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Keywords:

  • Primary graft nonfunction;
  • islet transplantation;
  • p38;
  • macrophage

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Nonspecific inflammation is associated with primary graft nonfunction (PNF). Inflammatory islet damage is mediated at least partially by pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) produced by resident islet macrophages. The p38 pathway is known to be involved in cytokine production in the cells of the monocyte–macrophage lineage. Therefore, inhibition of the p38 pathway may prevent pro-inflammatory cytokine production by resident islet macrophages and possibly reduce the incidence of PNF. Our present study has demonstrated that inhibition of the p38 pathway by a chemical p38 inhibitor, SB203580, suppresses IL-1β and TNF-α production in human islets exposed to lipopolysaccharide (LPS) and/or inflammatory cytokines. Although IL-1β is predominantly produced by resident macrophages, ductal cells and islet vascular endothelial cells were found to be another cellular source of IL-1β in isolated human islets. SB203580 also inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the treated islets. Furthermore, human islets treated with SB203580 for 1 h prior to transplantation showed significantly improved graft function. These results suggest that inhibition of the p38 pathway may become a new therapeutic strategy to improve graft survival in clinical islet transplantation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Resident islet macrophages play an important role in the initiation of beta cell destruction during the development of type1 diabetes (1–3). Previous reports have shown that these cells also play a major role in primary islet graft nonfunction (PNF) or early graft failure following islet transplantation (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 early graft failure/PNF. PNF occurs even with syngeneic or autologous islet transplants (6,7). The beta cell death associated with nonspecific inflammation appears to be mediated, at least in part, by production of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the grafted islets (4,5,8). These cytokines can also stimulate the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which are believed to be mediators of cytokine-induced islet damage. In fact, iNOS or COX-2 inhibitors have been shown to prevent cytokine-mediated islet cell damage both in vitro and in vivo (9–12). Therefore, the suppression of pro-inflammatory cytokine production in the islets should, similar to iNOS or COX-2 inhibitors, reduce early cell loss in the transplant and preserve the islet cell mass required for controlling hyperglycemia.

Pro-inflammatory cytokines are generally known to be produced by macrophages and monocytes upon stimulation with lipopolysaccharide (LPS) and/or cytokines. LPS activates mitogen-activated protein kinases (MAPKs) including c-Jun NH2-terminal kinase (JNK), p38 kinase (p38) and extracellular signal-regulated kinase (ERK). Other investigators reported the involvement of MAPKs in LPS-induced cytokine production (13,14). Particularly, it has been well documented that inhibition of the p38 pathway by chemical p38 inhibitors, such as SB203580, prevents IL-1β, TNF-α and IL-6 production by macrophages (15,16). These inhibitors also suppress iNOS and COX-2 expression in certain types of cells (17,18). However, it remains unknown whether p38 inhibitors are capable of suppressing cytokine production and iNOS and COX-2 expression in isolated human islets.

Besides macrophages, iNOS is expressed by pancreatic beta cells, ductal cells and endothelial cells following cytokine (such as IL-1β and IFN-γ) stimulation, and mediates beta cell damage (19–21). COX-2 is also induced by various stimuli, including cytokines and high glucose, and leads to prostaglandin E2 production that causes beta cell dysfunction (22,23). Moreover, it has recently been reported that beta cells are the source of IL-1β in the islets of type 2 diabetic patients (24). In the present study, we have examined, using human islets, whether inhibition of the p38 pathway by SB203580 suppresses islet production of inflammatory mediators and reduces PNF after transplantation. We also investigated the cellular source of IL-1β and TNF-α in human islet preparations.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Materials

LPS (from Escherichia coli 0111:B4) was purchased from Sigma (St. Louis, MO). Human recombinant IL-1β, TNF-α and IFN-γ were purchased from R&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 α-tubulin was from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse and goat anti-IL-1β antibodies were from R&D Systems Inc. and mouse anti-COX-2 antibody was purchased from BD Biosciences (San Jose, CA).

