American Journal of Transplantation 2005; 5: 484–493Blackwell Munksgaard

Copyright C© Blackwell Munksgaard 2004

doi: 10.1111/j.1600-6143.2004.00716.x

Inhibition of p38 Pathway Suppresses Human IsletProduction of Pro-Inflammatory Cytokines andImproves Islet Graft Function

Takeru Matsudaa,b, Keiko Omoria, Tommy

Vuonga, Michael Pascuala, Luis Valientea, Kevin

Ferreria, Ivan Todorova, Yoshikazu Kurodac,

Craig V. Smitha,b, Fouad Kandeela

and Yoko Mullena,b,∗

aSouthern California Islet Cell Resources Center,Department of Diabetes, Endocrinology and Metabolism,City of Hope National Medical Center/Beckman ResearchInstitute, Duarte, CA, USAbDepartment of Surgery, University of California at LosAngeles, Los Angeles, CA, USAcDepartment of Gastroenterological Surgery, GraduateSchool of Medical Sciences, Kobe University,Kobe, Japan∗Corresponding author: Yoko Mullen, ymullen@coh.org

Nonspecific inflammation is associated with primarygraft nonfunction (PNF). Inflammatory islet damageis mediated at least partially by pro-inflammatorycytokines, such as interleukin-1b (IL-1b ) and tumornecrosis factor-a (TNF-a ) produced by resident isletmacrophages. The p38 pathway is known to be in-volved in cytokine production in the cells of themonocyte–macrophage lineage. Therefore, inhibitionof the p38 pathway may prevent pro-inflammatory cy-tokine production by resident islet macrophages andpossibly reduce the incidence of PNF. Our presentstudy has demonstrated that inhibition of the p38pathway by a chemical p38 inhibitor, SB203580, sup-presses IL-1b and TNF-a production in human isletsexposed to lipopolysaccharide (LPS) and/or inflamma-tory cytokines. Although IL-1b is predominantly pro-duced by resident macrophages, ductal cells and isletvascular endothelial cells were found to be another cel-lular source of IL-1b in isolated human islets. SB203580also inhibited the expression of inducible nitric oxidesynthase (iNOS) and cyclooxygenase-2 (COX-2) in thetreated islets. Furthermore, human islets treated withSB203580 for 1 h prior to transplantation showed sig-nificantly improved graft function. These results sug-gest that inhibition of the p38 pathway may become anew therapeutic strategy to improve graft survival inclinical islet transplantation.

Key words: Primary graft nonfunction, islet trans-plantation, p38, macrophage

Received 4 July 2004, revised 20 September 2004 andaccepted for publication 12 October 2004

Introduction

Resident islet macrophages play an important role in theinitiation of beta cell destruction during the developmentof type1 diabetes (1–3). Previous reports have shown thatthese cells also play a major role in primary islet graft non-function (PNF) or early graft failure following islet trans-plantation (4,5). Within a short period after transplantation,inflammatory responses occur in and around the grafts,causing beta cell dysfunction and death that lead to earlygraft failure/PNF. PNF occurs even with syngeneic or autol-ogous islet transplants (6,7). The beta cell death associatedwith nonspecific inflammation appears to be mediated, atleast in part, by production of pro-inflammatory cytokinessuch as interleukin-1b (IL-1b) and tumor necrosis factor-a(TNF-a) in the grafted islets (4,5,8). These cytokines canalso stimulate the expression of inducible nitric oxide syn-thase (iNOS) and cyclooxygenase-2 (COX-2), which are be-lieved to be mediators of cytokine-induced islet damage. Infact, iNOS or COX-2 inhibitors have been shown to preventcytokine-mediated islet cell damage both in vitro and in vivo(9–12). Therefore, the suppression of pro-inflammatory cy-tokine production in the islets should, similar to iNOS orCOX-2 inhibitors, reduce early cell loss in the transplantand preserve the islet cell mass required for controllinghyperglycemia.

Pro-inflammatory cytokines are generally known to be pro-duced by macrophages and monocytes upon stimulationwith lipopolysaccharide (LPS) and/or cytokines. LPS acti-vates mitogen-activated protein kinases (MAPKs) includ-ing c-Jun NH2-terminal kinase (JNK), p38 kinase (p38) andextracellular signal-regulated kinase (ERK). Other investi-gators reported the involvement of MAPKs in LPS-inducedcytokine production (13,14). Particularly, it has been welldocumented that inhibition of the p38 pathway by chem-ical p38 inhibitors, such as SB203580, prevents IL-1b,TNF-a and IL-6 production by macrophages (15,16). Theseinhibitors also suppress iNOS and COX-2 expression in cer-tain types of cells (17,18). However, it remains unknownwhether p38 inhibitors are capable of suppressing cytokineproduction and iNOS and COX-2 expression in isolated hu-man islets.

