Cross-Talk between Oxidative Stress and Pro-Inflammatory Cytokines in Acute Pancreatitis: A Key Role...

Current Pharmaceutical Design, 2009, 15, 000-000 1

1381-6128/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.

Cross-Talk between Oxidative Stress and Pro-Inflammatory Cytokines in Acute Pancreatitis: A Key Role for Protein Phosphatases

Javier Escobar1, Javier Pereda

1, Alessandro Arduini

1, Juan Sandoval

2, Luis Sabater

3, Luis Aparisi

4,

Gerardo López-Rodas2 and Juan Sastre

1,*

1Department of Physiology,

2Department of Biochemistry & Molecular Biology, University of Valencia, Spain;

3Department of Surgery and

4Laboratory of Pancreatic Function, Universitary Clinic Hospital, Valencia, Spain

Abstract: Acute pancreatitis is an acute inflammatory process localized in the pancreatic gland that frequently involves

peripancreatic tissues. It is still under investigation why an episode of acute pancreatitis remains mild affecting only the

pancreas or progresses to a severe form leading to multiple organ failure and death. Proinflammatory cytokines and oxida-

tive stress play a pivotal role in the early pathophysiological events of the disease. Cytokines such as interleukin 1beta and

tumor necrosis factor alpha initiate and propagate almost all consequences of the systemic inflammatory response syn-

drome. On the other hand, depletion of pancreatic glutathione is an early hallmark of acute pancreatitis and reactive oxy-

gen species are also associated with the inflammatory process. Changes in thiol homestasis and redox signaling decisively

contribute to amplification of the inflammatory cascade through mitogen activated protein kinases (MAP kinases) path-

ways. This review focuses on the relationship between oxidative stress, pro-inflammatory cytokines and MAP

kinase/protein phosphatase pathways as major modulators of the inflammatory response in acute pancreatitis. Redox sen-

sitive signal transduction mediated by inactivation of protein phosphatases, particularly protein tyrosin phosphatases, is

highlighted.

Key Words: Acute pancreatitis, oxidative stress, nitrosative stress, glutathione, TNF- , MAP kinases, protein tyrosin phospha-tases.

1. ACUTE PANCREATITIS

Acute pancreatitis (AP) is an initially localized inflam-mation of the pancreatic gland that frecuently involves peri-pancreatic tissues which may lead to local and systemic complications. The etiology of the disease varies but alcohol and gallstones are the most important causes. The incidence of AP in the European Union and USA varies widely de-pending on the country and the type of study from 5 to 30 cases/100 000 /year, [1-3] and it is increasing during the last few years, mainly due to alcoholic consumption [4, 5]. The overall mortality in patients with acute pancreatitis is ap-proximately 5%, but this percentage increases up to 17% in patients with necrotizing pancreatitis due to multiple organ failure [6]. The percentage of mortality has diminished by improving antibiotic therapy, intensive care units and sur-gery [7, 8]. However, no specific effective treatment has been reported so far in clinical trials.

The precise mechanisms by which the etiological factors induce an attack of AP are still unclear, but when initiated, common inflammatory and repair pathways seem to be in-volved. Numerous inflammatory mediators such as activated pancreatic enzymes, cytokines, chemokines, free radicals, Ca

2+, platelet activating factor, adenosine, substance P and

other neurogenic factors have been involved in the patho-genesis of acute pancreatitis [9-18].

*Address correspondence to this author at the Department of Physiology,

School of Pharmacy, University of Valencia, Avda. Vicente Andrés Estellés

s/n, 46100 Burjasot (Valencia), Spain; Tel: 34-963543815; Fax: 34-

963543395; E-mail: [email protected]

It is also unknown why an episode of AP remains mild or progresses to a severe form. Extensive pancreatic damage and necrosis lead to activation of pathophysiological mecha-nisms involved in the systemic inflammatory response, being cytokines and oxidative stress components of major impor-tance [19]. Mortality in AP is produced by multiple organ failure due to systemic inflammatory response. Thus, AP should be considered as another pathological condition which together with sepsis, trauma, burns, and surgery, may lead to the systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) [20]. In all these inflammatory diseases cytokines and oxy-gen free radicals certainly play a key role as initiators, en-hancers and damaging agents (see Fig. 1) [21].

Main differences between acute pancreatitis and other inflammatory disease may rely on the role of acinar cell ne-crosis to determine severity if extensive or infected. Pancre-atic necrosis during acute pancreatitis is a key factor predic-tive of outcome [22-24] and infection of necrotic tissue is the most serious complication in severe acute pancreatitis [25].

Pancreatic digestive enzymes contribute at an early stage to necrosis of acinar cells and consequently to the inflamma-tion of the pancreas. Nevertheless, they are not responsible for the conversion of a local inflammatory process into a systemic inflammatory response. Consequently, no satisfac-tory results in terms of mortality have emerged from clinical trials inhibiting digestive enzymes [26]. Cytokines and oxi-dative stress seems to be the major actors for development of SIRS.

2 Current Pharmaceutical Design, 2009, Vol. 15, No. 00 Escobar et al.

2. LOCAL EARLY EVENTS IN ACUTE PANCREATI-

TIS

Knowledge of the early events initiating an attack of acute pancreatitis, and the mechanism of progression from mild AP to a severe necrotizing form has been the aim of numerous studies. The pancreas synthesizes a great amount of digestive enzymes which in normal conditions are stored as inactive zymogen precursors to avoid autodigestion. Many mechanisms maintain zymogens inactive, such as non opti-mal pH, presence of the trypsin inhibitor and presence of proteases degrading low levels of active forms.

An early event in AP is intrapancreatic activation of zy-mogen, and consequently, activation of trypsinogen and other zymogen enzymes [27-29]. One interesting theory of zymogen activation is the colocalization hypothesis, which suggests that pancreatic zymogens colocalize with lysosomal enzymes during the early events in AP. This phenomenon occurs in different animal models of AP and precedes typical early features of pancreatitis such as hyperamylasemia and pancreatic edema [30]. Lysosomal cathepsin B seems to be involved in activating trypsinogen since induction of AP in cathepsin B knockout mice showed 80% reduced tripsin ac-tivity compared with wild-type [31].

