safety and acceptable pharmacokinetics characteristics, warranting further investigation 16. There were no adverse effects associated with single doses of this drug. While the preliminary pharmacokinetic data suggest a twice-daily dosing regimen, our data show significant effects at low concentrations. More research upon the effects of p38 MAPK inhibition on other cell types involved in inflammation will establish its applicability as drug in the near future. The results presented in this study are very promising. ACKNOWLEDGMENTS We gratefully acknowledge dr. Fred Breukelman and dr. Lex Boerboom for delivery of the synovial tissues. We thank dr. Marco Harmsen and dr. Sigga Asgeirsdottir for assistance with the PCR experiments and dr. Miek van Leeuwen for critically reading of the manuscript. We are grateful to mrs. Berber Doornbos-van der Meer for her excellent technical assistance. This work was supported by the Dutch Rheumatology Foundation and Johnson and Johnson Pharmaceutical Research and Development, Raritan, New Jersey, USA. REFERENCES 1 Pap T, Muller-Ladner U, Gay RE, Gay S. Fibroblast biology. Role of synovial fibroblasts in the

pathogenesis of rheumatoid arthritis. Arthritis Res. 2000; 2: 361-7. 2 Firestein GS, Manning AM. Signal transduction and transcription factors in rheumatic disease.

Arthritis Rheum. 1999; 42: 609-21. 3 Cobb MH, Goldsmith EJ. How MAP kinases are regulated. J.Biol.Chem. 1995; 270: 14843-6. 4 Dong C, Davis RJ, Flavell RA. MAP kinases in the immune response. Annu.Rev.Immunol.

2002; 20: 55-72. 5 Suzuki M, Tetsuka T, Yoshida S et al. The role of p38 mitogen-activated protein kinase in IL-6

and IL-8 production from the TNF-alpha- or IL-1beta-stimulated rheumatoid synovial fibroblasts. FEBS Lett. 2000; 465: 23-7.


7 Ravanti L, Hakkinen L, Larjava H et al. Transforming growth factor-beta induces collagenase-3 expression by human gingival fibroblasts via p38 mitogen-activated protein kinase. J.Biol.Chem. 1999; 274: 37292-300.

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8 Yamanishi Y, Boyle DL, Clark M et al. Expression and regulation of aggrecanase in arthritis: the role of TGF- beta. J.Immunol. 2002; 168: 1405-12.

9 Guan Z, Baier LD, Morrison AR. p38 mitogen-activated protein kinase down-regulates nitric oxide and up- regulates prostaglandin E2 biosynthesis stimulated by interleukin-1beta. J.Biol.Chem. 1997; 272: 8083-9.

10 Lasa M, Brook M, Saklatvala J, Clark AR. Dexamethasone destabilizes cyclooxygenase 2 mRNA by inhibiting mitogen-activated protein kinase p38. Mol.Cell Biol. 2001; 21: 771-80.

11 Badger AM, Cook MN, Lark MW et al. SB 203580 inhibits p38 mitogen-activated protein kinase, nitric oxide production, and inducible nitric oxide synthase in bovine cartilage- derived chondrocytes. J.Immunol. 1998; 161: 467-73.

12 Branger J, van den BB, Weijer S et al. Anti-inflammatory effects of a p38 mitogen-activated protein kinase inhibitor during human endotoxemia. J.Immunol. 2002; 168: 4070-7.

13 Haddad JJ. VX-745. Vertex Pharmaceuticals. Curr.Opin.Investig.Drugs 2001; 2: 1070-6. 14 Wadsworth SA, Cavender DE, Beers SA et al. RWJ 67657, a potent, orally active inhibitor of

p38 mitogen-activated protein kinase. J.Pharmacol.Exp.Ther. 1999; 291: 680-7. 15 Fijen JW, Zijlstra JG, de Boer P et al. Suppression of the clinical and cytokine response to

endotoxin by RWJ-67657, a p38 mitogen-activated protein-kinase inhibitor, in healthy human volunteers. Clin.Exp.Immunol. 2001; 124: 16-20.


17 van Leeuwen MA, Westra J, Limburg PC, van Riel PL, van Rijswijk MH. Interleukin-6 in relation to other proinflammatory cytokines, chemotactic activity and neutrophil activation in rheumatoid synovial fluid. Ann.Rheum.Dis. 1995; 54: 33-8.

18 de Bont ES, Vellenga E, Swaanenburg JC, Fidler V, Visser-van Brummen PJ, Kamps WA. Plasma IL-8 and IL-6 levels can be used to define a group with low risk of septicaemia among cancer patients with fever and neutropenia. Br.J.Haematol. 1999; 107: 375-80.

19 Posthumus MD, Limburg PC, Westra J, van Leeuwen MA, van Rijswijk MH. Serum matrix metalloproteinase 3 levels during treatment with sulfasalazine or combination of methotrexate and sulfasalazine in patients with early rheumatoid arthritis. J.Rheumatol. 2002; 29: 883-9.

20 Klein D. Quantification using real-time PCR technology: applications and limitations. Trends Mol.Med. 2002; 8: 257-60.

21 van den Berg WB, Bresnihan B. Pathogenesis of joint damage in rheumatoid arthritis: evidence of a dominant role for interleukin-I. Baillieres Best.Pract.Res.Clin.Rheumatol. 1999; 13: 577-97.

22 Reunanen N, Li SP, Ahonen M, Foschi M, Han J, Kahari VM. Activation of p38 alpha MAPK enhances collagenase-1 (matrix metalloproteinase (MMP)-1) and stromelysin-1 (MMP-3) expression by mRNA stabilization. J.Biol.Chem. 2002; 277: 32360-8.

23 Posthumus MD, Limburg PC, Westra J, van Leeuwen MA, van Rijswijk MH. Serum matrix metalloproteinase 3 in early rheumatoid arthritis is correlated with disease activity and radiological progression. J.Rheumatol. 2000; 27: 2761-8.

24 Lark MW, Bayne EK, Flanagan J et al. Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints. J.Clin.Invest 1997; 100: 93-106.

25 Little CB, Flannery CR, Hughes CE et al. Aggrecanase versus matrix metalloproteinases in the catabolism of the interglobular domain of aggrecan in vitro. Biochem.J. 1999; 344 Pt 1: 61-8.

26 Guan Z, Buckman SY, Pentland AP, Templeton DJ, Morrison AR. Induction of cyclooxygenase-2 by the activated MEKK1 --> SEK1/MKK4 --> p38 mitogen-activated protein kinase pathway. J.Biol.Chem. 1998; 273: 12901-8.

27 Faour WH, He Y, He QW et al. Prostaglandin E(2) regulates the level and stability of cyclooxygenase-2 mRNA through activation of p38 mitogen-activated protein kinase in interleukin-1 beta-treated human synovial fibroblasts. J.Biol.Chem. 2001; 276: 31720-31.

28 Lasa M, Mahtani KR, Finch A, Brewer G, Saklatvala J, Clark AR. Regulation of cyclooxygenase 2 mRNA stability by the mitogen-activated protein kinase p38 signaling cascade. Mol.Cell Biol. 2000; 20: 4265-74.


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29 Westra J, Doornbos-Van Der Meer B, de Boer P, van Leeuwen MA, van Rijswijk MH, Limburg PC. Strong inhibition of TNF-alpha production and inhibition of IL-8 and COX-2 mRNA expression in monocyte-derived macrophages by RWJ 67657, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Arthritis Res.Ther. 2004; 6: R384-R392.

30 Bijl M, Horst G, Bijzet J, Bootsma H, Limburg PC, Kallenberg CG. Serum amyloid P component binds to late apoptotic cells and mediates their uptake by monocyte-derived macrophages. Arthritis Rheum. 2003; 48: 248-54.

4

Strong inhibition of TNF-α production and inhibition of

IL-8 and COX-2 mRNA expression in monocyte-derived

macrophages by RWJ 67657, a p38 mitogen activated

protein kinase (MAPK) inhibitor

Johanna Westra1

Berber Doornbos-van der Meer1

Peter de Boer3

Miek A van Leeuwen1


Pieter C Limburg1,2

From the Departments of 1 Rheumatology, 2 Pathology and Laboratory Medicine,

University Medical Center Groningen, The Netherlands, and 3 Pharmaceutical Research and Development, Johnson and Johnson,

Saunderton, United Kingdom

Arthritis Research and Therapy 2004; 6 (4): R384 – R392

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ABSTRACT In inflammatory processes the p38 mitogen activated protein kinase (MAPK) signal transduction route regulates production and expression of cytokines and other inflammatory mediators. Tumor necrosis factor α (TNF-α) is a pivotal cytokine in rheumatoid arthritis (RA) and its production in macrophages is under control of the p38 MAPK route. Inhibition of the p38 MAPK route may inhibit production not only of TNF-α, but also of other inflammatory mediators produced by macrophages, and indirectly of inflammatory mediators by other cells induced by TNF-α stimulation. Here we investigate the effects of RWJ 67657, a p38 MAPK inhibitor, on mRNA expression and protein production of TNF-α and other inflammatory mediators, in monocyte-derived macrophages (MDM). A strong inhibition of TNF-α was seen at pharmacological relevant concentrations of RWJ 67657, but also inhibition of mRNA expression of IL-1β, IL-8 and COX-2 was shown. Furthermore, it was shown that monocyte-derived macrophages have a high constitutive production of MMP-9, which is not affected by p38 MAPK inhibition. The results presented here may have important implications for the treatment of rheumatoid arthritis. Key words. COX-2, matrix metalloproteinase, monocyte-derived macrophage, p38 MAPK inhibitor, TNF-α

p38 MAPK inhibition in MDM

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INTRODUCTION Rheumatoid arthritis (RA) is characterized by chronic inflammation of synovial tissue and destruction of cartilage and bone in the joints 1. Macrophages play an important role in RA, as the rheumatoid synovium is intensively infiltrated by macrophages and their numbers correlate with clinical scores 2 and articular destruction in RA 3. RA patients with active disease display a faster generation of CD14+ myelomonocytic cells from the bone marrow and faster differentiation into HLA-DR+ cells than control individuals do 4. Activation of the monocytic lineage in inflammatory disease is not only restricted to synovial macrophages, but extends to circulating monocytes and other cells of the mononuclear phagocyte system 5. The activation state of monocytes /macrophages is characterized by increased expression and transcription of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), but also of other pro-inflammatory and regulatory cytokines and growth factors 6. Highly specific therapeutics have been developed to target these cytokines, like monoclonal antibodies, soluble receptors, binding proteins, and receptor antagonists. TNF-α blockade has been the major breakthrough in the therapy of RA during the past ten years. However more than half of the patients do not achieve adequate responses, remissions are rare and these drugs do have side effects 7;8. The importance of mitogen-activated protein kinases (MAPK) in cell biology has been reported in many studies concerning different inflammatory diseases. These MAPK belong to three families: the extracellular signal regulated kinases (ERK), the c-Jun N-terminal or stress activated protein kinases (JNK/SAPK) and the p38 MAPK. All three families have been shown to become activated in macrophages using a variety of stimuli both in primary cells and in cell lines 9. In the RA synovium p38 MAPK is predominantly activated in endothelial cells and in the lining layer 10. Inhibition of p38 MAPK therefore could provide an interesting target for intervention in inflammation as it occurs in the synovia in rheumatoid arthritis. In vitro stimulation of macrophages with LPS (lipopolysaccharide) leads to activation of MAPK cascades through the LPS receptor (CD14) or Toll-like receptors 9. The ability of bacterial toxins or super-antigens to induce pro-inflammatory responses leading to the production of TNF-α and IL-1 is relevant in view of the possible micro-organism aetiology in RA 11. Stimulation of monocytes with LPS induces a number of matrix metalloproteinases (MMPs), including two prominent monocytic MMPs: interstitial collagenase (MMP-1) and gelatinase-B (MMP-9). These enzymes are involved in the connective-tissue loss associated with chronic inflammatory diseases. In vivo a significant part of macrophage effector responses occur through cell-contact -dependent signalling with several inflammatory cells, mainly T-cells and fibroblasts. A few soluble stimuli are known to have a stimulatory effect on macrophages, like IL-15 and IL-17. It has also been reported that IL-17 induces the production of MMP-9 and cyclo-oxygenase-2 (COX-2, which is the rate-limiting enzyme in prostaglandin and leukotriene synthesis) in monocytes/macrophages 12. The p38 MAPK inhibitor RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol) has been shown to inhibit the release of TNFα from LPS-treated human peripheral blood mononuclear cells with an IC50 of 3 nM 13. Moreover this compound effectively inhibited endotoxin-induced clinical effects and cytokine release in normal healthy volunteers14. Furthermore a report was