484

p38 Pathway Inhibition in Human Islets

Besides macrophages, iNOS is expressed by pancreaticbeta cells, ductal cells and endothelial cells followingcytokine (such as IL-1b and IFN-c ) stimulation, and me-diates beta cell damage (19–21). COX-2 is also inducedby various stimuli, including cytokines and high glucose,and leads to prostaglandin E2 production that causes betacell dysfunction (22,23). Moreover, it has recently been re-ported that beta cells are the source of IL-1b in the islets oftype 2 diabetic patients (24). In the present study, we haveexamined, using human islets, whether inhibition of thep38 pathway by SB203580 suppresses islet production ofinflammatory mediators and reduces PNF after transplan-tation. We also investigated the cellular source of IL-1b andTNF-a in human islet preparations.

Materials and Methods

Materials

LPS (from Escherichia coli 0111:B4) was purchased from Sigma (St. Louis,MO). Human recombinant IL-1b, TNF-a and IFN-c were purchased fromR&D systems Inc. (Minneapolis, MN). SB203580, a selective p38 inhibitor,was from Calbiochem (San Diego, CA). Rabbit polyclonal antibodies to p38,phospho-p38 and phospho-HSP27 were from Cell Signaling Technology(Beverly, MA). Rabbit polyclonal antibody to a-tubulin was from Santa CruzBiotechnology (Santa Cruz, CA). Mouse and goat anti-IL-1b antibodies werefrom R&D Systems Inc. and mouse anti-COX-2 antibody was purchasedfrom BD Biosciences (San Jose, CA).

Human islets

Human islets were provided for research use by the Southern California IsletCell Resources Center, City of Hope (Duarte, CA). Islets were isolated bythe two-step digestion method (25) and cultured in serum-free CMRL1066medium (Mediatech, Inc., Holly Hill, FL) for 2 days prior to being releasedfor research use. These islets were then cultured in Ham’s F12 mediumsupplemented with 10 mM HEPES, antibiotic antimycotic, and 10% fetalbovine serum (all from Sigma) for an additional 1–2 days before being usedin experiments. All experiments started within 4 days after isolation. Theislet numbers were expressed as the number equivalent to islets 150 lmin diameter (IEQ). Only islets with greater than 80% purity, as determinedby dithizone staining, were used. Otherwise, islets were hand-picked.

Treatment of human islets

Islets were treated with 10 lM SB203580 or vehicle alone (DMSO) in culturemedium for 30 min, followed by stimulation with either 10 lg/mL LPS alone,10 lg/mL LPS, 50 ng/mL TNF-a and 750 U/mL IFN-c (LPS + TNF-a + IFN-c ), or 75 U/mL IL-1b and 750 U/mL IFN-c (IL-1b + IFN-c ), and then culturedfor an indicated period.

Assessment of islet cell viability

The viability of treated islets was determined by 3-(4,5-dimethylthiazolyl-2)2,5-diphenyltetrazolium bromide (MTT) assay as described previously (26).Islets were plated in a 96-well plate as six replicates (40 IEQ/well), andthe viability was assessed after 2- and 4-day culture following LPS and/orcytokine stimulation. Viability was expressed by the percentage of the MTTabsorbance of the untreated islets.

Semiquantitative PCR

Cultured islets were collected 8 h after stimulation with LPS and/or cy-tokines and stored at −80◦C until use. Total RNA was extracted from isletsusing TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OH). Atotal of 2.0 lg of RNA from each sample was then reverse-transcribedinto first-strand cDNA in 20-lL solution using SuperscriptRNase H− reverse