Calcium is also related with tripsinogen activation and seems a pivotal key in early AP, but remains controversial if

is directly involved in protease activation or mediates indi-rect mechanisms for zymogen activation [32, 33]. In acinar cells, Ca

2+ spiking occurs mainly in the apical zone and it

regulates fluid and enzyme secretion.

Another early mechanism in AP is up-regulation of heat shock proteins, which are a family of proteins that protects cells against inflammation or stress. Thus, Hsp27, Hsp60 and Hsp70, are up-regulated in the pancreas during AP as protec-tive mechanisms in response to acinar cell injury [34-36]. It has been demonstrated that up-regulation of HSPs prevents zymogen activation [35]. Thus, there is spontaneous activa-tion of pancreatic trypsinogen in Hsp70.1 knockout mice [37]. More recently, it has been reported that overexpression of Hsp27, a regulator of actin polymerization, preserved the F-actin microfilaments, reduced the severity and protected against the systemic inflammatory response in cerulein-induced AP [38]. The protective effects of HSP are also me-diated through other inflammatory mediators such as NF-kB [38, 39].

The type of acinar cell death is crucial in AP. Severe forms of acute pancreatitis are associated with extended ne-crosis whereas mild attackts of acute pancreatitis exhibit apoptosis and little necrosis. Thus, shifting the pattern of death responses of pancreatitis towards apoptosis and away from necrosis could be of therapeutic value [24, 40]. Re-cently, it has been described that Ca

2+exert a dual response

Fig. (1). Pathophysiological mechanisms of the systemic inflammatory response in acute pancreatitis. Abbreviations: ICAM: intercellular

adhesion molecule; IL- 1 : interleukin 1 ; MODS: Multiple organ dysfunction syndrome; PAF: platelet activating factor; PLA2: phospholi-

pase A2; ROS: Reactive oxygen species; TNF- : tumor necrosis factor ; XO : xanthine oxidase; VCAM : vascular adhesion molecule.

Cross-Talk between Oxidative Stress and Pro-Inflammatory Cytokines Current Pharmaceutical Design, 2009, Vol. 15, No. 00 3

on cytochrome c release from isolated mitochondria. Ca2+

per se stimulates cytochrome release but Ca

2+-induced depo-

larization inhibits it. ROS play a pivotal role in apoptosis through releasing cytochrome C from mitochondria [41]. Thus, Ca

2+ is presented as a modulator of apoptosis-necrosis

shift. Other authors have shown that the oscillatory global rises of cytosolic Ca

2+ may induce apoptosis [42] while sus-

tained elevations promote necrosis [43, 44].

Extracellular factors such as microvascular circulation are also important in formation of edema and inflammatory response. Microvascular circulation is compromised in se-vere forms of acute pancreatitis, leading to ischaemia and circulatory stasis. It has been observed that overall pancreatic blood flow decreases very early in AP [45, 46]. Functional capillary density, which is a measure of the proportion of capillaries that are perfused, is also significantly reduced [47]. The severity of microcirculatory disorders correlates with severity of the disease, suggesting that microperfusion is a key event in necrosis [48]. Several mediators are in-volved in microcirculatory disorders, including endothelin-1, ROS, endothelial nitric oxide synthase, substance P as well as cytokines and chemokines [6].

Cytokine up-regulation and oxidative stress are key early events in AP that activate intracellular signal pathways lead-ing to edema, inflammation, epigenetic modulation, and/or cell death. Their role in integrating other inflammatory me-diators, provoking local damage, activating cell signals and amplifying the systemic inflammatory response is discussed below.

3. CYTOKINES AND OTHER MEDIATORS OF IN-

FLAMMATION IN ACUTE PANCREATITIS

Cytokines are low molecular weight soluble proteins produced during stress or injury in numerous cell types as means of cell-to-cell communication [49, 50]. Activated leu-kocytes are the main source of cytokines, which are conse-quently essential components of the inflammatory cascade. The primary members of the cytokine inflammatory family, particularly interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF- ), induce their own expression as well as ex-pression of other cytokines, leading to amplification of the inflammatory response [50]. These cytokines initiate and propagate almost all the consequences of the systemic in-flammatory response syndrome [50, 51]. Since dexametha-sone treatment does not affect tripsinogen activation, it seems that cytokines and inflammation occurs immediately after trypsinogen activation [52].

Acute pancreatitis is characterized initially by interstitial edema together with migration of macrophages and neutro-phils towards the pancreatic tissue [53, 54]. Initially, macro-phages and neutrophils were the only cells thought to be in-volved in the inflammatory response. Nevertheless, more recent evidence clearly demonstrates that resident paren-chymal and mesenchymal cells may secrete chemokines, cytokines and may induce adhesion molecules. Thus, acinar cells respond, produce and release cytokines, chemokines and adhesion molecules [9, 55-60]. Consequently, acinar cells behave as inflammatory cells [59], and together with macrophages induce leukocyte infiltrate and trigger inflam-matory response [61].

Leukocyte recruitment within the inflamed pancreas be-gins as early as 3 hours after AP induction with rolling and adhesion of the circulating leukocytes to the endothelium [61]. This process is carried out via adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) which is markedly up-regulated during inflammation [62]. Infiltrated neutrophils, attracted by chemokines, cytokines, and oxida-tive stress, amplify the inflammatory response. Conse-quently, neutrophil depletion by anti-neutrophils antibodies partially diminished the experimental AP severity [63]. Therefore, neutrophilic NADPH oxidase may mediate intra-pancreatic trypsin activation, which in turn, aggravates AP.

Activated macrophages release pro-inflammatory cytoki-nes, such as IL-1, IL-6 and TNF- , in response to the local damage of the pancreas [64]. As indicated above, local cells contribute to increase serum levels of IL-1, IL-6 and TNF- in experimental acute pancreatitis. These levels correlate with the degree of pancreatic inflammation [65-67].

Monocytes from patients with systemic complications in acute pancreatitis exhibit an increased production of TNF- , IL-6 and IL-8 in comparison with those from patients with-out them [68]. Similar results were found regarding the re-lease of these cytokines by peripheral blood mononuclear cells from patients with severe AP [69]. Thus, peripheral blood monocyte and neutrophil counts correlate with plasma inflammatory cytokines and TNF- soluble receptors in AP [70].