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published in which pharmacokinetics and pharmacodynamics of RWJ 67657 were presented, showing that the compound has acceptable safety to warrant further investigation 15. Our group recently showed that RWJ 67657 significantly inhibited IL-6, IL-8, MMP-3 and COX-2 mRNA expressed by IL-1β and/or TNF-α stimulated rheumatoid synovial fibroblasts 16. It has been shown that differences occur in signalling pathways in myeloid cells in relation to the maturation stage of macrophages 17. Strong inhibition of TNF-α in monocytes has been reported due to p38 MAPK inhibition 13, but the effects in matured macrophages are not fully known. Therefore in this study we investigated the effects of the p38 MAPK inhibitor RWJ 67657 on the mRNA expression and production of inflammatory cytokines and MMPs by stimulated monocyte-derived macrophages (MDM). We investigated macrophages, differentiated in medium with pooled serum or by the method described by Plesner and co-workers 18 and compared MDMs from healthy controls and rheumatoid arthritis patients. Strong inhibition of mRNA expression and production of TNF-α by RWJ 67657 was found, as well as inhibition of IL-1β, IL-8 and COX-2 mRNA expression. MATERIALS AND METHODS Reagents RWJ 67657 was provided by Johnson and Johnson (R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey, USA). Antibodies for flowcytometry were obtained from IQProducts, Groningen, The Netherlands. Lipopolysaccharide was purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands). Recombinant human macrophage colony stimulating factor (M-CSF) and ELISA antibodies were from R&D Systems (Minneapolis, Minnesota, USA). Foetal calf serum (FCS) and RPMI 1640 culture medium were obtained from Biowhittaker (Verviers, Belgium). All reagents for RNA isolation and reverse transcriptase reaction were purchased from Invitrogen, Life Technologies (Gaithersburg, Maryland, USA). Reagents for real-time RT-PCR were obtained from Applied Biosystems (Foster City, California, USA). Specific antibodies to p38 MAPK, phospho-p38 MAPK and phospho-MAPKAPK-2 were purchased from Cell Signalling Technologies (Beverly, Massachusetts, USA) and detecting antibody peroxidase-swine-anti-rabbit was from DAKO (Glostrup, Denmark). Macrophage culture Blood was obtained from RA patients who had given informed consent, and from healthy laboratory workers. Peripheral mononuclear cells were isolated by Lymphoprep density gradient centrifugation from citrated blood. Cells were suspended in RPMI with gentamycin at 106 cells/ml and seeded in 6-well plates (Costar, Badhoevedorp, The Netherlands) in 3 ml or 0.5 ml in 24-well plates and cultured at 37°C in a 5% CO2 atmosphere. After 2 hours non-adherent cells were discarded and adherent monocytes were allowed to differentiate into macrophages in RPMI containing gentamycin, 50 ng/ml M-CSF + 1% FCS 18, while medium was refreshed at day 2. To compare methods monocytes were also differentiated in RPMI with gentamycin + 2% pooled human serum; on day 2 and 5 fresh medium was added to the wells. Cells were cultured for 5 or 7 days. We found no marked differences between


49

differentiation methods and decided to perform further experiments with macrophages differentiated with M-CSF and FCS. Flowcytometric analysis Expression of surface markers CD14 (LPS receptor), HLA-DR, CD18 (β2 integrin subunit), CD36 (GPIIIb, GPIV) and CD83 (dendritic cell marker) on monocytes in the PBMC-fraction and on differentiated macrophages was detected by flowcytometric analysis in an Epics-Elite Flowcytometer (Coulter Electronics, Mijdrecht, The Netherlands). Phosphorylation studies Phosphorylation of p38 MAPK in MDM was analyzed by western blotting. Cells were cultured in RPMI containing gentamycin, 50 ng/ml M-CSF + 1% FCS for 5 days and stimulated with 50 ng/ml LPS for various periods of time. Cell extracts were prepared by lysing the cells with 1x SDS sample buffer (containing 2% SDS, 10% glycerol, 50 mM dithiothreitol, 62.5 mM Tris-HCl (pH=6.8) and 0.01% brome-phenol blue). Cells were scraped off the wells and the lysates were subsequently sonicated for 5-10 seconds and boiled for 5 minutes. After centrifugation the samples were loaded onto a 10% SDS-PAGE gel and resolved by running at 200 V and 15 Watt constant. Semidry-blotting was performed onto nitrocellulose membrane and immunodetection was with anti-phospho-p38 MAPK and peroxidase-anti-rabbit-immnoglobulins. Enhanced chemiluminescence (ECL) detection was performed according to the manufacturers guidelines (Lumi-Lightplus, Roche Diagnostics, Mannheim, Germany). To determine the effect of RWJ 67657 on phosphorylation of p38 MAPK and its downstream substrate MAPK-activating protein kinase-2 (MAPKAPK-2) a concentration range of the p38 MAPK inhibitor (0, 0.01, 0.1, 1, 10 µM) was added 1 hour before stimulation with 50 ng/ml LPS for 30 minutes. Blotting experiments were performed with specific antibodies to p38 MAPK, phospho-p38 MAPK, and phospho-MAPKAPK-2. Determination of TNF-α, IL-1β, IL-6, IL-8, MMP-1, MMP-9 and TIMP-1 levels in cell culture supernatants MDM from 8 healthy controls and 9 RA patients, who were not treated with steroids, were cultured in 24 well-plates in RPMI containing gentamycin, 50 ng/ml M-CSF + 1% FCS for 5 days. The cells were subsequently pre-treated with increasing concentrations of RWJ 67657 (stocksolution 10 mM in dimethylsulfoxide [DMSO]), from 0. 01 µM -10 µM for 1 hour prior to stimulation with 50 ng/ml LPS for 24 hours. Cytokine (TNFα, IL-1β, IL-6 and IL-8), MMP-1, MMP-9 and tissue inhibitor of matrix metalloproteinases (TIMP)-1 levels were measured in cell supernatants by ELISA, using matched antibody pairs for ELISA and recombinant proteins as standards from R&D Systems. Detection limits for all cytokine ELISAs was 20 pg/ml. For optimal determination of MMP-1, MMP-9 and TIMP-1 96 well plates (Greiner M129A) were precoated with F(ab)2 fragments of goat-anti-mouse IgG-Fc (Jackson, West Grove, Pennsylvania, USA) in 0.1M carbonate buffer (pH=9.6) for at least 48 hours before coating of the capture antibody. After sample incubation and binding of the biotinylated detection antibodies, the color reaction was performed with

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streptavidin-poly-HRP (Sanquin, Amsterdam, The Netherlands) and tetramethyl-benzidin (TMB, Roth, Karlsruhe, Germany). The detection limit for MMP-1 and TIMP-1 was 1 ng/ml and for MMP-9, 0.1 ng/ml. RNA isolation and real-time RT-PCR Monocyte-derived macrophages from healthy controls were cultured in 6 well-plates in RPMI containing gentamycin, 50 ng/ml M-CSF + 1% FCS for 5 days. First macrophages were stimulated with 50 ng/ml LPS for different periods of time to determine optimal mRNA expression of TNF-α, IL-1β, IL-6, IL-8, MMP-9 and COX-2 genes. At the optimal time point the effect of p38 MAPK inhibition on LPS-stimulated MDM was determined by pre-treatment of the cells with increasing concentrations of RWJ 67657 for 1 hour. Total RNA was isolated from the cells with TRIzol reagent according to the manufacturers instructions (Life Technologies). After DNase treatment (Ambion DNA-free, Austin, Texas, USA) cDNA was synthesized from 1.0 µg of total RNA using M-MLV Reverse Transcriptase and oligo (dT)24 (Life Technologies). For detection of mRNA expression a fluorescence based real-time RT-PCR was performed, which allows relative quantification of steady-state mRNA. The amount of emitted fluorescence is proportional to the amount of PCR product and enables the monitoring of the PCR reaction. For the measurement of IL-1β, TNF-α, IL-6, IL-8, MMP-9, COX-2 and glyceraldehyde-3-phosphate dehydro-genase (GAPDH), 1µl of cDNA in triplicate was used for amplification by the real-time quantitative PCR system (ABI Prism 7900HT Sequence Detection System, Applied Biosystems) with specific Taqman primers/probes. The Assay-on-Demand numbers for the genes were: IL-1β: Hs00174097_m1, TNFα: Hs00174128_m1, IL-6: Hs00174131_m1, IL-8: Hs00174103_m1, MMP-9: Hs00234579_m1, COX-2: Hs00153133_m1, and GAPDH: Hs99999905_m1. The amount of target, normalized to an endogenous reference (GAPDH) and relative to a control sample, is given by: 2-∆∆CT in which CT is the threshold cycle. The results are expressed as fold induction relative to untreated samples. STATISTICS Paired T-tests were performed using GraphPad Prism version 3.00 for Windows, GraphPad Software (San Diego, CA, USA). RESULTS Macrophage differentiation Expression of surface markers on monocytes and macrophages was measured by flowcytometry. In figure 1 the mean fluorescence intensity (MFI) of CD14 and HLA-DR on monocytes (left panel) and macrophages differentiated in 50 ng/ml M-CSF and 1% FCS after 5 days (right panel) is shown, the number of cells measured in the flowcytometer. Macrophages differentiated in this way showed lower expression of CD14 compared to monocytes relative to the isotype control, whereas the HLA-DR expression did not differ between both cell types.


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Figure 1. Expression of CD14 (bold grey line) and HLA-DR (bold black line) on monocytes (left graph) and macrophages, differentiated with 50 ng/ml macrophage-colony-stimulating factor and 1% FCS after 5 days (right panel), measured by flow cytometry and expressed in mean fluorescence intensity (MFI). The isotype control is an IgG2a antibody (dashed line), the blank is in solid fill. The mean fluorescence intensity (MFI) is shown on the x-axis, while on the y-axis the number of cells measured in the flowcytometer is expressed. PE = phycoerythrin.

Expression of CD18 increased with differentiation, while expression of CD36 decreased (data not shown). Expression of CD83 was not seen on matured macrophages, excluding differentiation into dendritic cells. For further experiments monocytes differentiated in medium with 50 ng/ml M-CSF and 1% FCS for 5 days were used. Effect of RWJ 67657 on phosphorylation of p38 MAPK and MAPKAPK-2 In figure 2A a representative example is shown of phosphorylation of p38 MAPK in MDMs after stimulation with 50 ng/ml LPS. Phosphorylation occurs already after 15 minutes, is maximal at 30-60 minutes and diminishes after 2 hours. p38 MAPK is constitutively expressed in the cells as is demonstrated in the control blot. The effect of RWJ 67657 on phosphorylation of p38 MAPK and its direct downstream substrate MAPKAPK-2 measured after 30 minutes of stimulation with 50 ng/ml LPS is shown in figure 2B. RWJ 67657 does not inhibit phosphorylation of p38 MAPK, but does inhibit its activity, as can be seen from the strong inhibition at 0.01 µM and the complete inhibition of MAPKAPK-2 phosphorylation at 0.1 µM RWJ 67657. The solvent, 0.1% DMSO, did not affect phosphorylation of either kinase. Effect of RWJ 67657 on cytokine and MMP production Stimulation of MDM with 50 ng/ml LPS for 24 hours resulted in increased production of TNF-α, IL-6, IL-8 and MMP-9 both in control macrophages and in rheumatoid arthritis macrophages (figure 3A). Production of IL-1β, MMP-1 and TIMP-1 was too low for detection. Before stimulation, TNF-α production was below detection limit, while after stimulation the mean production in control MDM was 2.204 (± 1.993) ng/ml and in RA-MDM 2.150 (± 1.816) ng/ml.

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Figure 2. Effect of RWJ 67657 on phosphorylation of p38 MAPK and MAPKAPK-2. (A) Representative presentation of phosphorylation of p38 mitogen-activated protein kinase (MAPK) in monocyte-derived macrophages after stimulation with lipopolysaccharide (LPS) at various time points. Phosphorylation was measured by western blotting using specific antibodies to p38 MAPK and phospho-p38 MAPK. (B) Effect of RWJ 67657 on phosphorylation of the direct substrate of p38 MAPK, MAPKAPK-2 (MAPK-activating protein kinase-2), measured after 30 minutes of stimulation. DMSO = dimethylsulfoxide. Pre-treatment of the macrophages with increasing concentrations of RWJ 67657 showed a dose-dependent decrease of protein production for TNF-α and IL-8. Inhibition of TNFα production was seen with an IC50 of 0.015 µM for control cells and 0.03 µM for RA cells (figure 3B). For IL-8 production the IC50 was 0.3 µM for control cells and 1.2 µM for RA cells. IL-6 and MMP-9 production was inhibited only at concentrations between 1 and 10 µM RWJ 67657. Pre-treatment of MDMs with 0.1% DMSO had no significant effect on protein production (data not shown). Effects of RWJ 67657 on mRNA expression To determine optimal stimulation time points for the genes involved in this study MDM of 2 healthy controls were stimulated during 0.5, 2, 4, 8, 12, and 24 hours with 50 ng/ml LPS. mRNA expression of TNF-α, IL-1β, IL-6, IL-8 and COX-2 was measured with real-time RT-PCR as depicted in figure 4. A significant increase of mRNA expression of all but MMP-9 was found after stimulation with LPS. After 4 and 8 hours of stimulation most genes were highly expressed, IL-8 mRNA expression was still increased after 12 and 24 hours. For measurement of the effects of the p38 MAPK inhibitor MDMs were stimulated for 4 hours.


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Figure 3. Protein production of tumor necrosis factor (TNF)-α, IL-6, IL-8 and matrix metalloproteinase-9 (MMP-9) by monocyte-derived macrophages from healthy controls (n=8, open squares) and rheumatoid arthritis patients (n=9, filled squares). Cells were stimulated with LPS for 24 hours and treated one hour beforehand with RWJ 67657 at various concentrations. Protein production was measured in supernatants by ELISA and expressed in ng/ml (A). Inhibition was calculated against the stimulated control and depicted in figure B. Bars show mean and SEM. (* p< 0.05, ** p<0.001, paired T-test, calculated against the stimulated control). Unst = unstimulated.

A B

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Figure 4. Time course of induction of mRNA expression of tumor necrosis factor (TNF)-α, IL-1β, IL-6, IL-8 and cyclo-oxygenase-2 (COX-2). Monocyte-derived macrophages from healthy controls (n=2) were stimulated for increasing periods of time with lipopolysaccharide (50 ng/ml). mRNA expression was determined with real-time RT-PCR and results were calculated as fold induction in comparison to unstimulated cells (fold induction=1).