transcriptase (Invitrogen, Carlsbad, CA). The final cDNA products were thendiluted by adding 80 lL of H2O. PCR was performed using the FailSafe PCRSystem (EPICENTRE, Madison, WI). A standard 25 lL PCR reaction solutioncontained 1.5 lL of cDNA, 30 pmol each of forward and reverse primers,12.5 lL of PremixD and 0.25 lL of Enzyme Mix. The following primers wereused: human IL-1b (204 bp) forward 5′-CCT GTG GCC TTG GGC CTC AA-3′,reverse 5′-GGT GCT GAT GTA CCA GTT GGG-3′; human TNF-a (325 bp) for-ward 5′-CAG AGG GAA GAG TTC CCC AG-3′, reverse 5′-CCT TGG TCT GGTAGG AGA CG-3′; human iNOS (236 bp) forward 5′-ACA TTG ATC AGA AGCTGT CCC AC-3′, reverse 5′-CAA AGG CTG TGA GTC CTG CAC-3′; humanGAPDH (600 bp) forward 5′-CCA CCC ATG GCA AAT TCC ATG GCA-3′, re-verse 5′-TCT AGA CGG CAG GTC AGG TCC ACC-3′. The cycle number wasdetermined to be in the linear range of amplification for each primer pair.PCR using 1/2 diluted concentrations of cDNA template verified that PCRproduct yields were proportional to the initial concentrations of cDNA tem-plate. Electrophoresis of PCR products were performed in 1.5% agarosegel containing 0.5mg/mL of ethidium bromide and analyzed with an AlphaInnotech densitometer.

Western blotting

After LPS and/or cytokine stimulation, 800 IEQ were harvested from eachsample at an indicated time, washed twice with ice-cold phosphate bufferedsaline (PBS) and stored at −80◦C until use. Islet cell lysis and Westernblotting were performed as described previously (27).

Determination of cytokine and nitrite levels in culture media

Islets (600 IEQ/600 lL of culture media) were cultured in duplicate for48 h after stimulation with LPS and/or cytokines and then the culture super-natants were collected for cytokine and nitrite determination. Nitrite produc-tion in the supernatant was measured immediately after collection using aGriess Reagent Kit (Molecular Probes, Eugene, OR). Samples for cytokinedetermination were stored at −20◦C, and the levels of IL-1b and TNF-awere measured using Quantikine kits (R&D systems Inc.).

Immunocytochemistry

Forty-eight hours after LPS and/or cytokine stimulation, islets (800IEQ/sample) were collected, washed with PBS and gently agitated in CellDissociation Buffer (Invitrogen) for 10 min at 37◦C. Cells were then col-lected, washed once with PBS and plated on poly-L-Lysine-coated coverslips. Cells were fixed with 100% methanol for 20 min at −20◦C, thendipped in acetone. The cover slips were stored at −20◦C until cells werestained. After washing with PBS, the cover slips were blocked with 5% nor-mal donkey serum and then incubated with goat anti-IL-1b (1:30 dilution;R&D Systems Inc.) for 1 h at 37◦C, followed by 1 h incubation with mouseanti-CD68 (1:10 dilution; DAKO, Carpinteria, CA), mouse anti-cytokeratin-7 (1:15 dilution; DAKO), or rabbit anti-von Willebrand factor (1:100 dilu-tion; DAKO). Following incubation, the cover slips were washed four timeswith PBS, and incubated for 1 h at room temperature with FITC-conjugateddonkey anti-goat and Texas Red-conjugated donkey anti-mouse (or rabbit)antibodies (1:100 dilution; Jackson Immuno Research Laboratories, WestGrove, PA).

Transplantation of a marginal islet mass

into diabetic athymic mice

Immunodeficient male athymic mice (Swiss-background, 8–10 weeks old)were used as recipients of human islets. Mice were rendered diabetic by anintraperitoneal injection of 250 mg/kg streptozotocin (STZ) (Sigma) freshlydissolved in citrate buffer. A marginal number (1500 IEQ) of human isletswas treated with 10 lM SB203580 or vehicle (DMSO) alone in culturemedium for 1 h, and then transplanted under the kidney capsule as de-scribed previously (28). Four different islet preparations were used in this

American Journal of Transplantation 2005; 5: 484–493 485

Matsuda et al.

study. Animals were anesthetized by an intraperitoneal injection of ketamine(120 mg/kg) and xylazine (10 mg/kg).

Monitoring islet graft function

Nonfasting blood glucose levels of <200 mg/dL that continued for three con-secutive days were taken as the reversal of diabetes. The period requiredfor diabetes reversal was defined as the number of days from the day oftransplantation to the first day that blood glucose was <200 mg/dL. After30 days of observation, nephrectomy of the graft-bearing kidney was per-formed in all glucose-normalized mice to confirm the diabetes reversal wasdue to the islet graft. Animal procedures followed the protocols approved bythe Animal Care Committee of the City of Hope Medical Center/BeckmanResearch Institute.