Obesity is a prognostic factor for severity in the evolution of acute pancreatitis since systemic complications are more frequent in obese patients than in non-obese ones, and pa-tients with severe acute pancreatitis exhibit higher percent-age of fat than those with mild acute pancreatitis [71-75]. The mechanism responsible for more severity in pancreatitis in obese subjects is not clear yet. It is worth noting that obe-sity is a pro-inflammatory condition [76] associated with oxidative and nitrosative stress [77]. Obese subjects and animals exhibit high serum and tissue levels of pro-inflammatory cytokines, such as TNF- and interleukin 6 [78]. The levels of pro-inflammatory interleukin IL-18 are also elevated in obese subjects, and simultaneous treatment with IL-12 and IL-18 causes severe acute pancreatitis in obese mice but edematous pancreatitis in control mice [79]. A decrease in adiponectin levels is a feature of obese animals and it might contribute to the severity of pancreatitis since adiponectin exhibits anti-inflammatory properties and a defi-ciency in adiponectin causes severe pancreatitis in mice fed a high-fat diet, whereas its over-expression protects against tissue damage [80].

Despite all the evidence implicating pro-inflammatory cytokines in the progression of AP, no clinical trials pertain-ing to cytokine modulation show clear beneficial effects [26]. The discovery of specific inhibitors of proinflammatory cytokines may allow the development of an effective anti-inflammatory therapy [8].

3.1. TNF- and IL-1

TNF- and IL-1 are pivotal cytokines in AP and both exhibit synergic effects in amplification of the inflammatory response. Consequently, attenuation of severity in AP is ob-served when IL-1 and TNF- receptors are blocked and mor-

https://www.researchgate.net/publication/5985301_Acute_Pancreatitis_Bench_to_the_Bedside?el=1_x_8&enrichId=rgreq-be756918-9871-49ff-a066-d850c669a583&enrichSource=Y292ZXJQYWdlOzI2ODEyMjQ2O0FTOjEyNjkzMDgyOTQ1MTI2NEAxNDA3Mjc0MDc2Mjg1

https://www.researchgate.net/publication/11770333_Optimising_Outcomes_in_Acute_Pancreatitis?el=1_x_8&enrichId=rgreq-be756918-9871-49ff-a066-d850c669a583&enrichSource=Y292ZXJQYWdlOzI2ODEyMjQ2O0FTOjEyNjkzMDgyOTQ1MTI2NEAxNDA3Mjc0MDc2Mjg1

https://www.researchgate.net/publication/13834879_Chemokine_gene_expression_in_rat_pancreatic_acinar_cells_is_an_early_event_associated_with_acute_pancreatitis?el=1_x_8&enrichId=rgreq-be756918-9871-49ff-a066-d850c669a583&enrichSource=Y292ZXJQYWdlOzI2ODEyMjQ2O0FTOjEyNjkzMDgyOTQ1MTI2NEAxNDA3Mjc0MDc2Mjg1


tality is also dramatically reduced in IL-1 and TNF- knock-out mice after AP [81-83].

TNF- is released from different tissues in the course of acute pancreatitis. There is an induction of its mRNA and protein in pancreas [66, 84]. Although acinar cells may pro-duce TNF- , leukocytes from the inflammatory infiltrate within the pancreatic tissue are considered as the predomi-nant source [50]. Macrophages increase TNF- secretion in response to acinar cell necrosis in vitro [85]. Acinar cells contribute to TNF- production, following activation by the ascitic fluid present in AP [57]. A few hours after the in-crease in TNF- expression in pancreas, an induction of its mRNA and protein also occurs in lung, liver and spleen [84, 86, 87].

TNF- triggers cell death signaling through divergent mechanisms mediated by protein kinase C and causes NF-kB activation leading to pro-inflammatory up-regulation [88, 89]. Etanercept, which inhibits TNF- production showed beneficial effects in experimental AP, diminishing pancreatic apoptosis via TNFR1 [90]. Beneficial effects were also re-ported with thalidomide, an immunomodulatory agent that inhibits TNF- and angiogenesis [91]. We found that inhibi-tion of TNF- production by pentoxifylline markedly dimin-ished leukocyte infiltrate, edema and glutathione depletion in pancreas as well as reduced serum lipase activity after ce-rulein-induced pancreatitis in rats [92]. In knockout mice deficient in TNF- receptors, the rate of mortality due to necrotizing AP decreased because the systemic response was restrained, although there was no reduction in the severity of the pancreatic damage [12].

The production of IL-1ß in AP is also pancreatic and extrapancreatic. As with TNF- , a few hours after the in-crease in IL-1ß expression in pancreas, both its mRNA and protein are induced in lungs and liver [84]. Leukocytes from the inflammatory infiltrate within the pancreatic tissue ap-pear to be the predominant source of IL-1ß [50]. IL-1 pro-duction is closely associated with induction of other genes within the same gene-family, such as that encoding for inter-leukin 1ß-converting enzyme (ICE), which is necessary for cleavage of pro-IL-1 protein into its active form [50].

IL-1 exhibits similar actions to TNF- . Thus, it induces the release of other cytokines, such as IL-2 by T-helper lym-phocytes and cellular adhesion molecules, which extend the inflammatory response [64]. IL-1 seems to be a pivotal inflammatory mediator in cell death associated sterile in-flammation [93], which is an important early event in acute pancreatitis.

IL-1 RA, the receptor antagonist of IL-1 , blocks the action of this cytokine and diminishes the injury in distant organs in necrotizing acute pancreatitis [94, 95]. Administra-tion of ghrelin, which reduce the release of IL-1 , attenuates pancreatic damage and the severity of pancreatitis in rats [96]. Moreover, inhibition of type IV phosphodiesterase by rolipram, which attenuates the production of inflammatory mediators by increasing intracellular cyclic AMP levels, re-duced IL-1 production and ameliorated AP [97].

3.2. IL-6 and IL-8

Serum levels of IL-6 correlate with the severity of the disease in patients with acute pancreatitis, thus IL-6 has been

proposed as a marker of severity of the disease [68, 98, 99]. A clinical study comparing mild AP vs. severe AP and measuring cytokine levels showed that IL-6 presented the highest differences between groups [100].