Human islets

Human islets were provided for research use by the Southern California Islet Cell Resources Center, City of Hope (Duarte, CA). Islets were isolated by the two-step digestion method (25) and cultured in serum-free CMRL1066 medium (Mediatech, Inc., Holly Hill, FL) for 2 days prior to being released for research use. These islets were then cultured in Ham's F12 medium supplemented with 10 mM HEPES, antibiotic antimycotic, and 10% fetal bovine serum (all from Sigma) for an additional 1–2 days before being used in experiments. All experiments started within 4 days after isolation. The islet numbers were expressed as the number equivalent to islets 150 μm in diameter (IEQ). Only islets with greater than 80% purity, as determined by dithizone staining, were used. Otherwise, islets were hand-picked.

Treatment of human islets

Islets were treated with 10 μM SB203580 or vehicle alone (DMSO) in culture medium for 30 min, followed by stimulation with either 10 μg/mL LPS alone, 10 μg/mL LPS, 50 ng/mL TNF-α and 750 U/mL IFN-γ (LPS + TNF-α+ IFN-γ), or 75 U/mL IL-1β and 750 U/mL IFN-γ (IL-1β+ IFN-γ), and then cultured for 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), and the viability was assessed after 2- and 4-day culture following LPS and/or cytokine stimulation. Viability was expressed by the percentage of the MTT absorbance of the untreated islets.

Semiquantitative PCR

Cultured islets were collected 8 h after stimulation with LPS and/or cytokines and stored at −80°C until use. Total RNA was extracted from islets using TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OH). A total of 2.0 μg of RNA from each sample was then reverse-transcribed into first-strand cDNA in 20-μL solution using SuperscriptRNase H reverse transcriptase (Invitrogen, Carlsbad, CA). The final cDNA products were then diluted by adding 80 μL of H2O. PCR was performed using the FailSafe PCR System (EPICENTRE, Madison, WI). A standard 25 μL PCR reaction solution contained 1.5 μL of cDNA, 30 pmol each of forward and reverse primers, 12.5 μL of PremixD and 0.25 μL of Enzyme Mix. The following primers were used: human IL-1β (204 bp) forward 5′-CCT GTG GCC TTG GGC CTC AA-3′, reverse 5′-GGT GCT GAT GTA CCA GTT GGG-3′; human TNF-α (325 bp) forward 5′-CAG AGG GAA GAG TTC CCC AG-3′, reverse 5′-CCT TGG TCT GGT AGG AGA CG-3′; human iNOS (236 bp) forward 5′-ACA TTG ATC AGA AGC TGT CCC AC-3′, reverse 5′-CAA AGG CTG TGA GTC CTG CAC-3′; human GAPDH (600 bp) forward 5′-CCA CCC ATG GCA AAT TCC ATG GCA-3′, reverse 5′-TCT AGA CGG CAG GTC AGG TCC ACC-3′. The cycle number was determined to be in the linear range of amplification for each primer pair. PCR using 1/2 diluted concentrations of cDNA template verified that PCR product yields were proportional to the initial concentrations of cDNA template. Electrophoresis of PCR products were performed in 1.5% agarose gel containing 0.5mg/mL of ethidium bromide and analyzed with an Alpha Innotech densitometer.

Western blotting

After LPS and/or cytokine stimulation, 800 IEQ were harvested from each sample at an indicated time, washed twice with ice-cold phosphate buffered saline (PBS) and stored at −80°C until use. Islet cell lysis and Western blotting were performed as described previously (27).

Determination of cytokine and nitrite levels in culture media

Islets (600 IEQ/600 μL of culture media) were cultured in duplicate for 48 h after stimulation with LPS and/or cytokines and then the culture supernatants were collected for cytokine and nitrite determination. Nitrite production in the supernatant was measured immediately after collection using a Griess Reagent Kit (Molecular Probes, Eugene, OR). Samples for cytokine determination were stored at −20°C, and the levels of IL-1β and TNF-α were measured using Quantikine kits (R&D systems Inc.).