As can been seen in figure 5 there is a dose-dependent decrease in mRNA expression with increasing RWJ 67657 concentration. TNF-α mRNA expression is inhibited by 48.4% at 0.01 µM and 65.8% at 0.1 µM p38 MAPK inhibitor. IL-1β, IL-8 and COX-2 mRNA expression was reduced by 40.2%, 56.6% and 65.0% respectively at 1 µM. MMP-9 mRNA expression is not induced and not inhibited and proved to be constitutively expressed at a high level. Control incubations with 0.1% DMSO had no significant effect on mRNA expression, as can been seen in figure 5. DISCUSSION In this study we have shown the significant inhibition of TNF-α production in MDMs by the p38 MAPK inhibitor RWJ 67657. The strong inhibition was seen both at the level of mRNA expression and protein production, so inhibition is already apparent at the level of transcription. p38 MAPK activity in RA is found predominantly in the synovial lining layer and in endothelial cells in the synovium 10. The lining layer consists mainly of fibroblasts and macrophages, both important players in the process of inflammation by the production of cytokines and degrading enzymes. Recently our group demonstrated strong inhibition of IL-6, IL-8, COX-2 and MMP-3 expression in rheumatoid synovial fibroblasts by the p38 MAPK inhibitor RWJ 67657 19. Studying macrophages in vitro raises some difficulties, because isolation and culture of macrophages from synovial tissue is disturbed by the overgrowth of fibroblasts. Therefore, macrophages differentiated from peripheral blood monocytes are widely used for in vitro studies. By


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Figure 5. mRNA expression of tumor necrosis factor (TNF)-α, IL-1β, cyclo-oxygenase-2 (COX-2), IL-6, IL-8 and matrix metalloproteinase-9 (MMP-9) of monocyte-derived macrophages from healthy controls (n=5). Cells were stimulated with LPS for 4 hours and pre-treated with a concentration range of RWJ 67657. mRNA expression was determined with real-time RT-PCR and results were expressed as fold induction compared to unstimulated cells (fold induction=1). Bars show means and SEM (* p<0.05, paired T- test, calculated against the stimulated control) using M-CSF and low FCS concentrations macrophages were generated, with high HLA-DR expression, that were not activated 18. Current treatment strategies in RA, including TNF-α and IL-1- blocking agents, alone or in combination with, for example methotrexate, still have limited efficacy in a substantial proportion of patients. Recently, Redlich and co-workers reviewed the multiple pathogenesis pathways involved in RA, and the possible targets for therapies, and stressed the importance of aiming at the interference of both with the pathways leading to inflammation and with those ultimately leading to destruction 8. Inhibition of signal transduction cascades may fit in this concept, because they are involved in the activation of pro-inflammatory cytokines as well as of MMP genes. The p38 MAPK inhibitor RWJ 67657 has been reported to be specific for p38 α and β, and has no activity for other kinases 13. A first-in-human study investigating the pharmacokinetics and pharmacodynamics of RWJ 67657 demonstrated that a single

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oral intake of 0.25 ranging up to 30 mg/kg resulted in plasma levels of 0.01 µM to 6 µM 15. Furthermore the study showed that at the doses tested there were no significant adverse effects. p38 MAPK acts mainly through phosphorylation of its down-stream substrate MAPKAPK-2 and a variety of transcription factors 20. Mice that lack MAPKAPK-2 show increased stress resistance and survive LPS-induced endotoxic shock, due to a reduction of 90% in the production of TNF-α 21. The level and stability of TNF-α mRNA was not reduced in these mice, so the inhibition was at the post-transcriptional level. With western blotting we demonstrated complete inhibition of phosphorylation of MAPKAPK-2 at 0.1 µM RWJ 67657, and strong inhibition at 0.01 µM. In our study TNFα production was significantly inhibited at nanomolar concentrations RWJ 67657, and mRNA expression was also decreased, by nearly 50% at 10 nM. Inhibition of p38 MAPK activity leads to reduced TNF-α mRNA expression and therefore reduced TNFα production, while inhibition of MAPKAPK-2, as in MAPKAPK-2 knock-out mice, leads to reduced TNF-α production without affecting the mRNA levels. IL-6 production and mRNA expression were not inhibited by p38 MAPK inhibition, but IL-8 production and mRNA expression were inhibited for more than 50% at 1 µM RWJ 67657. Bhattacharyya and co-workers showed that LPS from Helicobacter pylori stimulates IL-8 release from cells of the monocytic lineage through activation of NF-κB and MAPK cascades 22, and we demonstrated that in MDM the p38 MAPK route plays an important role in this process. Already in 1992 Herzyk and co-workers published findings showing that macrophage and monocyte IL-1β regulation differs at multiple sites23. Macrophages did not differ from monocytes in LPS sensitivity but had limitations in IL-1β release. Our study shows mRNA expression of IL-1β in MDM, as well as a reduction of mRNA expression at 1 µM RWJ 67657, but protein production in MDMs was below the detection limit of the measurement. We found a reduction of 65% in COX-2 mRNA expression after treatment with 1 µM RWJ 67657. It was previously demonstrated that p38 MAPK plays a role in transcription and stabilisation of COX-2 mRNA 24. However, Caivano and Cohen showed that both p38 MAPK and ERK influence COX-2 mRNA expression through activation of mitogen- and stress-activated protein kinase-1 (MSK-1) 25, an activator of important transcription factors like ATF-2 and CREB. Our results indicate an important role for p38 MAPK in COX-2 mRNA expression in MDM. In synovial tissue, the presence of macrophages is often seen together with the expression of MMPs and TIMP-1 26. In our study MMP-1 and TIMP-1 could not be detected in the cell supernatants of MDMs after LPS stimulation, in contrast to high production of MMP-9. MMP-9 is associated with macrophages and peripheral blood mononuclear cells and has a broad substrate specificity and may contribute together with collagenases to the degradation of fibrillar collagens, basement membrane-components and stromal ECM molecules 27. Stimulation with LPS induced a two-fold induction of MMP-9 levels, which could not be inhibited by pretreatment with RWJ 67657 at low concentrations. Also, mRNA expression of MMP-9 could not be induced by LPS stimulation, and no inhibition by p38 MAPK inhibitor was observed. This latter finding is in accordance with the study by Lai and co-workers, which showed


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that LPS induction of MMP-9 in monocytes is mainly regulated by the ERK1/2 pathway and not the p38 MAPK pathway 28. p38 MAPK inhibitors have effects on different cell types, thereby possibly enhancing the therapeutic effects, but increasing the risk of side effects. One of the reasons for undesirable effects might be the cross-reactivity against other kinases, which was not the case for RWJ 67657 13. The preliminary pharmacokinetic data suggest a twice-daily dosing regimen 15, while our data show significant effects at low concentrations. CONCLUSIONS A significant inhibition of TNF-α production and mRNA expression in LPS-stimulated monocyte-derived macrophages was observed after pre-treatment with RWJ 67657, a p38 MAPK inhibitor at pharmacological relevant concentrations. Inhibition of mRNA expression of IL-1β, IL-8 and COX-2 was also detected. MMP-9 was found to be constitutively produced at high levels and not inhibited by RWJ 67657. The results presented here could have important implications for the treatment of RA, since the drug used in this study has already proven to be safe in an in-human study 15. More research upon the effects of p38 MAPK inhibition on other cell types involved in inflammation will establish its applicability as a drug in the near future. ACKNOWLEDGEMENTS This work was supported by the Dutch Rheumatology Foundation and Johnson and Johnson Pharmaceutical Research and Development, Raritan, New Jersey, USA.

REFERENCES 1 Choy EH, Panayi GS. Cytokine pathways and joint inflammation in rheumatoid arthritis.

N.Engl.J.Med. 2001; 344: 907-16. 2 Tak PP, Smeets TJ, Daha MR et al. Analysis of the synovial cell infiltrate in early rheumatoid

synovial tissue in relation to local disease activity. Arthritis Rheum. 1997; 40: 217-25. 3 Mulherin D, Fitzgerald O, Bresnihan B. Synovial tissue macrophage populations and articular

damage in rheumatoid arthritis. Arthritis Rheum. 1996; 39: 115-24. 4 Hirohata S, Yanagida T, Itoh K et al. Accelerated generation of CD14+ monocyte-lineage cells

from the bone marrow of rheumatoid arthritis patients. Arthritis Rheum. 1996; 39: 836-43. 5 Burmester GR, Stuhlmuller B, Keyszer G, Kinne RW. Mononuclear phagocytes and rheumatoid

synovitis. Mastermind or workhorse in arthritis? Arthritis Rheum. 1997; 40: 5-18. 6 Kinne RW, Brauer R, Stuhlmuller B, Palombo-Kinne E, Burmester GR. Macrophages in

rheumatoid arthritis. Arthritis Res. 2000; 2: 189-202. 7 Smolen JS, Steiner G. Therapeutic strategies for rheumatoid arthritis. Nat.Rev.Drug Discov.

2003; 2: 473-88. 8 Redlich K, Schett G, Steiner G, Hayer S, Wagner EF, Smolen JS. Rheumatoid arthritis therapy

after tumor necrosis factor and interleukin-1 blockade. Arthritis Rheum. 2003; 48: 3308-19. 9 Rao KM. MAP kinase activation in macrophages. J.Leukoc.Biol. 2001; 69: 3-10. 10 Schett G, Tohidast-Akrad M, Smolen JS et al. Activation, differential localization, and

regulation of the stress- activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis. Arthritis Rheum. 2000; 43: 2501-12.

11 Ebringer A, Wilson C. HLA molecules, bacteria and autoimmunity. J.Med.Microbiol. 2000; 49: 305-11.

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12 Jovanovic DV, Martel-Pelletier J, Di Battista JA et al. Stimulation of 92-kd gelatinase (matrix metalloproteinase 9) production by interleukin-17 in human monocyte/macrophages: a possible role in rheumatoid arthritis. Arthritis Rheum. 2000; 43: 1134-44.


14 Fijen JW, Zijlstra JG, de Boer P et al. Suppression of the clinical and cytokine response to endotoxin by RWJ-67657, a p38 mitogen-activated protein-kinase inhibitor, in healthy human volunteers. Clin.Exp.Immunol. 2001; 124: 16-20.


16 Westra J, Limburg PC, de Boer P, van Rijswijk MH. Effects of RWJ 67657, a p38 mitogen activated protein kinase (MAPK) inhibitor, on the production of inflammatory mediators by rheumatoid synovial fibroblasts. Ann.Rheum.Dis. 2004; 63: 1453-9.

17 Lucas DM, Lokuta MA, McDowell MA, Doan JE, Paulnock DM. Analysis of the IFN-gamma-signaling pathway in macrophages at different stages of maturation. J.Immunol. 1998; 160: 4337-42.

18 Plesner A, Greenbaum CJ, Lernmark A. Low serum conditions for in vitro generation of human macrophages with macrophage colony stimulating factor. J.Immunol.Methods 2001; 249: 53-61.

19 Lanyon P, Muir K, Doherty S, Doherty M. Influence of radiographic phenotype on risk of hip osteoarthritis within families. Ann.Rheum.Dis. 2004; 63: 259-63.

20 Kumar S, Boehm J, Lee JC. p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases. Nat.Rev.Drug Discov. 2003; 2: 717-26.

21 Kotlyarov A, Neininger A, Schubert C et al. MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nat.Cell Biol. 1999; 1: 94-7.

22 Bhattacharyya A, Pathak S, Datta S, Chattopadhyay S, Basu J, Kundu M. Mitogen-activated protein kinases and nuclear factor-kappaB regulate Helicobacter pylori-mediated interleukin-8 release from macrophages. Biochem.J. 2002; 368: 121-9.

23 Herzyk DJ, Allen JN, Marsh CB, Wewers MD. Macrophage and monocyte IL-1 beta regulation differs at multiple sites. Messenger RNA expression, translation, and post-translational processing. J.Immunol. 1992; 149: 3052-8.

24 Dean JL, Brook M, Clark AR, Saklatvala J. p38 mitogen-activated protein kinase regulates cyclooxygenase-2 mRNA stability and transcription in lipopolysaccharide-treated human monocytes. J.Biol.Chem. 1999; 274: 264-9.

25 Caivano M, Cohen P. Role of mitogen-activated protein kinase cascades in mediating lipopolysaccharide-stimulated induction of cyclooxygenase-2 and IL-1 beta in RAW264 macrophages. J.Immunol. 2000; 164: 3018-25.

26 Smeets TJ, Barg EC, Kraan MC, Smith MD, Breedveld FC, Tak PP. Analysis of the cell infiltrate and expression of proinflammatory cytokines and matrix metalloproteinases in arthroscopic synovial biopsies: comparison with synovial samples from patients with end stage, destructive rheumatoid arthritis. Ann.Rheum.Dis. 2003; 62: 635-8.

27 Murphy G, Knauper V, Atkinson S et al. Matrix metalloproteinases in arthritic disease. Arthritis Res. 2002; 4 Suppl 3: S39-S49.

28 Lai WC, Zhou M, Shankavaram U, Peng G, Wahl LM. Differential regulation of lipopolysaccharide-induced monocyte matrix metalloproteinase (MMP)-1 and MMP-9 by p38 and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases. J.Immunol. 2003; 170: 6244-9.