IL- 8 is a pro-inflammatory cytokine member of the so-called chemokines, released by activated macrophages or endothelial cells that is involved in neutrophil chemotaxis, activation, and degranulation [64]. At present, both IL-6 and IL-8 are considered markers of severity of AP, although they are not the driving force for initiation and propagation of the systemic inflammatory response [50].

3.3. Platelet Activating Factor

Another mediator linked to the systemic inflammatory response in AP is platelet activating factor (PAF), a lipid that functions as a pro-inflammatory cytokine, since it induces platelet activation and aggregation, neutrophil and monocyte activation, chemotaxis, vasodilatation as well as an increase in vascular permeability [50, 101]. PAF levels are closely related to TNF-alpna and IL-1 levels since each one in-creases the production of the others [50]. In contrast to TNF-

and IL-1 , PAF itself may cause AP [102]. Furthermore, PAF antagonists exhibit beneficial effects in experimental AP: diminishing plasma IL-1 levels and permeability of pan-creatic capillary endothelium [103] and reducing the severity of systemic inflammation [104]. PAF modulates gut barrier dysfunction, since inhibiting PAF with lexipafant in AP re-duced severity of pancreatitis-associated intestinal dysfunc-tion, associated with a diminish in systemic concentrations of IL-1 and local leukocyte recruitment [105]. Thus, bacte-rial translocation is diminished using specific PAF inhibitors [106].

3.4. IL-10 and PAP

Much attention has been focused in potential anti-inflammatory mediators such as IL-10 and PAP. IL-10 exerts some of its anti-inflammatory properties by inhibiting the production of IL-1ß and TNF- [107]. Clinically, IL-10 plasma levels were highest on the day of admission for hos-pitalization in patients with AP and stayed high in severe cases of AP, but decreased rapidly during the following days in mild cases of AP [108]. Accordingly, neutralization of endogenous IL-10 by specific antibodies increased the sever-ity of pancreatitis and associated lung injury as well as TNF-

expression in experimental AP [109]. On the other hand, administration of IL-10 in AP decreased lipase, amylase and pancreatic damage score [110]. Consequently, IL-10 might play a crucial role determining the evolution of AP towards the severe or to the mild form of the disease, and this prompted its use in the treatment of AP. Moreover, IL-6/IL-10 ratio may be an important value for prognosis [111].

Pancreatitis-associated proteins (PAP) are members of the Reg gene family (14 to 17 kDa) secretory proteins which have been shown to be strongly induced during acute pan-creatitis [112] and other inflammatory diseases [113]. In rats, there are three homologous PAP isoforms, referred to as PAP1, PAP2, and PAP3 [112]. The role for PAPs include cellular apoptosis, mediators of cell regeneration and prolif-eration, carcinogenesis, immunity, and inflammation [113]. Functional similarities to IL-10 suggest that PAP I could


play a role similar to this anti-inflammatory cytokine in epithelial cells. PAP I inhibits the inflammatory response by blocking NF-kappaB activation [114]. Accordingly, PAP prevents TNF- induced NF-kappaB activation and down-regulates cytokine production and adhesion molecule expres-sion [115, 116]. Hence, it may represent an important anti-inflammatory mechanism in AP. Indeed, inhibition of PAP expression by antisense oligodeoxyribonucleotides signifi-cantly worsened edema and fat necrosis and elevated leuko-cyte infiltration in AP [117]. However, the caerulein-induced acute pancreatitis in PAP I knock-out mice showed increased pancreatic apoptosis, inflammation and little necrosis. Sur-prisingly, these mice suffered the typical symptoms of AP attenuated compared with wild type [118]. Consequently, PAP seems to modulate apoptosis and inflammation but its role as protector in AP needs to be revised.

4. OXIDATIVE STRESS IN ACUTE PANCREATITIS

Oxidative stress was first defined as an unbalance be-tween pro-oxidant and antioxidants in favour of the formers [119]. During last decades several authors have highlighted the role of oxidative stress in the inflammatory response, particularly in the pancreatic injury associated with acute pancreatitis [17, 57, 120-123].

In the eighties the beneficial effects of pre-treatments with antioxidants such as superoxide dismutase (SOD), cata-lase (CAT) provided an indirect proof for the involvement of oxidative stress in acute pancreatitis [124, 125]. Indeed, these antioxidants diminished hyperamilasemia and pancre-atic edema in three different models of AP (ischemic, fatty acid infusion and duct obstruction plus hyperstimulation with secretine) [125]. In addition, the activities of pro-oxidant enzymes such as xanthine oxidase (XO) increase in acute pancreatitis, and allopurinol, an inhibitor of xanthine oxidase activity exhibited beneficial effects in this disease [125].

Later several groups found glutathione depletion together with an increase in the production of reactive oxygen species and lipid peroxidation in the pancreatic tissue and in acinar cells during the initial course of acute pancreatitis [92, 120, 126, 127]. By using cerium capture of oxygen free radicals, Telek and co-workers demonstrated oxygen free radical for-mation in the pancreas during pancreatitis in rats and humans

[128, 129]. The early free radical formation in acinar cells occurs in parallel with an up-regulation of P-selectin and ICAM expression [129]. Clinical studies have verified the presence of oxidative stress during the outcome of acute pancreatitis [130]. Indeed, lipid peroxidation, mieloperoxi-dase activity and protein carbonyls increase in plasma of patients with severe acute pancreatitis [131, 132].

The source of reactive oxygen species (ROS) in acute pancreatitis may differ depending on the experimental model. In mild acute pancreatitis caused by overstimulation with caerulein, free radical generation would be mainly asso-ciated with infiltration by activated neutrophils, whereas in necrotic acute pancreatitis induced by taurocholate retro-grade perfusion it would be mainly due to the conversion of XO deshidrogenase (DXH) to XO [133]. Other pro-oxidant enzymes that contribute to pancreatic inflammation are cyto-chrome P450 (CYP) [134, 135] and NADPH oxidase [136].