Immunocytochemistry

Forty-eight hours after LPS and/or cytokine stimulation, islets (800 IEQ/sample) were collected, washed with PBS and gently agitated in Cell Dissociation Buffer (Invitrogen) for 10 min at 37°C. Cells were then collected, washed once with PBS and plated on poly-l-Lysine-coated cover slips. Cells were fixed with 100% methanol for 20 min at −20°C, then dipped in acetone. The cover slips were stored at −20°C until cells were stained. After washing with PBS, the cover slips were blocked with 5% normal donkey serum and then incubated with goat anti-IL-1β (1:30 dilution; R&D Systems Inc.) for 1 h at 37°C, followed by 1 h incubation with mouse anti-CD68 (1:10 dilution; DAKO, Carpinteria, CA), mouse anti-cytokeratin-7 (1:15 dilution; DAKO), or rabbit anti-von Willebrand factor (1:100 dilution; DAKO). Following incubation, the cover slips were washed four times with PBS, and incubated for 1 h at room temperature with FITC-conjugated donkey anti-goat and Texas Red-conjugated donkey anti-mouse (or rabbit) antibodies (1:100 dilution; Jackson Immuno Research Laboratories, West Grove, 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 an intraperitoneal injection of 250 mg/kg streptozotocin (STZ) (Sigma) freshly dissolved in citrate buffer. A marginal number (1500 IEQ) of human islets was treated with 10 μM SB203580 or vehicle (DMSO) alone in culture medium for 1 h, and then transplanted under the kidney capsule as described previously (28). Four different islet preparations were used in this 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 consecutive days were taken as the reversal of diabetes. The period required for diabetes reversal was defined as the number of days from the day of transplantation to the first day that blood glucose was <200 mg/dL. After 30 days of observation, nephrectomy of the graft-bearing kidney was performed in all glucose-normalized mice to confirm the diabetes reversal was due to the islet graft. Animal procedures followed the protocols approved by the Animal Care Committee of the City of Hope Medical Center/Beckman Research Institute.

Statistical analysis

Data are expressed as mean ± SE. Differences between groups were analyzed by the unpaired Student's t-test. Time required for recovery from diabetes was calculated using Kaplan–Meier life tables, and differences between groups were assessed by a log-rank test. A p < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The p38 inhibitor, SB203580, inhibits the expression and release of IL-1β induced by LPS and/or cytokines

LPS is well known to stimulate IL-1β mRNA expression and production in macrophages. It has also been reported that IL-1β is produced by human islets treated with LPS + TNF-α+ IFN-γ (3). Using semiquantitative PCR, we examined whether or not a p38 inhibitor, SB203580, is capable of suppressing LPS- and LPS + TNF-α+ IFN-γ-induced IL-1β mRNA expression in human islets. Both LPS and LPS + TNF-α+ IFN-γ-induced IL-1β mRNA expression in the islets within 8 h after stimulation. Treatment of islets with SB203580 prior to stimulation had, essentially, no suppressive effect on the IL-1β mRNA expression (Figure 1A). Levels of IL-1β released from human islets into the culture medium were measured 48 h after stimulation with or without SB203580 pre-treatment. As shown in Figure 1B, SB203580 significantly suppressed LPS + TNF-α+ IFN-γ-induced IL-1β release (p < 0.05). LPS alone also induced IL-1β release, but at far lower levels than those induced by LPS + TNF-α+ IFN-γ. Despite varying levels of LPS and/or cytokines-induced IL-1β release from different islet preparations, SB203580 consistently suppressed the IL-1β release to 10–30% of the corresponding control level. Western blot analysis showed that the levels of pro-IL-1β protein (precursor molecule of IL-1β) induced in response to LPS and LPS + TNF-α+ IFN-γ were decreased by SB203580 treatment (Figure 1C). These results suggest that SB203580 inhibits LPS- and LPS + TNF-α+ IFN-γ-induced IL-1β production by islets at the post-transcriptional level.

image

Figure 1. Effect of SB203580 on LPS- and LPS + TNF-α+ IFN-γ-induced IL-1β expression and release by human islets. Human islets were treated with 10 μM SB203580 or vehicle (DMSO) alone for 30 min, then stimulated with 10 μg/mL LPS or 10 μg/mL LPS + 50 ng/mL TNF-α+ 750 U/mL IFN-γ, and subsequently cultured for 8 h (A, C) or 48 h (B). A: Total RNA was isolated and IL-1β mRNA expression was determined by RT-PCR. B: The levels of IL-1β released into the culture medium were measured by ELISA. C: pro-IL-1β expression was detected by Western blotting analysis. RT-PCR and Western blotting data represent three independent experiments using three separate human islet preparations. IL-1β release is expressed as mean ± SE of five independent experiments using separate islet preparations, each performed in duplicate. *p < 0.05 islets treated without vs. with SB203580.