Statistical analysis

Data are expressed as mean ± SE. Differences between groups were an-alyzed by the unpaired Student’s t-test. Time required for recovery fromdiabetes was calculated using Kaplan–Meier life tables, and differences be-tween groups were assessed by a log-rank test. A p < 0.05 was consideredsignificant.

Results

The p38 inhibitor, SB203580, inhibits the expression

and release of IL-1b induced by LPS and/or cytokines

LPS is well known to stimulate IL-1b mRNA expressionand production in macrophages. It has also been reportedthat IL-1b is produced by human islets treated with LPS +TNF-a + IFN-c (3). Using semiquantitative PCR, we exam-ined whether or not a p38 inhibitor, SB203580, is capableof suppressing LPS- and LPS + TNF-a + IFN-c -inducedIL-1b mRNA expression in human islets. Both LPS andLPS + TNF-a + IFN-c -induced IL-1b mRNA expression inthe islets within 8 h after stimulation. Treatment of isletswith SB203580 prior to stimulation had, essentially, nosuppressive effect on the IL-1b mRNA expression (Fig-ure 1A). Levels of IL-1b released from human islets intothe culture medium were measured 48 h after stimula-tion with or without SB203580 pre-treatment. As shown inFigure 1B, SB203580 significantly suppressed LPS + TNF-a + IFN-c -induced IL-1b release (p < 0.05). LPS alone alsoinduced IL-1b release, but at far lower levels than thoseinduced by LPS + TNF-a + IFN-c . Despite varying levelsof LPS and/or cytokines-induced IL-1b release from differ-ent islet preparations, SB203580 consistently suppressedthe IL-1b release to 10–30% of the corresponding con-trol level. Western blot analysis showed that the levelsof pro-IL-1b protein (precursor molecule of IL-1b) inducedin response to LPS and LPS + TNF-a + IFN-c were de-creased by SB203580 treatment (Figure 1C). These resultssuggest that SB203580 inhibits LPS- and LPS + TNF-a+ IFN-c -induced IL-1b production by islets at the post-transcriptional level.

SB203580 suppresses TNF-a expression and release

by human islets stimulated with LPS or IL-1b + INF-cTNF-a is known to cause beta cell dysfunction and deathwhen it is used to treat human islets in vitro in combina-

DMSOLPSLPS+TNF-α+IFN-γSB203580

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Figure 1: Effect of SB203580 on LPS- and LPS + TNF-a + IFN-

c -induced IL-1b expression and release by human islets. Hu-man islets were treated with 10 lM SB203580 or vehicle (DMSO)alone for 30 min, then stimulated with 10 lg/mL LPS or 10 lg/mLLPS + 50 ng/mL TNF-a + 750 U/mL IFN-c , and subsequently cul-tured for 8 h (A, C) or 48 h (B). A: Total RNA was isolated and IL-1bmRNA expression was determined by RT-PCR. B: The levels ofIL-1b released into the culture medium were measured by ELISA.C: pro-IL-1b expression was detected by Western blotting analy-sis. RT-PCR and Western blotting data represent three indepen-dent experiments using three separate human islet preparations.IL-1b release is expressed as mean ± SE of five independent ex-periments using separate islet preparations, each performed induplicate. ∗p < 0.05 islets treated without vs. with SB203580.

tion with IL-1b and IFN-c (29). TNF-a, together with IL-1b,is produced locally in the islet graft site in vivo shortly afterislet transplantation, and these cytokines are consideredas the major pro-inflammatory mediators (5,8). LPS treat-ment stimulated TNF-a mRNA expression and release inhuman islets as shown in Figure 2. IL-1b + IFN-c -inducedhigher levels of TNF-a release than LPS alone, although

486 American Journal of Transplantation 2005; 5: 484–493

p38 Pathway Inhibition in Human Islets

DMSOLPSIL-1β+IFN-γSB203580

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Figure 2: Effect of SB203580 on LPS- and IL-1b +IFN-c -

induced TNF-a expression and release by human islets. Hu-man islets were treated with 10 lM SB203580 or vehicle (DMSO)alone for 30 min, then stimulated with 10 lg/mL LPS or 75 U/mLIL-1b + 750 U/mL IFN-c , and subsequently cultured for 8 h (A)or 48 h (B). A: Total RNA was isolated and TNF-a mRNA expres-sion was determined by RT-PCR. B: The levels of TNF-a releasedinto the culture medium were measured by ELISA. RT-PCR dataare representative of three independent experiments each usingthree separate human islet preparations. TNF-a release resultsare expressed as mean ± SE of five independent experimentsperformed in duplicate using five separate isolations. ∗p < 0.05vs. islets without SB203580 treatment. ∗∗p < 0.0001 vs. isletswithout SB203580.