5

Monocytes and monocyte-derived macrophages differ

in regulation of signal transduction pathways

Johanna Westra1

Sander H Diks3

Berber Doornbos-van der Meer1

Karina Wessel1

Maikel P Peppelenbosch3


Pieter C Limburg1,2

From the Departments of 1Rheumatology, 2Pathology and Laboratory Medicine, and 3Cell Biology, University Medical Center Groningen, The Netherlands

In preparation

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ABSTRACT Background. In rheumatoid arthritis (RA) macrophages play a key role in the inflammatory process. It has been reported that inhibitors of signal transduction pathways interfere with cell differentiation. As p38 MAPK (mitogen-activated protein kinase) inhibitors are now in phase II clinical trials in RA, it is relevant to know whether these inhibitors differ with respect to their effects on monocytes and macrophages, and whether they influence the differentiation process. Objective. Aim of the study was to investigate whether p38 MAPK inhibition has a different effect on mediators produced by LPS-stimulated monocytes or monocyte-derived macrophages (MDM). In addition we investigated whether p38 MAPK inhibition (by RWJ 67657) influences the process of differentiation of monocytes into macrophages. Methods. Monocytes and MDM were stimulated with 50 ng/ml LPS with or without pretreatment of 1 µM RWJ 67657. IL-1β, TNF-α, IL-8 and MMP-9 mRNA expression and protein production were measured with real-time RTPCR and ELISA. Furthermore, monocytes were left to differentiate in the presence of 0, 0.1 or 1 µM RWJ 67657. Cells were stimulated with LPS at day 1, 2, 3, 4, and 5, and cytokine and MMP-9 mRNA and protein levels were measured. Lysates of stimulated and unstimulated monocytes and MDM were also tested in a kinase array to identify which kinases were activated. Results. Monocytes produced more cytokines than MDM, but less MMP-9. Inhibition with the p38 MAPK inhibitor RWJ 67657 was more effective in monocytes than in MDM. Differentiation of monocytes into macrophages in the presence of the p38 MAPK inhibitor significantly reduced TNF-α, IL-8 and MMP-9 protein production by macrophages. Conclusions. It was demonstrated that monocytes respond better to p38 MAPK inhibition than monocyte-derived macrophages, and that differentiation is influenced by p38 MAPK inhibition. With kinome profiling we found that several kinases involved in the p38 MAPK pathway are activated both in stimulated monocytes and MDM. Differences in kinase activity were found between monocytes and MDM, that need further investigation.

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INTRODUCTION Macrophages are known to play an important role in inflammatory diseases such as rheumatoid arthritis. Rheumatoid synovium is intensively infiltrated by macrophages and their numbers correlate well with articular destruction 1 and clinical scores 2. Macrophages perform many functions including phagocytosis, killing of pathogens, and the release of cytokines, proteinases and other inflammatory mediators. In response to inflammatory conditions, monocytes migrate from the bonemarrow into the vasculature, and subsequently into extravascular tissues. Transmigration of monocytes is accompanied by changes in morphological, biochemical, and functional characteristics. Circulating monocytes posses a markedly different functional phenotype as compared to tissue macrophages. It has been demonstrated that the route of monocyte differentiation determines their cytokine production and phenotype expression. Two cytokines that have been indentified as promoters of monocyte differentiation are macrophage-colony stimulating factor (M-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF)3. In vitro maturation of monocytes into macrophages by M-CSF with only 1% FCS has been shown to yield a maximum of macrophages without inducing proliferation or activation 4. CSFs have been found to be present at elevated levels in the synovial fluid of RA patients. Also in collagen-induced arthritis models in mice, M-CSF has been shown to exacerbate the arthritis 5. Because of the crucial role synovial macrophages have in the inflammatory process in RA, they are target of therapy themselves, for instance by clodrate-containing liposomes 6 and recently by anti-FcγRI (CD64) antibody to which the plant toxin ricin A (RTA) was chemically linked (CD64-RTA) 7. In vitro stimulation of monocytes and macrophages by a variety of agents causes activation of signal transduction pathways and in particular activation of mitogen-activated protein kinases (MAPK) 8. Cytokine production by monocytes and MDM has been reported to be significantly reduced by treatment with specific p38 MAPK inhibitors 9;10. Stimulation with lipopolysaccharide (LPS) is not only regulated by CD14 but also involves Toll-like receptors, leading to activation of stress-induced signal transduction pathways. Differentiation of macrophages with serum is reported to be under influence of the p38 MAPK pathway 11. In monocytic cell lines differentiation was shown to be potentiated by p38 MAPK inhibitors, with concomitant up-regulation of the c-jun N-terminal kinase (JNK) pathway 12. Between 10-15% of the proteins encoded by the human genome are involved in intracellular signal transduction. Nearly a third of all the different types of proteins inside the cells are subject to phosphorylation, usally at multiple sites 13. Recently progress has been made with the preparation of arrays exhibiting specific consensus sequences for protein kinases. This kind of array would allow faster and more extensive analysis of activity of various intracellular signaling pathways. Usefullnes of peptide arrays was recently tested by Diks et al 14, who demonstrated that with this array the enzymatic activities of a large group of kinases could be determined in LPS-stimulated PBMC. In this study we investigated whether p38 MAPK inihibition had a different effect on mediators produced by LPS-stimulated monocytes or monocyte-derived macrophages. Furthermore we investigated whether a p38 MAPK inhibitor (RWJ 67657) did

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influence M-CSF-induced differentiation of monocytes into macrophages. Finally we tested stimulated and unstimulated monocytes and MDM in the kinase array. MATERIAL AND METHODS Reagents The p38 MAPK specific inhibitor RWJ 67657 was provided by Johnson and Johnson (R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey, USA). Recombinant human macrophage colony stimulating factor (M-CSF) and ELISA antibodies were from R&D Systems (Minneapolis, Minnesota, USA). Foetal calf serum (FCS) and RPMI 1640 culture medium were obtained from Biowhittaker (Verviers, Belgium). All reagents for RNA isolation and reverse transcriptase reaction were purchased from Invitrogen, Life Technologies (Gaithersburg, Maryland, USA). Reagents for real-time RT-PCR were obtained from Applied Biosystems (Foster City, California, USA). Macrophage culture Blood was obtained from apparently healthy laboratory workers. Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep density gradient centrifugation from citrated blood. Cells were suspended in RPMI with gentamycin at 2.106 cells/ml and seeded in 3 ml in 6-well plates (Corning, Schiphol, The Netherlands) or 0.5 ml in 24-well plates and cultured at 37°C in a 5% CO2 atmosphere. The cells that adhered after two hours were used as monocytes or were allowed to differentiate into monocyte-derived macrophages (MDM) for five days in RPMI containing gentamycin, 50 ng/ml M-CSF + 1% FCS, while medium was refreshed at day two. Experimental set-up To investigate whether unstimulated or LPS stimulated monocytes and MDM responded differently to treatment with p38 MAPK inhibition, cells from four donors before and after differentiation were stimulated with 50 ng/ml LPS, with or without one hour pre-treatment with 1 µM RWJ 67657 during four hours for mRNA expression measurement or 24 hours for protein determination. After four hours stimulation cells were lysed in TRIzol reagent according to the manufacturers instructions (Life Technologies) and stored at -80°C until RNA isolation. For protein determination supernatants of treated cells were harvested and stored at -20°C until measurement. The effect of p38 MAPK inhibition on cell differentiation was investigated by incubating cells from four donors with 0, 0.1 or 1.0 µM RWJ 67657 during the entire differentiation period. At day 1, 2, 3, 4 and 5 cells were stimulated with 50 ng/ml LPS for four or 24 hours as described above. Before LPS stimulation RWJ 67657 was depleted from the medium by washing for 30 minutes, which experimentally has been shown to be sufficient (data not shown). RNA isolation and real-time RT-PCR RNA isolation was performed using TRIzol reagent according to the manufacturers instructions and mRNA expression of IL-1β, TNF-α, IL-8, MMP-9, and glyceralde-


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hyde-3-phosphate dehydrogenase (GAPDH), 1µl of cDNA in triplicate was used for amplification by the real-time quantitative PCR system (ABI Prism 7900HT Sequence Detection System, Applied Biosystems) with specific Taqman primers/probes as described earlier 10. In our experiments GAPDH and other genes of interest were always determined in the same RT-PCR run. Amplification was performed using standard conditions: denaturation at 95°C for 15 seconds, 40 cycles of amplification with annealing at 60°C for 1 minute, and extension at 50°C for 2 minutes. According to the comparative Ct (threshold cycle value) method described in the ABI manual, the resulting mRNA amount of the gene of interest was normalized to the housekeeping gene GAPDH, yielding the ∆Ct value. The ∆Ct value of unstimulated cells was subtracted from the average ∆Ct value of each sample, yielding the ∆∆Ct. The amount of target, normalized to an endogenous reference (GAPDH) and relative to the control sample, is given by: 2-∆∆CT. ELISA based determination of IL-1β, TNF-α, IL-8, and MMP-9 in cell culture supernatants Cytokine (TNF-α, IL-1β, and IL-8), and MMP-9 levels were measured in cell supernatants by ELISA, using matched antibody pairs for ELISA and recombinant proteins as standards from R&D Systems. Detection limits for all cytokine ELISAs was 50 pg/ml and for MMP-9 ELISA 100 pg/ml. Kinome profiling with kinase array To compare the activity of kinases between monocytes and MDM, kinase arrays were performed as described earlier 14. First it was investigated by western blotting whether monocytes were already activated by the isolation procedure and the adherence. Previously it was shown that unstimulated MDM had low phospho-p38 MAPK activity 10. PBMCs were isolated as described above and were left to adhere in 6-well plates at a concentration of 2.106 cells/ml (3 ml/well) in RPMI + 1% FCS. After 1, 2, 4, and 6 hours cells were either activated with 50 ng/ml LPS for 30 minutes and then lysed in 300 µl lysis buffer or were lysed unstimulated. Lysis buffer contained 2% SDS, 10% glycerol, 50 mM dithiothreitol, 62.5 mM Tris-HCl (pH=6.8) and 0.01% brome-phenol blue. Cells were scraped off the wells and the lysates were subsequently sonicated for 5-10 seconds and boiled for 5 minutes. After centrifugation the samples were loaded onto a 10% SDS-PAGE gel and resolved by running at 200 V and 15 Watt constant. Semidry blotting was performed onto nitrocellulose membrane and immunodetection with anti-phospho-p38 MAPK was performed, followed by incubation with peroxidase-anti-rabbit IgG. Enhanced chemi-luminescence (ECL) detection was performed according to the manufacturers guidelines (Lumi-Lightplus, Roche Diagnostics, Mannheim, Germany). Blots were exposed to HyperfilmTM (Amersham Biosciences, Roosendaal, The Netherlands) and developed. Subsequently, blots were stripped with RestoreTM Western Blot Stripping Buffer (Pierce, Rockford, IL, USA) and immunodetection with anti-p38 MAPK was performed.

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Peptide array analysis For kinase array 107 PBMC/well were plated in 4 ml RPMI + 1% FCS in 6-well plates, and were left to adhere or were differentiated to MDM with M-CSF for five days as described above. The following experiment was performed on both monocytes (after two hours of adherence) and MDM: one well was left unstimulated, one well was stimulated with 50 ng/ml LPS for 30 minutes. Stimulations were finalized by washing with ice-cold phosphate buffered saline. Next, cells were lysed in ice-cold kinase lysis buffer (100 µl/well): 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM NaF, 1 mM MgCl2, 1 µg/ml leupeptin, 1 µg/ml aprotin, 1 mM PMSF. The cell lysates were centrifuged at 10000 rpm for ten minutes. Peptide array incubation mix was produced by adding 10 µl of filter-cleared activation mix (50% glycerol, 50 µM [γ-33P]ATP, 0.05% v/v Brij-35, 0.25 mg/ml bovine serum albumin, [γ-33P]ATP (1000 kBq) to 50 µl of cell lysate. The lysate + peptide array mix was added onto the chips which consists of kinase substrate peptides on a glass slide (Pepscankinase, Pepscan Systems, Lelystad, The Netherlands) and the chips were kept at 37°C in a humidified stove for 75 minutes. The chips contain 1176 kinase substrate peptides in duplicate, and the consensus substrate peptides are approximately 9 peptides long. Subsequently the chips were washed twice with phosphate-buffered saline + 0.1% Triton, twice in 2 M NaCl, twice in demineralized H2O and then air dried. The chips were then exposed to a phosphor-imager plate for 72 hours, and the density of the spots was measured and analyzed with array software (ScanAlyze, Stanford University, Stanford, California, USA). STATISTICS Paired T-tests were performed using GraphPad Prism version 3.00 for Windows, GraphPad Software (San Diego, CA, USA). RESULTS Effects of p38 MAPK inhibition on mediator production by monocytes and monocyte-derived macrophages In table 1 the protein production (IL-1β, TNF-α, IL-8 and MMP-9) by monocytes and MDM before and after LPS stimulation and 1 µM RWJ 67657 pre-treatment is listed. The table shows that unstimulated monocytes did not produce TNF-α and IL-1β, and low IL-8 levels, whereas high levels were produced after LPS stimulation. Unstimulated MDM did not produce TNF-α and IL-1β, and low IL-8, but in contrast to unstimulated monocytes, produced unstimulated MDM high levels of MMP-9. In all situations there was inhibition due to pretreament with 1 µM RWJ 67657, but inhibition appeared to be more effective in monocytes than in MDM. Except for unstimulated monocytes MMP-9 production was less effectively inhibited compared to cytokines. mRNA expression was measured in stimulated monocytes and MDM, and mRNA expression in unstimulated cells was used as control (fold induction =1, table 2).


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In contrast to protein expression IL-1β mRNA was highly expressed in stimulated MDM. All cytokine genes were expressed at a higher level in MDM than in monocytes. MMP-9 mRNA was constitutively expressed in both monocytes and MDM and expression was not induced by stimulation (fold induction < 2). Again p38 MAPK inhibition was effective for cytokine mRNA expression, but not for MMP-9 expression.

Table 1. Protein production by monocytes and MDM with and without pre-treatment of 1 µM RWJ 67657. Cells were unstimulated or stimulated for 24 hours with 50 ng/ml LPS. Protein concentration of cytokines and MMP-9 was measured in supernatants by ELISA. BD = below detection, inh = % inhibition.