Oxidative stress is involved in cytokine/chemokine pro-duction, since N-acetylcysteine prevented overexpression of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC), and activation of p38MAPK, NF- B and STAT3 in mild AP [137]. How-ever, this effect was not observed in necrotizing AP, proba-bly because pro-oxidants overwhelmed antioxidants. Fur-thermore, treatment with antioxidants diminished TNF- and IL-6 production in neutrophils in response to 4beta-phorbol 12beta-myristate 13alpha-acetate [138]. In AR42J acinar cells, NADPH oxidase activity modulates cytokine expres-sion up-regulating IL-6 through NF-kB activation [139].

A relationship between the anti-inflammatory cytokine IL-10 and oxidative stress may exist. Antioxidants may modulate the ratio of anti-inflammatory to pro-inflammatory cytokines in experimental AP. Thus, administration of the antioxidant N-acetyl cysteine to rats with AP diminished the pancreatic injury, enhanced the ability of acinar cells to pro-duce IL-10 at early stages and increased the ratio IL-10/IL-6 [140, 141].

Oxidative stress seems to play a significant role since it is related to severity of the disease. The dramatic increase in ROS correlates with tissue injury in acute pancreatitis and it may be evidenced by the increase in malonaldehyde levels [142]. Superoxide radical and lipid peroxide levels also in-crease in blood of patients and animals with AP, and these changes correlate with the degree of severity of this disease disease [121, 143-145]. Increased levels of malonyldialde-hyde (MDA) has been associated to the pathogenesis of pan-creatitis associated Multiple Organ Dysfunction (MODS) [141]. Furthermore, markers of oxidative stress correlate with serum phospholipase A2 and plasma polymorphonu-clear elastase, two prognostic parameters in AP [144]. In addition, concentrations of antioxidant vitamins are reduced and are inversely related to the rise in C reactive protein level in AP [146].

The role of oxidative stress in death of acinar cells during acute pancreatitis is still controversial. On the one hand, the pancreatitis associated protein (PAP) is markedly up-regulated during the initial stage of pancreatitis; this induc-tion seems to be mediated at least in part by free radicals and PAP protects against apoptosis of acinar cells [147]. In con-trast, other proteins induced by stress which also increase their expression in pancreatitis promote cell death by apopto-sis [128]. Moreover, oxidative stress induces a loss of nu-clear DNA-repairing enzymes Ku70 and Ku80 in acinar AR42J cells leading to apoptosis [148]. Activation of NADPH oxidase and TNF- production by acinar cells play a key role in the oxidative stress and cell death associated with acute pancreatitis [58, 139]. Reactive oxygen species (ROS) have also been involved in acinar cell death by apop-tosis in mild acute pancreatitis [147, 148] as well as in death necrosis in severe acute pancreaititis [141, 149]. ROS seems to be a key mediator of CCK-induced apoptosis [41]. Indeed, ROS promotes cytochrome c release and apoptosis in acinar cells, but the Ca

2+-induced loss of m blocked cytochrome

c release by inhibition of mitochondrial ROS generation [41].

4.1. Glutathione and Acute Pancreatitis

Reduced glutathione (GSH) is the most abundant non protein thiol in mammal cells and its balance with oxidized


glutathione (GSSG) maintains the thiol/disulfide redox status inside cells. Thus, the GSSG/GSH ratio is a reliable indicator of oxidative stress because it reflects the balance between antioxidant status and pro-oxidant reactions in cells [150, 151]. GSH concentration in the pancreas is one of the largest in the body and this tissue exhibits active transulphuration pathway and GSH synthesis despite the relatively low activ-ity of the GCL [123].

GSH plays a central role as antioxidant in acute pan-creatitis. GSH depletion in the pancreatic tissue is a hallmark during the initial phase of acute pancreatitis [92, 120, 126, 152]. Pre-treatments with glutathione monoethyl ester, N-acetyl cysteine or pentoxifylline exhibited beneficial effects in acute pancreatitis by increasing pancreatic GSH levels, whereas inhibition of GSH synthesis with L-buthionine-(S,R)-sulfoximine (BSO) led to more pancreatic necrosis and reduced survival in rats with acute pancreatitis [152, 153]. Furthermore, it has been reported an association between certain genetic polimorfisms of glutathione S-transferase and severe acute pancreatitis [154]. Consequently, GSH deple-tion may contribute to the progression from mild to severe acute pancreatitis [154, 155].

It was suggested that the early GSH depletion could al-low a premature activation of digestive enzymes inside aci-nar cells triggering the inflammatory process [143]. How-ever, glutathione depletion itself cannot produce acute pan-creatitis [156, 157] nor perfusion with xanthine oxidase [158].

We found that glutathione depletion, but not glutathione oxidation, occurs initially in the pancreas in AP [92]. There-fore, ROS detoxification associated with the inflammatory process does not appear to be the major cause for the early glutathione depletion. Alternatively, Meister suggested that depletion of pancreatic glutathione may be due, at least in part, to the activation of proenzymes, since activated prote-ases such as carboxypeptidase may cleave GSH [159]. In fact, trypsinogen activation is accompanied by glutathione depletion in experimental acute pancreatitis [160].

Our group have recently shown that early up-regulation of glutamate cysteine ligase (GCL) expression occurs only in aedematous acute pancreatitis but not in necrotic acute pan-creatitis [155]. Accordingly, recovery of GSH levels only occurs in caerulein model while in the necrotic model the failure in up-regulation of GCL synthesis avoids the possible increase in GSH levels. A marked increase in cytosolic pan-creatic ribonuclease (RNase) during severe pancreatitis might be responsible for GCL mRNA degradation (see Fig. 2) [155].

4.2. Redox Homeostasis and Acute Pancreatitis

Knowledge of the thiol chemistry, represented by the central redox couples GSH/GSSG and cysteine/cystine (Cys/CysSS) together with the activities of the classical anti-oxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), as well as the re-dox regulators thioredoxins (TRXs), glutaredoxins (GRXs), sulforedoxins (SRXs) and peroxiredoxins (PRXs), have prompted the progress in understanding the physiological role of oxidative and nitrosative stress in many pathological

situations [161]. Reactive oxygen species integrate into cel-lular signal transduction through covalent modification of redox sensors [162]. Sulphur switches of sensitive targets, which include not only cysteine (cys) but also methionine (Met) residues, allow a transient oxidation of proteins to en-able transmission of a signal and subsequent enzymatic re-duction to their basal oxidation state.