these levels were much lower than the concentration thatis cytotoxic to human islets in vitro. SB203580 significantlyinhibited TNF-a mRNA expression, and almost completelyabolished TNF-a release induced by LPS or cytokines(Figure 2).

SB203580 suppresses iNOS and COX-2 expression

and reduces nitrite production in human islets

stimulated by LPS and/or cytokines

Reports from other laboratories have shown that thetreatment of human islets with LPS + TNF-a + IFN-cor IL-1b + IFN-c stimulates iNOS expression and nitriteproduction in human islets (3,30). We also found the in-duction of iNOS mRNA in LPS and/or cytokine stimulatedhuman islets. IL-1b + IFN-c -induced higher levels of iNOSmRNA than LPS + TNF-a + IFN-c when measured 8 h after

DMSOLPS+TNF-α+IFN-γIL-1β+IFN-γSB203580

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Figure 3: Effect of SB203580 on LPS + TNF-a + IFN-c - and

IL-1b + IFN-c -induced iNOS and COX-2 expression, nitrite

production and viability. Human islets were treated with 10lM SB203580 or vehicle (DMSO) alone for 30 min, then stimu-lated with 10 lg/mL LPS + 50 ng/mL TNF-a + 750 U/mL IFN-c or75 U/mL IL-1b + 750 U/mL IFN-c , and subsequently cultured for8 h (A), 48 h (B), 24 h (C) or 48 and 96 h (D). A: Total RNA wasisolated and iNOS mRNA expression was determined by RT-PCR.B: The level of nitrite production was measured in the culture me-dia using Griess reagent. C: Islets were collected and lysed, andWestern blotting was performed to detect COX-2 protein levels.D: Viability was determined by MTT assay. RT-PCR and Westernblotting data are representative of three independent experimentsusing three separate human islet preparations. Nitrite productionresults are expressed as mean ± SE of five independent experi-ments in duplicate using five separate human islet preparations.Viability results are expressed as mean ± SE of three independentexperiments in six replicate cultures using three separate prepa-rations. ∗p < 0.05 vs. untreated islets. ∗∗p < 0.05 vs. islets withSB203580.

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Figure 3: Continued.

stimulation. Islet treatment with SB203580 markedly inhib-ited both LPS + TNF-a + IFN-c - and IL-1b + IFN-c -inducediNOS mRNA expression (Figure 3A). These results wereconsistent with levels of nitrite produced in the culture me-dia (Figure 3B). LPS alone did not induce any detectableincrease in nitrite production.

Next, we examined whether SB203580 was capable ofmodifying the COX-2 induction by LPS and/or cytokines us-ing Western blotting. COX-2 expression was up-regulatedin islets treated for 24 h with LPS + TNF-a + IFN-c or IL-1b+ IFN-c , but not with LPS alone. Treatment with SB203580prevented up-regulation of COX-2 levels in cytokine stim-ulated islets with or without LPS (Figure 3C).

p38 inhibition prevents cell death in human islets in

response to LPS + TNF-a + IFN-cOur results consistently showed the suppressive effectof SB203580 on the production of pro-inflammatory medi-ators. On the basis of these results, we postulated thatSB203580 would prevent LPS and/or cytokine-inducedislet cell death. Therefore islet viability was determined byMTT assay 2 and 4 days after stimulation with LPS and/orcytokines, with or without SB203580 pre-treatment. Nei-ther LPS alone nor IL-1b + IFN-c induced any detectableislet cell death (data not shown, Figure 3D). In contrast,LPS + TNF-a + IFN-c caused a 15–20% decrease in viablecells. SB203580 significantly prevented this LPS + TNF-a + IFN-c -induced islet cell death as shown in Figure 3D(p < 0.05).