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SB203580 suppresses TNF-α expression and release by human islets stimulated with LPS or IL-1β+ INF

TNF-α is known to cause beta cell dysfunction and death when it is used to treat human islets in vitro in combination with IL-1β and IFN-γ (29). TNF-α, together with IL-1β, is produced locally in the islet graft site in vivo shortly after islet transplantation, and these cytokines are considered as the major pro-inflammatory mediators (5,8). LPS treatment stimulated TNF-α mRNA expression and release in human islets as shown in Figure 2. IL-1β+ IFN-γ-induced higher levels of TNF-α release than LPS alone, although these levels were much lower than the concentration that is cytotoxic to human islets in vitro. SB203580 significantly inhibited TNF-α mRNA expression, and almost completely abolished TNF-α release induced by LPS or cytokines (Figure 2).

image

Figure 2. Effect of SB203580 on LPS- and IL-1β+IFN-γ-induced TNF-α expression and release by human islets. Human islets were treated with 10 μM SB203580 or vehicle (DMSO) alone for 30 min, then stimulated with 10 μg/mL LPS or 75 U/mL IL-1β+ 750 U/mL IFN-γ, and subsequently cultured for 8 h (A) or 48 h (B). A: Total RNA was isolated and TNF-α mRNA expression was determined by RT-PCR. B: The levels of TNF-α released into the culture medium were measured by ELISA. RT-PCR data are representative of three independent experiments each using three separate human islet preparations. TNF-α release results are expressed as mean ± SE of five independent experiments performed in duplicate using five separate isolations. *p < 0.05 vs. islets without SB203580 treatment. **p < 0.0001 vs. islets without SB203580.

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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 the treatment of human islets with LPS + TNF-α+ IFN-γ or IL-1β+ IFN-γ stimulates iNOS expression and nitrite production in human islets (3,30). We also found the induction of iNOS mRNA in LPS and/or cytokine stimulated human islets. IL-1β+ IFN-γ-induced higher levels of iNOS mRNA than LPS + TNF-α+ IFN-γ when measured 8 h after stimulation. Islet treatment with SB203580 markedly inhibited both LPS + TNF-α+ IFN-γ- and IL-1β+ IFN-γ-induced iNOS mRNA expression (Figure 3A). These results were consistent with levels of nitrite produced in the culture media (Figure 3B). LPS alone did not induce any detectable increase in nitrite production.

imageimage

Figure 3. Effect of SB203580 on LPS + TNF-α+ IFN-γ- and IL-1β+ IFN-γ-induced iNOS and COX-2 expression, nitrite production and viability. Human islets were treated with 10 μM SB203580 or vehicle (DMSO) alone for 30 min, then stimulated with 10 μg/mL LPS + 50 ng/mL TNF-α+ 750 U/mL IFN-γ or 75 U/mL IL-1β+ 750 U/mL IFN-γ, and subsequently cultured for 8 h (A), 48 h (B), 24 h (C) or 48 and 96 h (D). A: Total RNA was isolated and iNOS mRNA expression was determined by RT-PCR. B: The level of nitrite production was measured in the culture media using Griess reagent. C: Islets were collected and lysed, and Western blotting was performed to detect COX-2 protein levels. D: Viability was determined by MTT assay. RT-PCR and Western blotting data are representative of three independent experiments using three separate human islet preparations. Nitrite production results are expressed as mean ± SE of five independent experiments in duplicate using five separate human islet preparations. Viability results are expressed as mean ± SE of three independent experiments in six replicate cultures using three separate preparations. *p < 0.05 vs. untreated islets. **p < 0.05 vs. islets with SB203580.

Next, we examined whether SB203580 was capable of modifying the COX-2 induction by LPS and/or cytokines using Western blotting. COX-2 expression was up-regulated in islets treated for 24 h with LPS + TNF-α+ IFN-γ or IL-1β+ IFN-γ, but not with LPS alone. Treatment with SB203580 prevented up-regulation of COX-2 levels in cytokine stimulated islets with or without LPS (Figure 3C).

p38 inhibition prevents cell death in human islets in response to LPS + TNF-α+ IFN

Our results consistently showed the suppressive effect of SB203580 on the production of pro-inflammatory mediators. On the basis of these results, we postulated that SB203580 would prevent LPS and/or cytokine-induced islet cell death. Therefore islet viability was determined by MTT assay 2 and 4 days after stimulation with LPS and/or cytokines, with or without SB203580 pre-treatment. Neither LPS alone nor IL-1β+ IFN-γ induced any detectable islet cell death (data not shown, Figure 3D). In contrast, LPS + TNF-α+ IFN-γ caused a 15–20% decrease in viable cells. SB203580 significantly prevented this LPS + TNF-α+ IFN-γ-induced islet cell death as shown in Figure 3D (p < 0.05).