The forward rate of the S-glutathionylation reaction can be influenced by glutathione S-transferase P (GSTP), whereas the reverse rate is affected by redox sensitive pro-teins including GRXs, TRXs and SRXs. In this regard, cyto-solic thioredoxin 1 has been proposed as a reliable oxidative-stress marker for the evaluation of AP severity in relation to oxidative stress. Moreover, reversible S-glutathionylation mediated by GRXs can be implicated in many inflammatory diseases [163]. Due to the GSH dependent activity of GTXSs and TRXs, further studies should be performed to clarify their possible role in GSH depletion in acute pancreatitis.

The increase in cytosolic ribonuclease (RNAse) activity that we found in severe acute pancreatitis might be related to its redox regulation. Indeed, RNase A has eight thiol groups and its most reduced state (-SH8) is the inactive status of the enzyme [164].

4.3. Ca2+

and Oxidative Damage During Early Stages of

Acute Pancreatitis

Ca2+

has been revealed as another key role player in pro-moting oxidative stress and injury in acinar cells during acute pancreatitis by activation of Ca

2+-dependent

proteases

and phospholipase A2, which may lead to cytoskeletal disup-tion and membrane damage [165]. The protein kinase C (PKC) family, which is formed by Ca

2+-dependent enzymes,

may activate NF-kB, cytokine production and ROS/RNS overproduction [166]. Substance P induces synthesis of chemokines MCP-1, MIP-1 and MIP-2 in mouse pancreatic acinar cells through an increase in Ca

2+ and activation of

PKC / II, ERK1/2 and JNK, targeting the transcription fac-tors NF- B and AP-1 [167]. During inflammation, the in-crease in intracellular Ca

2+ is also required for the neutrophil

respiratory burst and for activation of NADPH oxidase [168]. Cytosolic Ca

2+ synergized with ROS-induced altera-

tions in ultrastructure and energy metabolism of acinar cells during the early stages of acute pancreatitis [169].

Bile acids and non oxidative alcohol metabolites can in-duce Ca

2+ release from the RE resulting in abnormal cytoso-

lic Ca2+

signalling. Cytosolic Ca2+

overload causes premature digestive enzyme activation, vacuolization and necrosis [166]. It has been demonstrated in isolated pancreatic acinar cells incubated with high concentrations of ethanol, that fatty acids cause a large increase of cytosolic Ca

2+ sustained by

the presence of external Ca2+

but it was also irreversible upon removal of external Ca

2+ [43].

Pancreatic mitochondria are more sensitive to Ca2+

than other mitochondria, such as liver ones [41]. Ca

2+ exerted two

opposite effects on cytochrome c release in acinar cells: cy-tochrome c release, capspase activation and apoptosis are stimulated by Ca

2+ and ROS, but inhibited by the Ca

2+-

induced loss of mitochondrial membrane potential ( m) [41].


On the other hand, GSH could be the link between Ca2+

and redox signalling. It has been shown that in HL 60 cells, calcium release from the ER could lead to mitochondrial impairment and cell death by apoptosis whereas calcium overload led to necrosis, both orchestrated by S-glutathionylation of specific proteins. Accordingly, GSH levels together S-glutathionylation can modulate cytoplasmic calcium in-crease and ER Ca

2+ release in HL60 [170].

4.4. Nitrosative Stress in Acute Pancreatitis

Another enzyme that contributes to pancreatic inflamma-tion is inducible nitric oxydase syntetase (iNOS) [171-175].

Reactive especies of nitrogen (RNS) are also involved in the pathophysiology of acute pancreatitis. NO regulates normal pancreatic exocrine secretion, endocrine pancreatic insulin secretion and pancreatic microvascular blood flow. It has been suggested that certain amount of NO production has beneficial effects in experimental edematous acute pan-creatitis, but uncontrolled over-production of NO may be detrimental [176]. Endothelial NO synthase (eNOS) reduces the severity of the initial phase of experimental acute pan-creatitis [177]. NO production and NOS expression seems to be differentially regulated temporally and in magnitude in the pancreas and lungs in response to cerulein hyperstimula-

Fig. (2). Inefficient up-regulation of glutamate cysteine leads to maintained glutathione depletion in severe acute pancreatitis. Abbreviations:

ERK1/2: extracellular regulated kinase ; GCL: glutamate cysteine ligase; GSH: Reduced glutathione; RNApol: RNA polymerase II; RNAse: ribonuclease.


tion, which suggests differing roles for each NOS isoform [178]. Acute pancreatitis also provoked deleterious effects on endothelium-dependent relaxing response for acetilcho-line (Ach) in vitro and on haemodynamic disturbances, which were associated with high plasma NOx-levels as con-sequence of intense inflammatory responses [179].

Nevertheless, the role of nitric oxide also shows some controversy. Endogenous nitric oxide protects against oxida-tive damage since the inhibition of nitric oxide synthase by L-NAME increases lipid peroxidation and protein oxidation in some subcellular fractions [180]. However, lipid peroxida-tion, the expression of adhesion molecules and tissue dam-age are markedly restrained in mice deficient in inducible nitric oxide synthase (iNOS) with acute pancreatitis [171].

5. REDOX SIGNALING AND INFLAMMATORY RE-SPONSE IN ACUTE PANCREATITIS

Oxidative and nitrosative stress not only causes oxidative damage but also may act as intracellular signal in inflamma-tory processes [181, 182], particularly in the up-regulation of pro-inflammatory genes [123]. Indeed, reactive oxygen spe-cies act as inflammatory mediators through the activation, migration and adhesion of leukocytes, as wells as by enhanc-ing the expression of other mediators, such as cytokines, chemokines and adhesion molecules [17, 18, 129, 183, 184].

In pancreatic acinar cells ROS induce activation of nu-clear factor B (NF- B), one of the major transcription fac-tors responsible for the expression of pro-inflammatory genes [185, 186]. Furthermore, ROS generated by xanthine oxidase during acute pancreatitis induce up-regulation of P-selectin, an important mediator of neutrophil infiltration [183].

ROS participate in many transduction pathways involved in inflammation such as Ca

2+ signalling [187], the activator

protein (AP-1) [188], Janus kinases/ signal transducers and activators of transcription (JAK/STAT) [189-191], phospho-inositide 3-kinase (PI3K) [188, 192-194], mitogen activated protein kinases (MAPK) [188, 190, 192, 194] and NF B activation [188, 192, 195].