Monocytes, unstimulated

MDM, unstimulated

Monocytes, stimulated

MDM, stimulated

RWJ Mean (SEM) Inh Mean

(SEM) Inh Mean (SEM) Inh Mean

(SEM) Inh

- BD BD 2392(421) BD IL-1β pg/ml + BD BD 331 (63) 86% BD

- BD BD 2021(374) 1075(493) TNF-α pg/ml + BD BD 138 (39) 93% 587(305) 45%

- 1.7(0.6) 0.3(0.1) 116 (69) 11.7 (1.7) IL-8 ng/ml + 0.03(0.01) 98% 0.08(0.02) 74% 51.2(23.7) 56% 6.9(1.3) 41%

- 6.2(2.1) 79.8(19.2) 28.6(10.1) 97.0(13.5) MMP9 ng/ml + 1.1(0.5) 82% 52.3(13.0) 34% 22.7(9.1) 21% 61.4(11.0) 37%

Table 2. mRNA expression (fold induction) in stimulated monocytes and MDM with and without pre-treatment of 1 µM RWJ 67657. Cells were stimulated with 50 ng/ml LPS for 4 hours and mRNA expression of cytokines and MMP-9 was determined by real-time RT-PCR. Monocytes MDM

RWJ Mean (SEM) Inhibition Mean (SEM) Inhibition

- 341.6(140.9) 3434.0(1423.2) IL-1β + 84.9(42.9) 75% 1093.5(549.4) 68% - 175.7(28.0) 589.7(158. 6) TNF-α + 33.5(16.0) 81% 125.2(33.9) 79% - 67.8(30.2) 324.2(118.9) IL-8 + 15.3(10.0) 77% 207.9(89.6) 36% - 1.10(0.33) 1.50(0.12) MMP9 + 0.65(0.25) 42% 1.33(0.43) 11%

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Figure 1. Protein production by monocytes and MDM during differentiation in the presence of 0, 0.1, and 1.0 µM RWJ 67657. Cells were unstimulated or stimulated for 24 hours with 50 ng/ml LPS. Protein concentration of cytokines and MMP-9 was measured in supernatants by ELISA (∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.001, paired T-test, tested against MDM, untreated with RWJ 67657). Effects of p38 MAPK inhibition on differentiation As p38 MAPK inhibition might not only affect protein production and mRNA expression in cells but also might have an effect on the differentiation process, we left monocytes to differentiate in the presence of 0, 0.1 and 1.0 µM RWJ 67657. In figure 1 the protein production of cytokines and MMP-9 is shown in unstimulated and stimulated cells during the differentiation process. Figure 1 shows clearly that immediately after differentiation has started IL-1β production was decreased below detection limit of the assay, while TNF-α production was decreased by more than 70%. For all cytokines the presence of RWJ 67657 reduced the production during


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differentiation, statistically significant when tested against untreated MDM. MMP-9 production was increased during differentiation, and the presence of RWJ 67657 led to reduced protein production. Measurement of mRNA expression (figure 2) confirmed the protein data concerning reduced expression in cells differentiated in the presence of the p38 MAPK inhibitor.

Figure 2. mRNA expression of TNF-α, IL-1β, IL-8, and MMP-9 in stimulated monocytes and MDM during differentiation in the presence of 0 and 1 µM RWJ 67657. Cells were stimulated with 50 ng/ml LPS for 4 hours and mRNA expression of cytokines and MMP-9 was determined by real-time RT-PCR. (* p<0.05, paired T-test, tested against MDM, untreated with RWJ 67657).

Kinome profiling of monocytes and MDM To investigate the activation state of isolated and adhered monocytes, cell lysates were prepared from monocytes after isolation and different adherence time points. In figure 3 the results are shown of western blots with antibodies to p38 MAPK and phosphorylated p38 MAPK. After one hour of adherence the cells were still activated and showed phospho-p38 MAPK reactivity, but after two hours of adherence the activity was decreased. Cells were also stimulated with 50 ng/ml LPS for 30 minutes as control, which gave strong phospho-p38 MAPK bands. Next cell lysates were prepared of monocytes after two hours of adherence and of MDM, unstimulated and stimulated for 30 minutes. Kinase arrays were performed with the lysates, and radioactivity bound to the chips was detected with phosphorimager. With the ScanAlyze program the spots were quantified, and the correlations between the duplicates after correction for background were calculated. Correlations between the duplicates were well above 0.70, except for the kinase chip of the unstimulated monocytes, of which only one of the duplicates could be used.

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Figure 3. Western blot of monocytes after different timepoints of adherence. Activation status of cells was measured with phospho-p38 MAPK, p38 MAPK was used as control. In table 3 results are shown of kinases involving in the p38 MAPK pathway. The results under control and LPS represent the radioactivity incorporated in the substrates (in Phosphorimager/Aida quantification units) and are the average of 2 measurements, except for the unstimulated monocytes results. The ratio of LPS stimulated samples and controls is given in the next column. From the results it appears that all substrates involved in the p38 MAPK pathway are phosphorylated after stimulation with LPS, although differences in intensity are seen between monocytes and MDM.

Table 3. Monocyte and MDM kinase activity. Depicted are kinases and substrate involved in the p38 MAPK pathway. Results represent the radioactivity in corporated in the substrates (in phosphorimager /Aida quantification units). (MK2-MAPKAPK2, cPLA2-cytosolic phospholipase A2, eIF4E-eukaryotic translation initiation factor 4E).

monocytes MDM

Kinase Substrate Sequence phos.

site

contr LPS LPS/

contr

contr LPS LPS/

contr

Raf1 MAPKK1 QLIDSMANS S-217 1823 3045 1.67 1255 3229 2.57

MKK3/6 p38MAPK DDEMTGYVA T-180 4783 8962 1.87 889 4209 4.73

MAPK MAPKAPK2 KVPQTPLHT T-300 6335 11398 1.80 5032 8679 1.72

MAPK cPLA2 SYPLSPLSD S-505 11649 14853 1.28 8729 12028 1.38

MK2 hsp27 LRGPSWDPF S-15 807 9021 11.18 2906 5643 1.94

MK2 hsp27 SRALSRQLS S-78 9149 12325 1.35 14072 14168 1.01

Mnk1 elF4E TKSGSTTKN S-209 2417 3729 1.54 2270 6602 2.91

STAT-1 LLPMSPEEF S-727 7324 10388 1.42 3436 8360 2.43


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DISCUSSION In the present study it was shown that monocytes respond better to p38 MAPK inhibition than monocyte-derived macrophages, and also that cell differentiation is influenced by p38 MAPK inhibition. The importance of synovial macrophages in inflammation has been established in several studies, and recently it has been reported that the number of synovial sublining macrophages does correlate well with clinical improvement after antirheumatic treatment (MTX, leflunomide, Infliximab, prednisone) 15. In previous work from our group it has been shown that TNF-α production by monocyte-derived macrophages was effectively inhibited by the p38 MAPK inhibitor RWJ 67657 10. As p38 MAPK inhibitors are in phase II clinical trials in RA 16 knowledge of the exact effects of such drugs is important. TNF-α production was higher and more effectively inhibited by RWJ 67657 (93%) in monocytes than in MDM (45%), whereas IL-1β production was inhibited in monocytes (93%), but was not excreted by MDM. This latter finding is in concordance with the study by Herzyk et al 17 and is related to the difficulty of release of IL-1β by macrophages. IL-8 production was tenfold higher in monocytes, but effects of p38 MAPK inhibitor treatment were comparable. In contrast to cytokine production was MMP-9 abundantly produced by MDM, even in unstimulated cells. Inhibition of MMP-9 by p38 MAPK inhibitor treatment in monocytes was only 21%, and in MDM 37%, indicating that pathways other than the p38 MAPK pathway must be involved in MMP-9 regulation. Lai et al demonstrated a dominant role for the ERK1/2 pathway in MMP-9 regulation in human monocytes 18. With real-time RT-PCR we found higher expression of TNF-α, IL-1β and IL-8 mRNA levels in MDM compared to monocytes. Although protein production in monocytes was higher, mRNA levels seem to accumulate in MDM. It has been known that activation of p38 MAPK has a stabilizing effect on mRNA of inflammatory genes. This effect has been described for TNF-α both for primary human monocytes 19 as well as for macrophage-like cell lines 20. The results from our study show that reduction of mRNA expression of TNF-α and IL-1β by p38 MAPK inhibition was approximately the same for monocytes and MDM (81% and 79% (TNF-α), and 75% and 68% (IL-1β) respectively), whereas IL-8 and MMP-9 mRNA was more reduced in monocytes than MDM due to RWJ 67657 treatment (77% and 36% (IL-8), 42% and 11% (MMP-9) respectively). From the differentiation experiments it could be demonstrated that alreay after one day of differerentiation with M-CSF, the properties of the monocytes changed: IL-1β was no longer produced, TNF-α production was very low, whereas MMP-9 production increased Differentiation in the presence of RWJ 67657 significantly reduced TNF-α protein and mRNA expression and IL-8 and MMP-9 protein production. Ayala et al reported that p38 MAPK inhibition at concentrations, which were reported to inhibit MAPKAPK-2 activation, blocked monocyte differentiation and chemotaxis 11. From our data we can not conclude that differentiation is blocked, but rather that protein production is decreased due to the p38 MAPK inhibition. The results from the kinase array showed that indeed the substrates involved in the p38 MAPK pathway were phosphorylated by activation both in monocytes and in MDM. From the literature it is known that in vitro MKK3, MKK4, and MKK6 all show a

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strong preference for phosphorylation of the tyrosine residue of the Thr-Gly-Tyr motif in the p38 MAPK substrate 21 and this is in concordance with our findings. Recently it was reported that oxidative stress of resident vascular cells and macrophages potently enhances eIF4E phosphorylation by the activation of Mnk-1 22. Phosphorylation of eukaryotic translation initiation factor 4E is associated with increased activity of the translational machinery. With this array the activation status of several kinases in a single experiment can be investigated, but more research is needed to understand the interactions of the intracellular signal transduction pathways. REFERENCES 1 Mulherin D, Fitzgerald O, Bresnihan B. Synovial tissue macrophage populations and articular

damage in rheumatoid arthritis. Arthritis Rheum. 1996; 39: 115-24. 2 Tak PP, Smeets TJ, Daha MR et al. Analysis of the synovial cell infiltrate in early rheumatoid

synovial tissue in relation to local disease activity. Arthritis Rheum. 1997; 40: 217-25. 3 Bender AT, Ostenson CL, Giordano D, Beavo JA. Differentiation of human monocytes in vitro

with granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor produces distinct changes in cGMP phosphodiesterase expression. Cell Signal. 2004; 16: 365-74.

4 Plesner A, Greenbaum CJ, Lernmark A. Low serum conditions for in vitro generation of human macrophages with macrophage colony stimulating factor. J.Immunol.Methods 2001; 249: 53-61.

5 Campbell IK, Rich MJ, Bischof RJ, Hamilton JA. The colony-stimulating factors and collagen-induced arthritis: exacerbation of disease by M-CSF and G-CSF and requirement for endogenous M-CSF. J.Leukoc.Biol. 2000; 68: 144-50.

6 Barrera P, Blom A, Van Lent PL et al. Synovial macrophage depletion with clodronate-containing liposomes in rheumatoid arthritis. Arthritis Rheum. 2000; 43: 1951-9.

7 van Roon JA, Bijlsma JW, van de Winkel JG, Lafeber FP. Depletion of synovial macrophages in rheumatoid arthritis by an anti-Fc{gamma}RI-Calicheamicin immunoconjugate. Ann.Rheum.Dis. 2004.

8 Rao KM. MAP kinase activation in macrophages. J.Leukoc.Biol. 2001; 69: 3-10. 9 Wadsworth SA, Cavender DE, Beers SA et al. RWJ 67657, a potent, orally active inhibitor of

p38 mitogen-activated protein kinase. J.Pharmacol.Exp.Ther. 1999; 291: 680-7. 10 Westra J, Doornbos-Van Der Meer B, de Boer P, van Leeuwen MA, van Rijswijk MH, Limburg

PC. Strong inhibition of TNF-alpha production and inhibition of IL-8 and COX-2 mRNA expression in monocyte-derived macrophages by RWJ 67657, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Arthritis Res.Ther. 2004; 6: R384-R392.

11 Ayala JM, Goyal S, Liverton NJ, Claremon DA, O'Keefe SJ, Hanlon WA. Serum-induced monocyte differentiation and monocyte chemotaxis are regulated by the p38 MAP kinase signal transduction pathway. J.Leukoc.Biol. 2000; 67: 869-75.

12 Wang X, Studzinski GP. Inhibition of p38MAP kinase potentiates the JNK/SAPK pathway and AP-1 activity in monocytic but not in macrophage or granulocytic differentiation of HL60 cells. J.Cell Biochem. 2001; 82: 68-77.

13 Pelech S. Tracking cell signaling protein expression and phosphorylation by innovative proteomic solutions. Curr.Pharm.Biotechnol. 2004; 5: 69-77.

14 Diks SH, Kok K, O'Toole T et al. Kinome profiling for studying lipopolysaccharide signal transduction in human peripheral blood mononuclear cells. J.Biol.Chem. 2004; 279: 49206-13.

15 Haringman JJ, Gerlag DM, Zwinderman AH et al. Synovial tissue macrophages: highly sensitive biomarkers for response to treatment in rheumatoid arthritis patients. Ann.Rheum.Dis. 2004.

16 Nikas SN, Drosos AA. SCIO-469 Scios Inc. Curr.Opin.Investig.Drugs 2004; 5: 1205-12. 17 Herzyk DJ, Allen JN, Marsh CB, Wewers MD. Macrophage and monocyte IL-1 beta regulation

differs at multiple sites. Messenger RNA expression, translation, and post-translational processing. J.Immunol. 1992; 149: 3052-8.


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18 Lai WC, Zhou M, Shankavaram U, Peng G, Wahl LM. Differential regulation of lipopolysaccharide-induced monocyte matrix metalloproteinase (MMP)-1 and MMP-9 by p38 and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases. J.Immunol. 2003; 170: 6244-9.