Identification of the cellular source of IL-1β and TNF

To determine the cellular source(s) for IL-1β and TNF-α, islets cultured for 48 h in medium containing either LPS + TNF-α+ IFN-γ or IL-1β+ IFN-γ were examined by immunohistochemistry. Expectedly, most (80–90%) of the IL-1β-positive cells also stained positively for CD68 (a macrophage marker) (Figure 4). In addition, we found a small number (<10%) of IL-1β-positive cells that were also positive for cytokeratin-7 (a ductal cell marker) or von Willebrand factor (an endothelial cell marker), indicating that, in addition to macrophages, ductal cells and islet vascular endothelial cells are a source of IL-1β in human islet preparations. In contrast, TNF-α expression always colocalized with CD68-staining (data not shown). Neither IL-1β nor TNF-α expression was detected in any type of endocrine cells.

image

Figure 4. Colocalization of IL-1β and the cell markers in dispersed islet cells. Human islets were stimulated with 10 μg/mL LPS + 50 ng/mL TNF-α+ 750 U/mL IFN-γ for 48 h, and then dispersed into single cells and small cell clusters. IL-1β staining (green) and CD68, cytokeratin-7 or von Willebrand factor staining (red) were performed as described in Material and Methods. Cells double positive for IL-1β and other cell markers were identified yellow by merged images (Merge). Results are representative of four independent experiments each using four separate human islet preparations.

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SB203580 inhibits p38 pathway in human islets

Various stresses, including LPS and IL-1β, activate p38, which activates MAPKAP kinase-2 (MAPKAPK-2), which in turn phosphorylates HSP27 (31,32). SB203580 has been reported to inhibit p38 activity by binding to the ATP binding site of p38 and to block MAPKAPK-2 activation (16,33,34). To investigate the activation of p38 by LPS and/or cytokines and to confirm the p38 pathway inhibition by SB203580 in human islets, Western blotting analysis was performed. Treatment with IL-1β+ IFN-γ resulted in the maximum levels of p38 phosphorylation (p-p38) at 60 min after stimulation, which then returned to basal levels in 120 min (Figure 5A). In contrast, a rapid and prolonged phosphorylation of p38 was observed after stimulating with LPS + TNF-α+ IFN-γ (Figure 5B). Treatment with SB203580 at a 10 μM concentration had no or a marginal effect in inhibiting phosphorylation of p38 in response to either IL-1β+ IFN-γ or LPS + TNF-α+ IFN-γ. However, SB203580 almost completely suppressed phosphorylation of HSP-27 (p-HSP-27) in both cases, indicating the activation of MAPKAPK-2 was blocked by SB203580. This observation that SB203580 inhibits the activity but not the phosphorylation of p38 is consistent with the previous reports using other cell systems (34–36).

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Figure 5. p38 pathway inhibition by SB203580 in human islets activated by IL-1β+ IFN-γ and LPS + TNF-α+ IFN-γ. Human islets were treated with 10 μM SB203580 or vehicle (DMSO) alone for 30 min, and then stimulated with 75 U/mL IL-1β+ 750 U/mL IFN-γ (A) or 10 μg/mL LPS + 50 ng/mL TNF-α+ 750 U/mL IFN-γ (B) for the periods indicated. After stimulation, islets were collected and lysed, and Western blotting was performed to detect phosphorylated p38 (p-p38). The same samples were also used to detect total p38 (p38) and phosphorylated HSP27 (p-HSP27). Data are representative of three independent experiments each using three separate human islet preparations.