Amplification of the inflammatory cascade is triggered by the synergysm between oxidative stress and pro-inflammatory cytokines, particularly TNF- , that may lead to an uncontrolled inflammatory cascade [196-198]. TNF- in-duces oxidative stress through different mechanisms: i) con-version of xanthine dehydrogenase to xanthine oxidase [199], ii) increasing mitochondrial ROS production [200], iii) rapid and transient depletion of intracellular GSH due to glutathione oxidation [201, 202] iiii) promoting chemotaxis and activation of neutrophils [64]. Accordingly, N-acetylcysteine prevented NF- B activation and subsequently sup-pressed cytokine production in pancreatic acinar cells [203] and blockade of NF-kB activation using pyrrolidine dithio-carbamate abrogated the lypopolysaccharide-induced expres-sion of TNF- , cyclooxygenase-2 and intercellular adhesion molecule-1 (ICAM-1) mRNAs and proteins [204].

The cross-talk between oxidative stress and cytokines contributes to both local and systemic inflammation. Thus, lung cells also release inflammatory mediators, such as TNF-

, IL-1 and IL-8, in response to oxidative stress [205].

6- MAP KINASES: THE LINK BETWEEN OXIDA-

TIVE STRESS AND CYTOKINES IN ACUTE PAN-

CREATITIS

Oxidative stress together with pro-inflammatory cytoki-nes may activate intracellular signalling pathways mediated by protein kinases activated by mitogens (MAP kinases), which play a key role in inflammatory processes and cell death [206].

MAP kinase signalling cascades, which are regulated by phosphorylation and dephosphorylation on serine and/or threonine residues, respond to activation of receptor tyrosine kinases, other protein tyrosine kinases, receptors of cytoki-nes and growth factors, and heterotrimeric G protein-coupled receptors [207-210]. The primary pro-inflammatory cytoki-nes, TNF- and IL-1, activate JNK, p38 and NF- B leading to up-regulation of genes coding for cytokines, chemokines and other pro-inflammatory mediators [198, 211]. JNK and p38 activation elicits phosphorylation and the subsequent activation of the transcription factor AP-1, which induces the expression of pro-inflammatory genes [212]. Other cytoki-nes, such as IL-6, also contribute to amplification of the acute-phase response in the inflammatory process through activation of MAP kinase cascades [213]. One of the best examples for cytokine induction by MAP kinases and NF- B is the case of IL-8, which is up-regulated by a dual mecha-nism: transcriptional activation of its gene by NF- B and the JNK pathway, and stabilization of its mRNA by the p38 pathway [214]. In addition, MAP kinases activation may be involved in cell death. Thus, apoptosis signal-regulating kinase 1 activates both JNK and p38, leading to apoptotic cell death [215].

On the other hand, oxidative stress up-regulates pro-inflammatory genes, such as TNF- , IL-1 , IL-8 and iNOS [216-218] through activation of MAPK and NF- B [197, 198, 219-222].

During the onset of acute pancreatitis, a link between oxidative stress, MAP kinases and cytokine production has been demonstrated. Oxidative stress causes activation of p38 which in turn up-regulates TNF- expression [223]. Accord-ingly, inhibition of p38 reduced TNF- production decreas-ing markedly lung damage associated with experimental acute pancreatitis [217]. In addition, ROS can activate ERK1/2, JNK and p38 [224], and the activation of the MAP kinase cascades induces cytokine production in acute pan-creatitis [225].

In mild acute pancreatitis induced by caerulein, p38 and ERK exhibited basal activation, but not of JNK which was activated more faintly by higher doses of caerulein [226]. The severity of pancreatitis has been related to JNK which is activated by 4-hydroxynonenal, a product of lipid peroxida-tion [227]. Moreover, 4-hydroxynonenal was proposed as marker of pancreatitis severity [228].

The effect of ROS on MAP kinase activation and up-regulation of pro-inflammatory genes may differ between acinar and non acinar cells. Acinar overexpression of chemokines monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC) is associated with activation of p38, NF- B and STAT3 activa-tion [137]. This chemokine up-regulation is reduced by the


antioxidant N-acetyl cysteine in pancreatitis induced by bile-pancreatic duct obstruction, but other pancreatic cells still release these chemokines by antioxidant resistant mecha-nisms [137].

Our group reported that the combined treatment of ex-perimental acute pancreatitis with pentoxifylline, that inhib-its TNF- production, and oxypurinol, that inhibits xanthine oxidase, blocks simultaneously the three major MAP kinases (p38, JNK and ERK 1/2) in pancreas [229]. This blockade is associated with a remarkable reduction of the inflammatory response in pancreas and lung, as well as in ascites. Conse-quently, we proposed that oxidative stress enhances the local and systemic inflammatory response by acting together with TNF- towards the simultaneous activation of the three ma-jor MAP kinases [17].

All these results suggest that a cross-talk arises between oxidative stress and pro-inflammatory cytokines that greatly contributes to amplification of the uncontrolled inflamma-tory cascade and to tissue injury through MAP kinases and NF- B, and therefore it may be a key factor in the develop-ment of acute pancreatitis.

7. MODULATION OF PROTEIN PHOSPHATASES AS

REDOX SENSITIVE SIGNAL TRANSDUCTION IN

ACUTE PANCREATITIS

Elucidation of the mechanisms that regulate the syner-gism between oxidative stress and MAPK is presently un-derway. In this regard, protein phosphatases are major can-didates as mediators between oxidative stress and MAP kinase activation (see Fig. 3).

Protein phosphatases regulate cellular signalling by dephophorylation of kinase/substrates decreasing their acti-vation and turning them to a basal state. More than 500 pro-tein kinases have been identified so fat, but only 140 phos-phatases (Arena, et al., 2005). Phosphatase activity has a very important role in the negative regulation of MAPK ac-tion on gene expression in the immune response (Hunter et al., 1995).

Proteins can be phosphorylated on nine amino acids (ty-rosine, serine, threonine, cysteine, arginine, lysine, aspartate, glutamate and histidine) with serine, threonine and tyrosine phosphorylation being predominant in eukaryotic cells (Moorhead et al., 2009).