19 Wang SW, Pawlowski J, Wathen ST, Kinney SD, Lichenstein HS, Manthey CL. Cytokine mRNA decay is accelerated by an inhibitor of p38-mitogen-activated protein kinase. Inflamm.Res. 1999; 48: 533-8.

20 Brook M, Sully G, Clark AR, Saklatvala J. Regulation of tumour necrosis factor alpha mRNA stability by the mitogen-activated protein kinase p38 signalling cascade. FEBS Lett. 2000; 483: 57-61.

21 Fleming Y, Armstrong CG, Morrice N, Paterson A, Goedert M, Cohen P. Synergistic activation of stress-activated protein kinase 1/c-Jun N-terminal kinase (SAPK1/JNK) isoforms by mitogen-activated protein kinase kinase 4 (MKK4) and MKK7. Biochem.J. 2000; 352 Pt 1: 145-54.

22 Duncan RF, Peterson H, Sevanian A. Signal transduction pathways leading to increased eIF4E phosphorylation caused by oxidative stress. Free Radic.Biol.Med. 2005; 38: 631-43.

6

Chemokine production and E-selectin expression in


(mitogen activated protein kinase) inhibitor RWJ 67657

Johanna Westra1

Joanna M Kułdo2


Grietje Molema2

Pieter C Limburg1

From the Departments of 1 Rheumatology, and 2 Medical Biology section of Pathology

and Laboratory Medicine, University Medical Center Groningen, The Netherlands

International Immunopharmacology: in press

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ABSTRACT Endothelial cells play an important role in inflammatory diseases like rheumatoid arthritis by recruitment of inflammatory cells. The cytokines TNF-α and IL-1β are major inducers of endothelial cell activation and are stimulators of inflammatory signal transduction pathway involving p38 MAPK (mitogen-activated protein kinase). The present study investigated the effects of p38 MAPK inhibition on cell adhesion molecule (CAM) expression and chemokine production by endothelial cells both on mRNA and protein level. Pre-treatment of endothelial cells with the pharmacologically relevant concentration of 1 µM of the p38 MAPK inhibitor RWJ 67657 reduced TNF-α and IL-1β induced mRNA and membrane expression of E-selectin. Moderate inhibitory effects on ICAM-1 and VCAM-1 expression were found. Significant reduction of mRNA expression and protein production of the inflammatory cytokine IL-6 and the chemokines IL-8 and MCP-1 was demonstrated. Treatment with RWJ 67657 could lead to reduced leukocyte infiltration by the reduction of E-selectin expression and chemokine production. Key words. chemokine, endothelial cell, E-Selectin, MAPK inhibitor

p38 MAPK inhibition in EC

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INTRODUCTION Rheumatoid arthritis (RA) is an inflammatory disease characterized by hyperplasia, increased vascularity, and infiltration of inflammatory cells into the synovial membrane 1. It is now known that the endothelial cells (EC), which line the lumen of the blood vessels, are not passive bystanders but are active responders to stimuli like activated leukocytes and cytokines 2. After stimulation EC can produce a number of inflammatory mediators and express cellular adhesion molecules (CAMs). Most CAMs involved in endothelial activation belong to three families, i.e., the integrins, selectins, and immunoglobulin superfamilies 3. The adhesion of leukocytes to EC takes place in four steps. The first step is a weak adhesion (tethering and rolling) mediated by E-, P- and L-selectins. The next step is leukocyte activation and is a consequence of interaction between chemokines and their receptors on leukocytes. Then firm adhesion takes place, which is mediated mostly by interaction between integrins on the leukocytes and vascular adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 on the EC. The final step is the transendothelial migration, which is directed by secreted chemokines bound to endothelial heparan sulphate glycosaminoglycans. Essential chemokines are interleukin (IL)-8 which attracts neutrophils, and monocyte chemo-attractant protein (MCP)-1, the main attractant for mononuclear cells 2;3. The cytokines TNF (tumour necrosis factor)-α and IL-1β, produced by activated macrophages in the synovium, have been identified as the major inducers of EC activation in RA 4. Blockade of these cytokines has proven to be effective in the treatment of RA, although Redlich et al recently concluded that targeting individual molecules such as TNF-α may not be sufficient in interfering with both inflammation and joint destruction 5. Successful drug treatment of RA is associated by a decrease in cytokine production, but also by a decrease in E-selectin and ICAM-1 expression 6. In vitro studies demonstrated that in TNF-α and/or lipopolysaccharide (LPS) stimulated human umbilical vein endothelial cells (HUVEC) the nuclear factor-κB (NF-κB), and p38 mitogen-activated protein kinase (MAPK) pathways are important in controlling adhesion molecule expression 7;8. Activation of all three stress- and mitogen-activated protein kinases (SAPK/MAPK) was found throughout the RA synovial tissue, whereas activated p38 MAPK predominantly was located in the synovial lining layer and in synovial endothelial cells 9. Interest in protein kinases and in particular in p38 MAPK as drug targets has increased in the last years as recently reviewed by Kumar et al 10. The p38 MAPK inhibitor RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol) has been shown to be effective in inhibiting the release of TNF-α from LPS-treated human peripheral blood mononuclear cells with an IC50 of 3 nM 11. This compound was approximately 10-fold more potent than the reference standard p38 MAPK inhibitor SB 203580 in all p38 dependent in vitro systems tested. RWJ 67657 specifically inhibited the enzymatic activity of recombinant p38 α and β, but not of γ and δ in vitro, and had no significant activity against a variety of other kinases 11. We recently demonstrated that RWJ 67657 significantly inhibited IL-6, IL-8, MMP-3 and COX-2 mRNA expression by IL-1β and TNF-α stimulated rheumatoid synovial fibroblasts 12. Furthermore Fijen et al showed that this compound induced a dose-dependent suppression of leukocyte and endothelial cell response after endotoxin challenge in

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humans 13. Recent pharmaco-kinetic and pharmaco-dynamic studies of RWJ 67657 in humans showed that the compound has an acceptable safety profile to warrant further investigations 14. In the present study the effects of p38 MAPK inhibition in IL-1β or TNF-α stimulated HUVEC concerning adhesion molecule expression and chemokine production were studied at the level of mRNA expression as well as protein production. Pre-treatment of activated endothelial cells with 1 µM RWJ 67657 reduced E-selectin mRNA and protein expression, and significantly reduced IL-8, MCP-1 and IL-6 production and mRNA expression. These results indicate that treatment with RWJ 67657 could lead to reduced leukocyte infiltration and therefore could have an important therapeutic benefit. MATERIALS AND METHODS Reagents RWJ 67657 was provided by Johnson and Johnson (R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ). Recombinant TNF-α and IL-1β and ELISA antibodies for the detection of IL-8 and MCP-1 were obtained from R&D Systems (Minneapolis, MN). Antibodies for the detection of IL-6 were purchased from Sanquin (Amsterdam, The Netherlands). Specific antibodies to p38 MAPK, phospho-p38 MAPK and phospho-MAPK activated protein kinase (MAPKAPK)-2 were purchased from Cell Signalling Technologies (Beverly, MA) and detecting antibody peroxidase -swine-anti-rabbit was from DAKO (Glostrup, Denmark). Mouse anti-human E-selectin antibody (H18/7) was kindly provided by Dr. M. Gimbrone Jr., Harvard Medical School. Monoclonal anti-human VCAM-1 (CD106) and anti-human ICAM-1 (CD54), both labelled with phycoerytrin (PE) were from Beckton Dickinson (Bedford, MA). Monoclonal anti-human PECAM-1 (platelet endothelial cell adhesion molecule, CD31) was obtained from DAKO and PE-labeled goat-anti-mouse was from SBA (Southern Biotechnology Associates, Birmingham, AL). RNA isolation kit was from Stratagene (La Jolla, CA), quantitation of RNA was performed with Ribogreen RNA quantitation kit (Molecular Probes Europe BV, Leiden, The Netherlands). Other reagents for RNA isolation and reverse transcriptase reaction were purchased from Invitrogen (Breda, The Netherlands). Reagents, primers and probes for real-time RT-PCR were obtained from Applied Biosystems (Nieuwerkerk a/d IJssel, The Netherlands). Endothelial cell culture and stimulation HUVEC were obtained from the Endothelial Cell Facility at the UMCG (The Netherlands) as described previously 15. Primary isolates combined from 2 or 3 donors were cultured on 1% gelatin (Sigma-Aldrich, Zwijndrecht, The Netherlands) -precoated plastic tissue culture plates or flasks (Corning, Schiphol, The Netherlands) at 37º C under 5% CO2/95% air. The culture medium constisted of RPMI 1640 (Biowhittaker, Verviers, Belgium) supplemented with 20% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 5 U/ml heparin, 100 U/ml penicillin, 100 µg/ml streptomycin and 20 µg/ml endothelial cell growth factor, extracted from bovine brain according to the procedure described by Maciag 16. After attaining confluence, cells


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were detached from the surface by trypsin/EDTA (0.5/0.2 mg/ml) and split at a 1:3 ratio. In the experiments presented here HUVEC were used up to passage 4. In the experimental set-up HUVEC were grown to confluence in gelatin-coated 6-well plates. Fresh medium was added before cells were stimulated for 6 or 24 hours with 10 ng/ml TNF-α or IL-1β, with or 1 hour without pre-treatment with 1µM RWJ 67657 (stock solution 10 mM in DMSO = dimethylsulfoxide). Phosphorylation of p38 MAPK and MAPKAPK-2 in HUVEC HUVEC were pre-treated with 0, 0.1, 1, and 10 µM RWJ 67657 for 1 hour and stimulated for 30 minutes as mentioned above. Cell extracts were prepared by lysing the cells with 1X SDS sample buffer (containing 2% SDS, 10% glycerol, 50 mM dithiothreitol, 62.5 mM Tris-HCl (pH=6.8) and 0.01% brome-phenol blue). Cells were scraped off the wells and the lysates were subsequently sonicated for 5-10 seconds and boiled for 5 minutes. After centrifugation the samples were loaded onto a 10% SDS-PAGE gel and resolved by running at 200 V and 15 Watt constant. Semidry-blotting was performed onto a nitrocellulose membrane after which immunodetection with anti-p38 MAPK, anti-phospho-p38 MAPK, anti-phospho-MAPKAPK-2 and peroxidase labelled swine-anti-rabbit was performed. Enhanced chemi-luminescence (ECL) detection was performed according to the manufacturers guidelines (Lumi-Lightplus, Roche Diagnostics, Mannheim, Germany). RNA isolation and real-time RT-PCR HUVEC with or without 1 hour pre-treatment with 1µM RWJ 67657 were stimulated for 6 and 24 hours with 10 ng/ml TNF-α or IL-1β. Total RNA was isolated using the Absolutely RNA Microprep Kit according to the manufacturers guidelines. RNA was analysed qualitatively by gel electrophoresis and quantitatively by Ribogreen RNA Quantitation Kit. One microgram of total RNA was used for the synthesis of first strand cDNA using Superscript III RNase H- Transcriptase in 20 µl final volume containing 250 ng random hexamers and 40 units Rnase OUT inhibitor. For the measurement of mRNA for CD31, E-selectin, VCAM-1, ICAM-1, IL-8, MCP-1, IL-6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 1 µl of cDNA in triplicate was used for amplification by the Taqman real-time PCR system (ABI Prism 7900HT Sequence Detection System, Applied Biosystems) with specific Taqman primers/probes. In our experiments GAPDH and other genes of interest were always determined in the same RT-PCR run. Amplification was performed using standard conditions: denaturation at 95°C for 15 seconds, 40 cycles of amplification with annealing at 60°C for 1 minute, and extension at 50°C for 2 minutes. According to the comparative Ct (threshold cycle value) method described in the ABI manual, the resulting mRNA amount of the gene of interest was normalized to the housekeeping gene GAPDH, yielding the ∆Ct value. The ∆Ct value of unstimulated HUVEC was subtracted from the average ∆Ct value of each sample, yielding the ∆∆Ct. The amount of target, normalized to an endogenous reference (GAPDH) and relative to the control sample, is given by: 2-∆∆CT.

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Flow cytometric analysis HUVEC were stimulated for 6 and 24 hours as mentioned above and detached from the wells by short treatment with trypsin, and subsequently resuspend in FCS to neutralize the trypsin. After washing with PBS supplemented with 5% FCS, HUVEC were incubated with PE-labeled monoclonal antibodies against VCAM-1 (dilution 1:10) and ICAM-1 (1:25) for 45 minutes on ice or with monoclonal antibodies against CD31 (1:25) and E-selectin (undiluted) followed by incubation with PE-labeled goat-anti-mouse antibodies. Cells were fixed with 0.5% paraformaldehyde/PBS and adhesion molecule expression was detected by flow cytometric analysis in an Epics-Elite Flow cytometer (Coulter Electronics, Mijdrecht, The Netherlands). Non specific staining was assessed by staining with irrelevant isotype-matched monoclonal antibodies. The effects of RWJ 67657 on adhesion molecule expression was determined in 3 experiments with different HUVEC cultures. ELISA based determination of IL-8, MCP-1 and IL-6 in cell culture supernatants HUVEC were pre-treated with 1 µM RWJ 67657 for 6 and 24 hours and stimulated with 10 ng/ml TNF-α or IL-1β. IL-6 levels in cell supernatants were measured as described previously 17, IL-8 and MCP-1 were measured by ELISA, using matched antibody pairs for ELISA and recombinant proteins as standards from R&D Systems. In short, Corning high-binding ELISA plates were coated overnight with monoclonal antibodies in PBS. After blocking with 2% BSA (bovine serum albumin)/PBS diluted supernatants were added. Bound chemokines were detected with biotinylated polyclonal antibodies followed by incubation with peroxidase labelled Streptavidin (Sanquin, Amsterdam, The Netherlands). Colour reaction was performed with TMB (3’3’5’5’tetramethylbenzidin, Roth, Karlsruhe, Germany) and concentration of protein was determined with the SOFTmax PRO software (Molecular Devices, Sunnyvale, CA). Detection limits for ELISAs was 20 pg/ml for IL-6 and IL-8 and 50 pg/ml for MCP-1. STATISTICS Paired T-tests were performed using GraphPad Prism version 3.00 for Windows, GraphPad Software (San Diego, CA). RESULTS Effect of RWJ 67657 on phosphorylation of p38 MAPK and MAPKAPK-2 Upon stimulation with 10 ng/ml TNF-α and/or IL-1β p38 MAPK is rapidly phosphorylated within minutes, while after 60 minutes the level of phosphorylation is decreased (data not shown). In figure 1 the effect of RWJ 67657 on phosphorylation of p38 MAPK and its downstream substrate MAPKAPK-2 after 30 minutes of stimulation is shown. RWJ 67657 does not inhibit phosphorylation of p38 MAPK, but it does inhibit its activity as can be seen from the inhibition of MAPKAPK-2 phosphorylation at 0.1 µM. Complete inhibition was demonstrated at a concentration of 1 µM RWJ 67657. The solvent 0.01% DMSO did not affect phosphorylation of either kinase.