Identification of the cellular source of IL-1b and TNF-aTo determine the cellular source(s) for IL-1b and TNF-a, islets cultured for 48 h in medium containing eitherLPS + TNF-a + IFN-c or IL-1b + IFN-c were examinedby immunohistochemistry. Expectedly, most (80–90%) ofthe IL-1b-positive cells also stained positively for CD68(a macrophage marker) (Figure 4). In addition, we founda small number (<10%) of IL-1b-positive cells that werealso positive for cytokeratin-7 (a ductal cell marker) or vonWillebrand factor (an endothelial cell marker), indicating

that, in addition to macrophages, ductal cells and islet vas-cular endothelial cells are a source of IL-1b in human isletpreparations. In contrast, TNF-a expression always colocal-ized with CD68-staining (data not shown). Neither IL-1b norTNF-a expression was detected in any type of endocrinecells.

SB203580 inhibits p38 pathway in human islets

Various stresses, including LPS and IL-1b, activate p38,which activates MAPKAP kinase-2 (MAPKAPK-2), which inturn phosphorylates HSP27 (31,32). SB203580 has beenreported to inhibit p38 activity by binding to the ATP bindingsite of p38 and to block MAPKAPK-2 activation (16,33,34).To investigate the activation of p38 by LPS and/or cytokinesand to confirm the p38 pathway inhibition by SB203580 inhuman islets, Western blotting analysis was performed.Treatment with IL-1b + IFN-c resulted in the maximumlevels of p38 phosphorylation (p-p38) at 60 min after stim-ulation, which then returned to basal levels in 120 min(Figure 5A). In contrast, a rapid and prolonged phosphoryla-tion of p38 was observed after stimulating with LPS + TNF-a + IFN-c (Figure 5B). Treatment with SB203580 at a 10lM concentration had no or a marginal effect in inhibitingphosphorylation of p38 in response to either IL-1b + IFN-cor LPS + TNF-a + IFN-c . However, SB203580 almost com-pletely suppressed phosphorylation of HSP-27 (p-HSP-27)in both cases, indicating the activation of MAPKAPK-2 wasblocked by SB203580. This observation that SB203580 in-hibits the activity but not the phosphorylation of p38 is con-sistent with the previous reports using other cell systems(34–36).

Treatment of human islets with SB203580 prior to

transplantation improves graft function and increases

the diabetes-reversal rate in athymic mice

Our in vitro results consistently demonstrated the isletprotective effect of SB203580 from LPS and/or pro-inflammatory cytokine toxicity. We then tested if suchprotective effects displayed in vitro also extend in vivoto protect transplanted islets from PNF. We examinedwhether treatment of islets with SB203580 prior to trans-plantation improves graft function/survival by transplant-ing a marginal number of human islets treated withSB203580 or vehicle alone under the kidney capsule ofdiabetic athymic mice. A marginal islet number, that oth-erwise would not reverse diabetes, was determined tobe 1500 IEQ in our previous study (data not shown).When islets were pre-treated with SB203580, the pe-riod needed to achieve normoglycemia was significantlyreduced and a higher proportion of recipients reverseddiabetes within 30 days after transplantation (n = 8,all eight mice reversal, p = 0.006). When islets weretreated with vehicle alone, only four out of nine mice(n = 9) reversed diabetes by day 30 (Figure 6A,B). In allcases in which normoglycemia was achieved, the mice re-turned to hyperglycemia within 2 days after removal of thegrafts by nephrectomy.

488 American Journal of Transplantation 2005; 5: 484–493

p38 Pathway Inhibition in Human Islets

Figure 4: Colocalization of IL-1band the cell markers in dispersed

islet cells. Human islets were stimu-lated with 10 lg/mL LPS + 50 ng/mLTNF-a + 750 U/mL IFN-c for 48 h,and then dispersed into single cellsand small cell clusters. IL-1b stain-ing (green) and CD68, cytokeratin-7or von Willebrand factor staining (red)were performed as described in Ma-terial and Methods. Cells double pos-itive for IL-1b and other cell markerswere identified yellow by merged im-ages (Merge). Results are representa-tive of four independent experimentseach using four separate human isletpreparations.