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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 islet protective effect of SB203580 from LPS and/or pro-inflammatory cytokine toxicity. We then tested if such protective effects displayed in vitro also extend in vivo to protect transplanted islets from PNF. We examined whether treatment of islets with SB203580 prior to transplantation improves graft function/survival by transplanting a marginal number of human islets treated with SB203580 or vehicle alone under the kidney capsule of diabetic athymic mice. A marginal islet number, that otherwise would not reverse diabetes, was determined to be 1500 IEQ in our previous study (data not shown). When islets were pre-treated with SB203580, the period needed to achieve normoglycemia was significantly reduced and a higher proportion of recipients reversed diabetes within 30 days after transplantation (n = 8, all eight mice reversal, p = 0.006). When islets were treated with vehicle alone, only four out of nine mice (n = 9) reversed diabetes by day 30 (Figure 6A,B). In all cases in which normoglycemia was achieved, the mice returned to hyperglycemia within 2 days after removal of the grafts by nephrectomy.

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Figure 6. Reversal of diabetes in athymic mice by human islets treated with SB203580 prior to transplantation. A marginal number of human islets (1500 IEQ) were treated with 10 μM SB203580 (N = 8) or vehicle (DMSO) alone (N = 9) for 1 h and then transplanted under the kidney capsule of athymic mice made diabetic with streptozotocin. Nonfasting blood glucose levels were monitored (A) and the percent of mice that had reversed diabetes and days required for diabetes reversal (<200 mg/dL of blood glucose) were compared between SB203580-treated (open squares) and untreated (black squares) groups separately. *p = 0.006 vs. untreated islets

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Primary graft nonfunction or early graft failure is one of the main obstacles that need to be solved in order to consistently succeed with islet transplantation. Islet graft loss in the early stages of transplantation is caused primarily by nonspecific inflammation, but not specific immune responses or metabolic over dose, as shown by a number of animal studies with autologous and syngeneic islet transplantation (6,7). Clinical cases in which islets failed to function shortly after transplantation may also be attributable to nonspecific inflammation occurring within and around the graft. The report that PNF is associated with increased intra-islet cytokine production in syngeneic transplants suggests that such nonspecific inflammation reactions are at least partially mediated by the resident islet macrophages (4). Therefore, it is reasonable to think that the prevention of pro-inflammatory cytokine production by resident islet macrophages would improve islet graft survival as well as prevent or delay the development of autoimmune diabetes.

We reported here that inhibition of the p38 pathway by a chemical p38 inhibitor suppressed the production of IL-1β and TNF-α in isolated islets in response to LPS and/or cytokines and that pre-treatment of human islets with the p38 inhibitor improved islet graft function with regard to reversing diabetes. These results support our hypothesis that the prevention of resident islet macrophage activation by inhibition of the p38 pathway reduces PNF after islet transplantation.

Chemical p38 inhibitors, such as SB203580, have been widely used in various kinds of experiments using cultured cells. Their ability to suppress pro-inflammatory mediators, such as IL-1β, TNF-α and IL-6, has also been well documented in the cells of the monocyte–macrophage lineage (15,16,36). However, their applicability and effect on human islets have remained largely unknown and the available results are controversial (37,38). Our study is the first to report the inhibitory effect of a p38 inhibitor, SB203580, on the production of pro-inflammatory mediators in human islets.

We have shown that SB203580 does not reduce IL-1β mRNA levels in response to LPS and LPS + TNF-α+ IFN-γ stimulation, but inhibits pro-IL-1β protein expression and IL-1β release, suggesting that p38 regulates IL-1β production at post-transcriptional level in these settings. In the process of IL-1β release, pro-IL-1β produced in the cytosol of macrophages is cleaved into active IL-1β by IL-1-converting enzyme (ICE) or caspase-1 (39). Therefore, our findings also might be explained by the suppressive effect of p38 inhibitors on ICE levels as observed in chondrocytes (40). On the other hand, for TNF-α, the mRNA level was significantly decreased by SB203580 treatment prior to LPS alone or IL-1β+ IFN-γ-stimulation, and its release was completely abolished, suggesting that p38 is involved in TNF-α production at both transcriptional and translational levels.