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Figure 1. Phosphorylation of p38 MAPK and MAPKAPK-2 in stimulated HUVEC after pre-treatment with 10, 1 and 0.1 µM RWJ 67657. HUVEC were treated with TNF-α and/or IL-1β for 30 minutes with or without 1 hour pre-treatment with RWJ 67657. Phosphorylation was measured by Western blot using specific antibodies against p38 MAPK, phospho-p38 MAPK and its direct downstream substrate MAPKAPK-2. Control incubations were performed with 0.01% dmso, the vehicle solvent for RWJ 67657.

Figure 2. Effect of RWJ 67657 pre-treatment on mRNA expression of CD31, E-selectin, VCAM-1 and ICAM-1 in HUVEC. Cells were stimulated with TNF-α and/or IL-1β for 6 or 24 hours and pre-treated with 1µM RWJ 67657 for 1 hour. mRNA expression was determined with real-time RT-PCR and results are expressed as fold induction compared to unstimulated cells (fold induction=1). Bars show means (n=4-6) and SEM. (* p< 0.05, paired T-test calculated against the stimulated control).

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Effects of RWJ 67657 on mRNA expression of adhesion molecules With real-time RT-PCR mRNA expression of CD31, E-selectin, VCAM-1 and ICAM-1 was determined in HUVEC (n=4-6) after 6 and 24 hours of stimulation with 10 ng/ml TNF-α or IL-1β. The expression is measured as fold induction compared to the unstimulated sample. As can be seen from figure 2 mRNA expression of CD31 did not change during stimulation, in contrast to the strong induction of mRNA expression for E-selectin, VCAM-1 and ICAM-1. After 6 hours of stimulation the mRNA expression of these adhesion molecules was higher than after 24 hours of stimulation. Furthermore, IL-1β induced E-selectin mRNA expression to a higher extent than did TNF-α. Pre-treatment with 1 µM RWJ 67657 did not influence the expression of CD31 nor that of the induced VCAM-1 expression. E-selectin expression however was reduced to 78% and 65% respectively after TNF-α or IL-1β stimulation at 6 hours, and to 43% and 35% respectively after 24 hours of stimulation. ICAM-1 mRNA expression at 24 hours was reduced to 73% after TNF-α stimulation and 56% after IL-1β stimulation. Effects of RWJ 67657 on membrane expression of adhesion molecules Membrane adhesion molecule expression by HUVEC was determined at 6 and 24 hours after stimulation with 10 ng/ml TNF-α or IL-1β. E-selectin expression was higher after stimulation with IL-1β than with TNF-α. This was also the case for VCAM-1 expression except at 24 hours when TNF-α-induced expression was higher. Both cytokines induced high ICAM-1 expression, which increased with time (data not shown). The effect of pre-treatment with 1 µM RWJ 67657 on adhesion molecule expression was calculated as follows: the MFI (mean fluorescence intensity) of stimulated cells for each experiment was set to 100%, and the expression due to the treatment with RWJ 67657 was calculated relative to the 100% (Table 1). Pre-treatment with RWJ 67657 led to reduced expression of E-selectin under all conditions tested, of VCAM-1 after 6 hours, and of ICAM-1 after TNF-α stimulation for 6 hours (all not statistically significant). Table 1. Effect of RWJ pre-treatment on adhesion molecule expression by HUVEC (n=3). Adhesion molecule expression on TNF-α and IL-1β stimulated HUVEC was determined 6 and 24 hours after stimulation. The effect of 1 hour pre-treatment with 1µM RWJ 67657 was calculated as percentage compared to the MFI of stimulated cells (=100%).

TNF-α IL-1β 6 hr 24 hr 6 hr 24 hr % % % %

CD31 104 +/- 13 95 +/- 20 91 +/- 6 89 +/- 17

E-selectin 77 +/- 22 59 +/- 19 81 +/- 14 67 +/- 9

VCAM-1 66 +/- 15 105 +/- 37 73 +/- 17 108 +/- 18

ICAM-1 79 +/- 15 99 +/- 29 100 +/- 21 93 +/- 21


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In figure 3 one of the three experiments is shown for adhesion molecule expression after TNF-α and IL-1β stimulation, and after RWJ 67657 pre-treatment followed by cytokine stimulation. The MFI values and percentage bright positive cells are indicated in the figures. As can be seen from the figure there was an overall reduced expression of E-selectin after treatment with the p38 MAPK inhibitor and also the number of positive cells was decreased. This reduction however was not statistically significant. VCAM-1 and ICAM-1 were moderately reduced. Control experiments with 0.01% DMSO showed no significant effects of the solvent on adhesion molecule mRNA or protein expression (data not shown).

Figure 3. Effect of RWJ 67657 pre-treatment on adhesion molecule expression on HUVEC after 6 and 24 hours. HUVEC were stimulated with TNF-α or IL-1β. E-selectin, ICAM-1 and VCAM-1 expression was measured by flowcytometry (thick black line) and after pre-treatment with 1µM RWJ 67657 (grey line). The mean fluorescence intensity (MFI) is depicted in the graph: first line cytokine stimulated expression, second line cytokine + RWJ 67657 treated expression. In brackets the percentage of bright positive cells is indicated. The thin black line represents the isotype control.

Effects of RWJ 67657 on chemokine and cytokine mRNA expression and production Quantitative mRNA expression of IL-8, MCP-1 and IL-6 was determined in HUVEC (n=4-6) after 6 and 24 hours of stimulation with 10 ng/ml TNF-α or IL-1β in the presence or absence of RWJ 67657. Stimulation with IL-1β induced higher mRNA expression of both IL-8 and IL-6 compared to stimulation with TNF-α (figure 4A). At both time points a marked inhibition of IL-8 and IL-6 mRNA expression was seen due to pre-treatment with 1 µM RWJ 67657. This inhibition was statistically significant in

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nearly all cases. MCP-1 mRNA expression was induced equally by TNF-α and IL-1β, and decreased in time. The inhibition seen by the p38 MAPK inhibitor was not statistically significant. The production of IL-8, MCP-1 and IL-6 protein by HUVEC after 24 hours of TNF-α or IL-1β stimulation is shown in figure 4B. Protein production was induced at high levels, especially IL-8 and MCP-1. Pre-treatment with 1 µM RWJ 67657 induced a significant reduction of IL-8, MCP-1 an IL-6 production. No effect of 0.01% DMSO on cytokine production was observed (data not shown).

Figure 4. (A). Effect of RWJ 67657 pre-treatment on mRNA expression of IL-8, MCP-1 and IL-6 in HUVEC. Cells were stimulated with TNF-α and IL-1β for 6 or 24 hours in the absence or presence of 1µM RWJ 67657 pre-treatment for 1 hour. mRNA expression was determined with real-time RT-PCR. Results are expressed as fold induction compared to unstimulated cells (fold induction=1). Bars show means (n=4-6) and SEM. (* p< 0.05, ** p<0.001, paired T-test, calculated against the stimulated control). (B). Effects of RWJ 67657 pre-treatment on protein production of IL-8, MCP-1 and IL-6 by HUVEC (n=5). Cells were stimulated with TNF-α or IL-1β for 24 hours and pre-treated with 1µM RWJ 67657 (t=-1hr). Protein production was measured in supernatants by ELISA and expressed in ng/ml. Bars show mean and SEM. (* p< 0.05, ** p<0.001, paired T-test, calculated against the stimulated control).

A B


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DISCUSSION The effects of RWJ 67657, a p38 MAPK inhibitior, on TNF-α and IL-1β stimulated endothelial cells were investigated in this study. Complete inhibition of MAPKAPK-2 phosphorylation was demonstrated at 1 µM. At this concentration we found inhibition of E-selectin expression, both at the level of mRNA and protein production. A significant inhibition of production of the chemokines IL-8 and MCP-1 was found, and also production of the pro-inflammatory cytokine IL-6 was significant inhibited. In inflammatory diseases the accumulation of leukocytes in a given tissue can lead to varying degrees of cell damage, extra cellular matrix disruption and organ dysfunction. Several attempts have been made to therapeutically block leukocyte adhesion to endothelium and thus control inflammation. This has been done for instance by the use of specific monoclonal antibodies to adhesion molecules in animal models 18, and also in RA patients 19. Also currently used anti-rheumatic agents, for example metho-trexate, glucocorticosteroids and leflunomide interfere to varying degree with the expression or function of different CAMs 20;21. Therapeutic strategies aimed at blocking chemokines and their receptors have also been studied. Recently Haringman et al reported relevant biological effects in RA patients treated with chemokine receptor 1 antagonist 22. In rheumatoid synovial tissue p38 MAPK is predominantly expressed in the lining layer and in endothelial cells 9 and therefore we wanted to investigate the effects of the p38 MAPK inhibitor on adhesion molecule expression and chemokine production. In our study we found a complete inhibition of phosphorylation of MAPKAPK-2, the direct downstream substrate of p38 MAPK at a concentration of 1µM RWJ 67567. The study by Parasrampuria demonstrated that after a single oral dose ranging from 0.25 to 30 mg/kg a plasma concentration of 0.01 to 6 µM of the p38 MAPK inhibitor could be reached in humans 14. We therefore decided to perform our study with 1 µM RWJ 67567, which equals a dose of 5-10 mg/kg. We did not compare our p38 MAPK inhibitor with the literature standard SB 203580 for two reasons: first, RWJ 67657 is already described to be 10-fold more potent than SB 203580 in all p38-dependent systems tested 11. Secondly, it has been demonstrated that SB 203580 can also block protein kinase B (PKB) activity at 3-5 µM 23, as well as JNK activities at 3-10 µM 24. Therefore SB 203580 is now considered not to be a specific p38 MAPK inhibitor. Different subsets of leukocytes use different (combinations of) cell adhesion molecules (CAMs) depending on the inflammatory stimulus and the site of inflammation. Furthermore, the functional consequences of downregulating cell adhesion molecule expression on the endothelial surface can be at the level of leukocyte rolling, adhesion, and transmigration. McCafferty 25 reported that in postcapillary venules (studied in the cremaster muscle venules, size ~30 µm in diameter) E-selectin knock out could completely abrogate antigen challenge induced leukocyte recruitment: leukocyte rolling as well as adhesion and transmigration were completely abolished in the E/P-selectin k.o. mice, while in the P-selectin k.o. mice only leukocyte rolling was affected. In contrast, leukocyte recruitment induced by local TNF-α administration was neither affected in P-selectin k.o. nor in P-/E-selectin k.o.. In both the antigen induced and TNF-α induced inflammation, the number of cells infiltrating and the cell types making up the infiltrate were similar. The main difference between the two models was the expression of VCAM-1 in the venular endothelium of the TNF-α

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induced inflammation, indicating that VCAM-1 is possibly able to control leukocyte recruitment to sites of inflammation, irrespective whether P- or E-selectin is present. Norman and colleagues recently reported on the role of CAMs during immune complex-dependent inflammation in the mouse cremaster muscle 26. While E-selectin inhibition by antibody infusion could abrogate leukocyte rolling on the venular endothelium (venules were 25-40 µm in diameter), its effects on leukocyte migration were limited. In contrast, treatment with anti-VCAM-1 antibody could inhibit leukocyte adhesion more than 70%, with subsequent functional consequence being a more than 80% inhibition of leukocyte migration. In the current study we showed that pre-treatment of HUVEC with RWJ 67657 inhibited E-selectin expression to 77%/59% (TNF-α induced, 6 hrs resp. 24 hrs) and 81%/67% (IL-1β induced, 6 hrs resp. 24 hrs). Moreover, we showed that VCAM-1 protein expression was inhibited at the 6 hours time point, 66 % (TNF-α induced) versus 73 % (IL-1β induced), while also ICAM-1 was inhibited at 6 hours after start TNF-α activation. Reports on p38 MAPK inhibition on ICAM-1 expression after TNF-α stimulation in literature are contradicting 27;28, whereas results after IL-1β stimulation have not been reported. Our results concerning VCAM-1 mRNA and protein expression corroborate the data by Pietersma et al 29, who stated that p38 MAPK regulates endothelial VCAM-1 expression at the post-transcriptional level. Extensive in vivo studies will be needed to answer the question whether the anti-inflammatory effects in the ranges reported here will affect leukocyte rolling, adhesion, and subsequent recruitment to the diseased tissue. The fact that at 1 µM RWJ 67657 phosphorylation of MAPKAPK-2 was completely blocked, while adhesion molecule expression was partly inhibited, indicates that other signal transduction routes are also important in regulating adhesion molecule expression in HUVEC. Activation through p38 MAPK and NF-κB have been described to act synergistically in inducing adhesion molecule expression in endothelial cells 7;8. Recently Viemann et al 30 reported that the induction of cell-surface receptor expression measured with oligonucleotide microarray was highly depended on IKK2/NF-κB activation , whereas there was additional modulation by p38 MAPK. With real-time RT-PCR they found that for instance VCAM-1 and IL-8 were dependent of both pathways, where as ICAM-1 expression was dependent of NF-κB activation alone. So both pathways play a role in endothelial activation, with CAM expression being more regulated by the NF-κB pathway, and chemokine production more by the p38 MAPK pathway. In our study we demonstrated that p38 MAPK inhibition affects E-selectin expression both at the mRNA and the protein level in HUVEC. The study by Fijen et al demonstrated that RWJ 67657 at a single dose of 5, 10 and 20 mg/kg prevented endotoxin-induced increase of circulating ICAM-1 and circulating E-selectin and also of integrins on neutrophils 13. They demonstrated that also in vivo neutrophil and endothelial activation could be prevented by RWJ 67657. With quantitative real-time RT-PCR we found that induction of IL-8 and IL-6 mRNA was higher after stimulation with IL-1β than with TNF-α, and a significant inhibition by RWJ 67657 was seen after both stimuli. In our study RWJ 67657 was added 1 hour before stimulation of the cells, but also administration of the compound up till 4 hours after stimulation of HUVEC induced marked reduction in IL-8 and IL-6 mRNA levels