Discussion

Primary graft nonfunction or early graft failure is one ofthe main obstacles that need to be solved in order to con-sistently succeed with islet transplantation. Islet graft lossin the early stages of transplantation is caused primarilyby nonspecific inflammation, but not specific immune re-sponses or metabolic over dose, as shown by a number ofanimal studies with autologous and syngeneic islet trans-plantation (6,7). Clinical cases in which islets failed to func-tion shortly after transplantation may also be attributableto nonspecific inflammation occurring within and aroundthe graft. The report that PNF is associated with increasedintra-islet cytokine production in syngeneic transplants sug-gests that such nonspecific inflammation reactions are atleast partially mediated by the resident islet macrophages(4). Therefore, it is reasonable to think that the preventionof pro-inflammatory cytokine production by resident isletmacrophages would improve islet graft survival as wellas prevent or delay the development of autoimmune di-abetes.

We reported here that inhibition of the p38 pathway bya chemical p38 inhibitor suppressed the production of IL-1b and TNF-a in isolated islets in response to LPS and/orcytokines and that pre-treatment of human islets with thep38 inhibitor improved islet graft function with regard toreversing diabetes. These results support our hypothesisthat the prevention of resident islet macrophage activationby inhibition of the p38 pathway reduces PNF after islettransplantation.

Chemical p38 inhibitors, such as SB203580, have beenwidely used in various kinds of experiments using culturedcells. Their ability to suppress pro-inflammatory mediators,such as IL-1b, TNF-a and IL-6, has also been well docu-mented in the cells of the monocyte–macrophage lineage(15,16,36). However, their applicability and effect on hu-man islets have remained largely unknown and the avail-able results are controversial (37,38). Our study is the firstto report the inhibitory effect of a p38 inhibitor, SB203580,on the production of pro-inflammatory mediators in humanislets.

We have shown that SB203580 does not reduce IL-1bmRNA levels in response to LPS and LPS + TNF-a + IFN-c stimulation, but inhibits pro-IL-1b protein expression andIL-1b release, suggesting that p38 regulates IL-1b produc-tion at post-transcriptional level in these settings. In theprocess of IL-1b release, pro-IL-1b produced in the cy-tosol of macrophages is cleaved into active IL-1b by IL-1-converting enzyme (ICE) or caspase-1 (39). Therefore, ourfindings also might be explained by the suppressive effectof p38 inhibitors on ICE levels as observed in chondro-cytes (40). On the other hand, for TNF-a, the mRNA levelwas significantly decreased by SB203580 treatment priorto LPS alone or IL-1b + IFN-c -stimulation, and its releasewas completely abolished, suggesting that p38 is involvedin TNF-a production at both transcriptional and translationallevels.

The p38 inhibitor also suppressed iNOS and COX-2 ex-pression and nitric oxide production in the islets caused

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0 15 30 60 120

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Figure 5: p38 pathway inhibition by SB203580 in human islets

activated by IL-1b + IFN-c and LPS + TNF-a + IFN-c . Humanislets were treated with 10 lM SB203580 or vehicle (DMSO) alonefor 30 min, and then stimulated with 75 U/mL IL-1b + 750 U/mLIFN-c (A) or 10 lg/mL LPS + 50 ng/mL TNF-a + 750 U/mL IFN-c(B) for the periods indicated. After stimulation, islets were col-lected and lysed, and Western blotting was performed to detectphosphorylated p38 (p-p38). The same samples were also used todetect total p38 (p38) and phosphorylated HSP27 (p-HSP27). Dataare representative of three independent experiments each usingthree separate human islet preparations.

by both LPS + TNF-a + IFN-c and IL-1b + IFN-c stimu-lation. Previous studies using iNOS inhibitors, such as L-NG-monomethyl-arginine (L-NMMA), clearly showed thatcytotoxic effects of pro-inflammatory cytokines on betacells were at least partially mediated by activation of iNOS(9,21). Inhibition of beta cell function by COX-2 pathwayproducts, such as PGE2, also has been well documented(11,12). Therefore, the suppressive effect of SB203580 oniNOS and COX-2 expression may be beneficial to protectislets from various stimuli which activate these genes.

iNOS mRNA levels significantly increased in islets follow-ing IL-1b + IFN-c stimulation. Treatment with LPS + TNF-a+ IFN-c also up-regulated iNOS mRNA and nitrite levels,but these levels were much lower than those induced byIL-1b + IFN-c . Unexpectedly, however, IL-1b + IFN-c didnot induce distinct islet cell death as measured by MTT as-says, while treatment with LPS + TNF-a + IFN-c resulted