According to their residue dephosphorylation specificity, there are two major groups of protein phosphatases: ser-ine/threonine phosphatases (PPP) -such as PP1, PP2A, PP2B (calcineurin) or the metallo-dependent phosphatases (PPM) PP2C- and protein tyrosine phosphatases (PTPs) which in-cludes a diverse group in domain structure and substrate preference [96, 233]. This latter group is formed by mem-brane protein phosphatases (RPTPs), such as hematopoetic PTP (HePTP) and CD45, and by cytosolic members such as SHP1 and SHP2. An important subclass of the PTP super-gene family includes the dual specificity phosphatases (DSP), also called MAPK phosphatases (MKPs), which can dephos-phorylate phospho-tyr, phospho-ser and phospho-thr residues and appear to be critical for MAPK dephosphorylation [234, 235]. MKPs gene expression is strongly induced by growth factors and cellular stresses, providing a sophisticated tran-scriptional mechanism for targeted inactivation of selected MAP kinase activities.

Fig. (3). Redox signaling through the MAP kinase pathways. Abbreviations: MKKK: mitogen activated protein kinase kinase kinase; MKK:

mitogen activated protein kinase kinase; MAPK: mitogen activated protein kinase; NF-kB: nuclear factor kappa B; PK: protein kinase; PP: protein phosphatase.


At present oxidation is emerging as an important regula-tor of phosphatase activity (see Fig. 4). Thus, transitory oxi-dation of thiols in protein phosphatases leads to their inacti-vation by formation of intramolecular disulfides or by forma-tion of sulfoamides [236]. The PTP superfamily is character-ized by a conserved cysteine catalytic site with a low pKa [237, 238]. Oxidative stress can lead to reversible oxidation of the catalytic cysteine to sulfenic acid, whereas a greater oxidation can give rise to irreversible sulfinic and sulfonic acid formation [239]. Additionaly, dimerization of the RPTP by disulfide bridges between the catalityc cysteines leads to their inactivation [240]. Members of each PTPs subfamily can be oxidized by treatment with oxidizing agents, such as H2O2, leading to transient inactivation of PTPs, indicating that PTPs are important sensors of the cellular redox state [240, 241].

However, sensitivity to oxidative stress varies among PTPS being some of them even unsensitive to redox changes. Thus, some PTPs exhibit high or intermediate sensitivity to oxidation -such as PTEN or Sac1, and PTPL1/FAP-1, re-spectively-, but other PTPs such as the myotubularin lipid phosphatases are virtually unaffected by oxidation [242].

Oxidative stress may enhance the activation of MAP kinases through the inactivation of protein phosphatases. Hydrolysis of the phosphothreonine residue of ERK by PP2A seems to be a prerequisite for hydrolysis of the phos-photyrosine residue by PTPase activity, indicating that PP2A activity may be more important than PTPase activity in regu-lating ERK phosphorylation [243]. Among the numerous tyrosin phosphatases, SHP1 and SHP2 and CD45 should be highlighted because they are involved in the modulation of the inflammatory response by acting on NF-kB, MAP kinases and TNF- [60, 244]. SHP1 may play a relevant role as modulator of the inflammatory cascade through inhibition of NF-kB.

Redox changes can also affect PPPs favoring intra- e inter-molecular disulfide bonds which can diminish their activity. Thus, PP2A activity is inhibited in a DTT reversible manner by GSSG and H2O2 [245]. Moreover, cysteines of the active site in the catalytic subunit of PP2A (PP2Ac) can form intermolecular disulfide bonds with regulatory subunits [246] or even intramolecular bonds with vicinal thiols in the PP2Ac reducing the catalytic PP2A activity [247].

PP2B, also called calcinerin, is one of the best character-ized Ca

2+-regulatory proteins. Its affinity towards Ca

2+ and

its ability to activate target enzymes, such as phosphodi-esterases, can be decreased by intramolecular disulfide bonds formation [248]. Accordingly, calcineurin is sensitive to oxi-dative inactivation by H2O2 [249-251]. Methionine oxidation can also decrease calcineurin activity, being subunit A of the enzyme more redox sensitive than subunit B [252].

The redox regulation of protein phosphatases appears to be a key event in the inflammatory response in acute pan-creatitis. It has been reported an increase in the expression of tyrosin phosphatases SHP1 and SHP2 early in the course of pancreatitis [253]. However, the activity of tyrosin phospha-tases diminishes in the initial stage of pancreatitis [254]. SHP1 and SHP2 may be inactivated by oxidative stress [255] and the inhibition of tyrosin phosphatases is involved in the formation of oedema in pancreas during acute pancreatitis [254]. CD45 is a membrane tyrosin phosphatase and its ex-pression lowers in the course of acute pancreatitis in parallel with an increase in the production of TNF- , and both ef-fects are prevented by administration of the antioxidant N-acetyl cysteine [60]. In addition, we have recently found a marked decrease of PP2A activity early in the course of ne-crotizing acute pancreatitis associated with up-regulation of pro-inflammatory genes [256].

On the other hand, calcineurin mediates pancreatic zy-mogen activation in acinar cells [257]. Up-regulation of MKP-1, MKP-3, MKP-5 and protein tyrosine phosphatases SHP-1 and SHP-2 are an early event during acute pancreati-tis [253, 258]. Down-regulation of MAP kinase signaling by MKP induction provides protective effects by PAR2 activa-tion on caerulein-induced intrapancreatic damage [259]. The role of protein phosphatases in acute pancreatitis requires further research in order to elucidate the redox mechanisms that control the phosphatate activity and their involvement in the up-regulation up-regulation of pro-inflammatory genes through the MAPK pathways.

ACKNOWLEDGMENTS

The authors acknowledge the financial support obtained by Grants from Ministerio de Educación y Ciencia (SAF2006-06963 and Consolider CSD-2007-0020) to J. S.

Fig. (4). Thiol homeostasis in protein phosphatases as redox sensitive signal transduction. Abbreviation: PP: protein phosphatase.


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Received: April 15, 2009 Accepted: April 18, 2009

Cross-Talk between Oxidative Stress and Pro-Inflammatory Cytokines in Acute Pancreatitis: A Key Role...

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