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(unpublished results). MCP-1 mRNA and protein production was equally high after both stimuli. A significant inhibitory effect was demonstrated by RWJ 67657 treatment for IL-8, MCP-1 and IL-6 protein production. These observations corroborate data reported by others 31;32. Since chemokines play an essential role in maintaining the leukocyte-endothelial interactions after the initial interaction regulated by the selectins, the significant downregulation of IL-8 and MCP-1 could have an important effect on leukocyte infiltration in inflammatory disease. Previously we demonstrated significant inhibitory effects on inflammatory mediators produced by rheumatoid synovial fibroblasts 12 and we also showed significant inhibition of TNF-α production in monocyte-derived macrophages 33. p38 MAPK inhibitors have effects on different cell types, which could enhance the therapeutic effects, but also enlarge the risk of side effects. One of the reasons for undesirable effects might be the cross-reactivity against other kinases, which was not the case for RWJ 67657 11. Moreover, RWJ 67657 has been shown to have acceptable safety and acceptable pharmacokinetics to warrant further investigation 14. p38 MAPK inhibitors like RWJ 67657 have shown to effectively inhibit pro-inflammatory mediators in different cells in vitro, and also in vivo. Whether kinase inhibitors will have an important role in the treatment of RA will be dependent of safety issues, and will be investigated in the near future. ACKNOWLEDGEMENTS Annelie Rawee, Henk Moorlag and Berber Doornbos-van der Meer are gratefully acknowledged for excellent technical assistance. Supported by the Dutch Rheumatology Foundation, Johnson and Johnson Pharmaceutical Research and Development, Raritan, NJ, and the University Medical Center Groningen, The Netherlands.

REFERENCES

1 Choy EH, Panayi GS. Cytokine pathways and joint inflammation in rheumatoid arthritis. N.Engl.J.Med. 2001; 344: 907-16.

2 Szekanecz Z, Koch AE. Cell-cell interactions in synovitis. Endothelial cells and immune cell migration. Arthritis Res. 2000; 2: 368-73.

3 von Andrian UH, Mackay CR. T-cell function and migration. Two sides of the same coin. N.Engl.J.Med. 2000; 343: 1020-34.

4 Gonzalez-Amaro R, Diaz-Gonzalez F, Sanchez-Madrid F. Adhesion molecules in inflammatory diseases. Drugs 1998; 56: 977-88.

5 Redlich K, Schett G, Steiner G, Hayer S, Wagner EF, Smolen JS. Rheumatoid arthritis therapy after tumor necrosis factor and interleukin-1 blockade. Arthritis Rheum. 2003; 48: 3308-19.

6 Smith MD, Slavotinek J, Au V et al. Successful treatment of rheumatoid arthritis is associated with a reduction in synovial membrane cytokines and cell adhesion molecule expression. Rheumatology.(Oxford) 2001; 40: 965-77.

7 Jersmann HP, Hii CS, Ferrante JV, Ferrante A. Bacterial lipopolysaccharide and tumor necrosis factor alpha synergistically increase expression of human endothelial adhesion molecules through activation of NF-kappaB and p38 mitogen-activated protein kinase signaling pathways. Infect.Immun. 2001; 69: 1273-9.

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8 Read MA, Whitley MZ, Gupta S et al. Tumor necrosis factor alpha-induced E-selectin expression is activated by the nuclear factor-kappaB and c-JUN N-terminal kinase/p38 mitogen-activated protein kinase pathways. J.Biol.Chem. 1997; 272: 2753-61.





13 Fijen JW, Tulleken JE, Kobold AC et al. Inhibition of p38 mitogen-activated protein kinase: dose-dependent suppression of leukocyte and endothelial response after endotoxin challenge in humans. Crit Care Med. 2002; 30: 841-5.


15 Mulder AB, Blom NR, Smit JW et al. Basal tissue factor expression in endothelial cell cultures is caused by contaminating smooth muscle cells. Reduction by using chymotrypsin instead of collagenase. Thromb.Res. 1995; 80: 399-411.

16 Maciag T, Hoover GA, Weinstein R. High and low molecular weight forms of endothelial cell growth factor. J.Biol.Chem. 1982; 257: 5333-6.


18 Issekutz AC, Ayer L, Miyasaka M, Issekutz TB. Treatment of established adjuvant arthritis in rats with monoclonal antibody to CD18 and very late activation antigen-4 integrins suppresses neutrophil and T-lymphocyte migration to the joints and improves clinical disease. Immunology 1996; 88: 569-76.

19 Kavanaugh AF, Schulze-Koops H, Davis LS, Lipsky PE. Repeat treatment of rheumatoid arthritis patients with a murine anti-intercellular adhesion molecule 1 monoclonal antibody. Arthritis Rheum. 1997; 40: 849-53.

20 Dimitrijevic M, Bartlett RR. Leflunomide, a novel immunomodulating drug, inhibits homotypic adhesion of mononuclear cells in rheumatoid arthritis. Transplant.Proc. 1996; 28: 3086-7.

21 Wheller SK, Perretti M. Dexamethasone inhibits cytokine-induced intercellular adhesion molecule-1 up-regulation on endothelial cell lines. Eur.J.Pharmacol. 1997; 331: 65-71.

22 Haringman JJ, Kraan MC, Smeets TJ, Zwinderman KH, Tak PP. Chemokine blockade and chronic inflammatory disease: proof of concept in patients with rheumatoid arthritis. Ann.Rheum.Dis. 2003; 62: 715-21.

23 Lali FV, Hunt AE, Turner SJ, Foxwell BM. The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J.Biol.Chem. 2000; 275: 7395-402.

24 Clerk A, Sugden PH. The p38-MAPK inhibitor, SB203580, inhibits cardiac stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs). FEBS Lett. 1998; 426: 93-6.

25 McCafferty DM, Kanwar S, Granger DN, Kubes P. E/P-selectin-deficient mice: an optimal mutation for abrogating antigen but not tumor necrosis factor-alpha-induced immune responses. Eur.J.Immunol. 2000; 30: 2362-71.

26 Norman MU, Van De Velde NC, Timoshanko JR, Issekutz A, Hickey MJ. Overlapping roles of endothelial selectins and vascular cell adhesion molecule-1 in immune complex-induced leukocyte recruitment in the cremasteric microvasculature. Am.J.Pathol. 2003; 163: 1491-503.


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27 Fichtner F, Koslowski R, Augstein A, Hempel U, Rohlecke C, Kasper M. Bleomycin induces IL-8 and ICAM-1 expression in microvascular pulmonary endothelial cells. Exp.Toxicol.Pathol. 2004; 55: 497-503.

28 Ju H, Behm DJ, Nerurkar S et al. p38 MAPK inhibitors ameliorate target organ damage in hypertension: Part 1. p38 MAPK-dependent endothelial dysfunction and hypertension. J.Pharmacol.Exp.Ther. 2003; 307: 932-8.

29 Pietersma A, Tilly BC, Gaestel M et al. p38 mitogen activated protein kinase regulates endothelial VCAM-1 expression at the post-transcriptional level. Biochem.Biophys.Res.Commun. 1997; 230: 44-8.

30 Viemann D, Goebeler M, Schmid S et al. Transcriptional profiling of IKK2/NF-{kappa}B- and p38 MAP kinase-dependent gene expression in TNF-{alpha}-stimulated primary human endothelial cells. Blood 2004.

31 Goebeler M, Gillitzer R, Kilian K et al. Multiple signaling pathways regulate NF-kappaB-dependent transcription of the monocyte chemoattractant protein-1 gene in primary endothelial cells. Blood 2001; 97: 46-55.

32 Marin V, Farnarier C, Gres S et al. The p38 mitogen-activated protein kinase pathway plays a critical role in thrombin-induced endothelial chemokine production and leukocyte recruitment. Blood 2001; 98: 667-73.


6

Chemokine production and E-selectin expression in


(mitogen activated protein kinase) inhibitor RWJ 67657

Johanna Westra1

Joanna M Kułdo2


Grietje Molema2

Pieter C Limburg1

From the Departments of 1 Rheumatology, and 2 Medical Biology section of Pathology

and Laboratory Medicine, University Medical Center Groningen, The Netherlands

International Immunopharmacology: in press

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ABSTRACT Endothelial cells play an important role in inflammatory diseases like rheumatoid arthritis by recruitment of inflammatory cells. The cytokines TNF-α and IL-1β are major inducers of endothelial cell activation and are stimulators of inflammatory signal transduction pathway involving p38 MAPK (mitogen-activated protein kinase). The present study investigated the effects of p38 MAPK inhibition on cell adhesion molecule (CAM) expression and chemokine production by endothelial cells both on mRNA and protein level. Pre-treatment of endothelial cells with the pharmacologically relevant concentration of 1 µM of the p38 MAPK inhibitor RWJ 67657 reduced TNF-α and IL-1β induced mRNA and membrane expression of E-selectin. Moderate inhibitory effects on ICAM-1 and VCAM-1 expression were found. Significant reduction of mRNA expression and protein production of the inflammatory cytokine IL-6 and the chemokines IL-8 and MCP-1 was demonstrated. Treatment with RWJ 67657 could lead to reduced leukocyte infiltration by the reduction of E-selectin expression and chemokine production. Key words. chemokine, endothelial cell, E-Selectin, MAPK inhibitor


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INTRODUCTION Rheumatoid arthritis (RA) is an inflammatory disease characterized by hyperplasia, increased vascularity, and infiltration of inflammatory cells into the synovial membrane 1. It is now known that the endothelial cells (EC), which line the lumen of the blood vessels, are not passive bystanders but are active responders to stimuli like activated leukocytes and cytokines 2. After stimulation EC can produce a number of inflammatory mediators and express cellular adhesion molecules (CAMs). Most CAMs involved in endothelial activation belong to three families, i.e., the integrins, selectins, and immunoglobulin superfamilies 3. The adhesion of leukocytes to EC takes place in four steps. The first step is a weak adhesion (tethering and rolling) mediated by E-, P- and L-selectins. The next step is leukocyte activation and is a consequence of interaction between chemokines and their receptors on leukocytes. Then firm adhesion takes place, which is mediated mostly by interaction between integrins on the leukocytes and vascular adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 on the EC. The final step is the transendothelial migration, which is directed by secreted chemokines bound to endothelial heparan sulphate glycosaminoglycans. Essential chemokines are interleukin (IL)-8 which attracts neutrophils, and monocyte chemo-attractant protein (MCP)-1, the main attractant for mononuclear cells 2;3. The cytokines TNF (tumour necrosis factor)-α and IL-1β, produced by activated macrophages in the synovium, have been identified as the major inducers of EC activation in RA 4. Blockade of these cytokines has proven to be effective in the treatment of RA, although Redlich et al recently concluded that targeting individual molecules such as TNF-α may not be sufficient in interfering with both inflammation and joint destruction 5. Successful drug treatment of RA is associated by a decrease in cytokine production, but also by a decrease in E-selectin and ICAM-1 expression 6. In vitro studies demonstrated that in TNF-α and/or lipopolysaccharide (LPS) stimulated human umbilical vein endothelial cells (HUVEC) the nuclear factor-κB (NF-κB), and p38 mitogen-activated protein kinase (MAPK) pathways are important in controlling adhesion molecule expression 7;8. Activation of all three stress- and mitogen-activated protein kinases (SAPK/MAPK) was found throughout the RA synovial tissue, whereas activated p38 MAPK predominantly was located in the synovial lining layer and in synovial endothelial cells 9. Interest in protein kinases and in particular in p38 MAPK as drug targets has increased in the last years as recently reviewed by Kumar et al 10. The p38 MAPK inhibitor RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol) has been shown to be effective in inhibiting the release of TNF-α from LPS-treated human peripheral blood mononuclear cells with an IC50 of 3 nM 11. This compound was approximately 10-fold more potent than the reference standard p38 MAPK inhibitor SB 203580 in all p38 dependent in vitro systems tested. RWJ 67657 specifically inhibited the enzymatic activity of recombinant p38 α and β, but not of γ and δ in vitro, and had no significant activity against a variety of other kinases 11. We recently demonstrated that RWJ 67657 significantly inhibited IL-6, IL-8, MMP-3 and COX-2 mRNA expression by IL-1β and TNF-α stimulated rheumatoid synovial fibroblasts 12. Furthermore Fijen et al showed that this compound induced a dose-dependent suppression of leukocyte and endothelial cell response after endotoxin challenge in