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Figure 6: Reversal of diabetes in athymic mice by human

islets treated with SB203580 prior to transplantation. Amarginal number of human islets (1500 IEQ) were treated with10 lM SB203580 (N = 8) or vehicle (DMSO) alone (N = 9) for 1 hand then transplanted under the kidney capsule of athymic micemade diabetic with streptozotocin. Nonfasting blood glucose lev-els were monitored (A) and the percent of mice that had reverseddiabetes and days required for diabetes reversal (<200 mg/dL ofblood glucose) were compared between SB203580-treated (opensquares) and untreated (black squares) groups separately. ∗p =0.006 vs. untreated islets

in 10–15% decreased islet cell viability after 4 days in cul-ture, which was alleviated by SB203580 treatment. Theseresults suggest that other p38-related mechanisms in ad-dition to iNOS up-regulation are involved in the inductionof islet cell death caused by LPS + TNF-a + IFN-c .

Besides p38, two other MAPKs, JNKs and ERKs, have alsobeen implicated in LPS-induced cytokine production and

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p38 Pathway Inhibition in Human Islets

release. Especially, the involvement of ERKs in cytokineinduction and regulation has been well documented usingMEK/ERK inhibitors, such as U0126 and PD98059 (41,42).However, p38 appears to play a more important role in LPS-induced IL-1b and TNF-a production than ERKs or JNKssince LPS-induced TNF-a production in vivo is decreasedby 90% in mice that do not express MAPKAPK-2, the di-rect downstream target of p38 (43). Also, Hsu et al. demon-strated that p38 was more important in LPS-mediated IL-1bproduction by macrophages (36). However, there is still apossibility that ERKs and/or JNKs may play a greater rolethan p38 in the control of cytokine production in humanislets. Therefore, further investigation of the participationof JNKs and ERKs in cytokine regulation in human isletsmay give us an insight into the mechanisms of residentislet macrophage activation.

Arnush et al. reported in 1998 that CD68-positivemacrophages were the sole cellular source for IL-1b inhuman islets stimulated in vitro by LPS + TNF-a + IFN-c (3). However, in 2002, Maedler et al. showed highglucose-induced IL-1b production by beta cells and theyalso detected IL1b-expressing beta cells in the islets ofpoorly controlled type 2 diabetic patients (25). In the studyby Arnush et al., islets were analyzed by immunohisto-chemistry 4 h after stimulation. We were unable to findIL-1b-producing endocrine cells even 48 h after stimulation.However, in our study a small percentage of IL-1b-positivecells were found to co-stain with von Willebrand factoror cytokeratin-7, although the majority of IL-1b-positivecells were macrophages. Human islet preparations alwayscontain nonendocrine cells, such as ductal cells and en-dothelial cells. Endothelial cells, including human umbil-ical endothelia, are known to produce IL-1b, TNF-a andIL-6 (44–46). Previous reports have shown that islet en-dothelial cells, when activated by cytokines, can destroysyngeneic islet cells through nitric oxide (NO)-dependentmechanisms (47). Ductal cells are often located adjacentto islets and produce NO following IL-1b + IFN-c stimula-tion (48). On the basis of these data, ductal cells and isletvascular endothelial cells may also contribute to islet/betacell destruction in PNF following islet transplantation, byproducing IL-1b and NO.

The final goal of this study was to demonstrate the effec-tiveness of a p38 inhibitor in vivo for preventing PNF. Ourstudy with diabetes-induced athymic mice clearly demon-strated that treatment of human islets with a p38 in-hibitor, SB203580, prior to transplantation improves isletgraft function/survival. Presumably, the islet cell protec-tive effect of SB203580 is due to inhibition of the pro-duction of pro-inflammatory mediators, including IL-1b,TNF-a, iNOS and COX-2. Because SB203580 treatmentwas given only to the islets and not to the recipients, ourresults provide strong evidence supporting the role playedby resident islet macrophages in nonspecific inflammationand PNF. In addition, when islets are transplanted into theliver, resident liver macrophages (Kupffer cells) and vascu-

lar endothelium of the recipient are also a potential sourceof pro-inflammatory cytokines and iNOS (49). Therefore,systemic administration of a p38 inhibitor in islet recipi-ents should be even more effective in improving trans-plantation results. Taken together, inhibition of the p38pathway might be a new therapeutic approach to improveclinical islet transplantation results, if large animal studiesshow the similar efficacy and long-term safety of a p38inhibitor.

Acknowledgments

The authors acknowledge the editorial services provided by Elizabeth Stein,Ph.D. This work was supported by a grant from the Nora Eccles TreadwellFoundation.

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