The p38 inhibitor also suppressed iNOS and COX-2 expression and nitric oxide production in the islets caused by both LPS + TNF-α+ IFN-γ and IL-1β+ IFN-γ stimulation. Previous studies using iNOS inhibitors, such as L-NG-monomethyl-arginine (L-NMMA), clearly showed that cytotoxic effects of pro-inflammatory cytokines on beta cells were at least partially mediated by activation of iNOS (9,21). Inhibition of beta cell function by COX-2 pathway products, such as PGE2, also has been well documented (11,12). Therefore, the suppressive effect of SB203580 on iNOS and COX-2 expression may be beneficial to protect islets from various stimuli which activate these genes.

iNOS mRNA levels significantly increased in islets following IL-1β+ IFN-γ stimulation. Treatment with LPS + TNF-α+ IFN-γ also up-regulated iNOS mRNA and nitrite levels, but these levels were much lower than those induced by IL-1β+ IFN-γ. Unexpectedly, however, IL-1β+ IFN-γ did not induce distinct islet cell death as measured by MTT assays, while treatment with LPS + TNF-α+ IFN-γ resulted in 10–15% decreased islet cell viability after 4 days in culture, which was alleviated by SB203580 treatment. These results suggest that other p38-related mechanisms in addition to iNOS up-regulation are involved in the induction of islet cell death caused by LPS + TNF-α+ IFN-γ.

Besides p38, two other MAPKs, JNKs and ERKs, have also been implicated in LPS-induced cytokine production and release. Especially, the involvement of ERKs in cytokine induction and regulation has been well documented using MEK/ERK inhibitors, such as U0126 and PD98059 (41,42). However, p38 appears to play a more important role in LPS-induced IL-1β and TNF-α production than ERKs or JNKs since LPS-induced TNF-α production in vivo is decreased by 90% in mice that do not express MAPKAPK-2, the direct downstream target of p38 (43). Also, Hsu et al. demonstrated that p38 was more important in LPS-mediated IL-1β production by macrophages (36). However, there is still a possibility that ERKs and/or JNKs may play a greater role than p38 in the control of cytokine production in human islets. Therefore, further investigation of the participation of JNKs and ERKs in cytokine regulation in human islets may give us an insight into the mechanisms of resident islet macrophage activation.

Arnush et al. reported in 1998 that CD68-positive macrophages were the sole cellular source for IL-1β in human islets stimulated in vitro by LPS + TNF-α+ IFN-γ (3). However, in 2002, Maedler et al. showed high glucose-induced IL-1β production by beta cells and they also detected IL1β-expressing beta cells in the islets of poorly controlled type 2 diabetic patients (25). In the study by Arnush et al., islets were analyzed by immunohistochemistry 4 h after stimulation. We were unable to find IL-1β-producing endocrine cells even 48 h after stimulation. However, in our study a small percentage of IL-1β-positive cells were found to co-stain with von Willebrand factor or cytokeratin-7, although the majority of IL-1β-positive cells were macrophages. Human islet preparations always contain nonendocrine cells, such as ductal cells and endothelial cells. Endothelial cells, including human umbilical endothelia, are known to produce IL-1β, TNF-α and IL-6 (44–46). Previous reports have shown that islet endothelial cells, when activated by cytokines, can destroy syngeneic islet cells through nitric oxide (NO)-dependent mechanisms (47). Ductal cells are often located adjacent to islets and produce NO following IL-1β+ IFN-γ stimulation (48). On the basis of these data, ductal cells and islet vascular endothelial cells may also contribute to islet/beta cell destruction in PNF following islet transplantation, by producing IL-1β and NO.

The final goal of this study was to demonstrate the effectiveness of a p38 inhibitor in vivo for preventing PNF. Our study with diabetes-induced athymic mice clearly demonstrated that treatment of human islets with a p38 inhibitor, SB203580, prior to transplantation improves islet graft function/survival. Presumably, the islet cell protective effect of SB203580 is due to inhibition of the production of pro-inflammatory mediators, including IL-1β, TNF-α, iNOS and COX-2. Because SB203580 treatment was given only to the islets and not to the recipients, our results provide strong evidence supporting the role played by resident islet macrophages in nonspecific inflammation and PNF. In addition, when islets are transplanted into the liver, resident liver macrophages (Kupffer cells) and vascular endothelium of the recipient are also a potential source of pro-inflammatory cytokines and iNOS (49). Therefore, systemic administration of a p38 inhibitor in islet recipients should be even more effective in improving transplantation results. Taken together, inhibition of the p38 pathway might be a new therapeutic approach to improve clinical islet transplantation results, if large animal studies show the similar efficacy and long-term safety of a p38 inhibitor.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

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

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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