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1 Institut d’Investigació Biomèdica de Bellvitge (IDIBELL); Endocrine Unit, Hospital Universitari de Bellvitge, Barcelona, Spain
2 Department of Clinical Sciences, University of Barcelona, Barcelona, Spain
(Requests for offprints should be addressed to E Montanya who is now at Endocrine Unit (13-2), Hospital Universitari Bellvitge, Feixa Llarga, s/n, 08907 L’Hospitalet de Llobregat, Barcelona, Spain; Email: montanya{at}ub.edu)
* (M Montolio and M Biarnés contributed equally to this work) 
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Abstract |
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Introduction |
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Early islet cell dysfunction and damage take place before immunological rejection of the graft, and although it is more common in xenogeneic and allogeneic transplants, it has also been described in syngeneic islet transplants (Kaufman et al. 1990, Biarnes et al. 2002), confirming the involvement of non-immunological processes. The mechanism of early graft failure is probably multifactorial, and includes technical problems during the transplantation process (Kaufman et al. 1988), inadequate mass of islet tissue (Montaña et al. 1993), hypoxia of islets (Dionne et al. 1993), the metabolic condition of the recipient (Merino et al. 1997) and the absence of survival factors present in the non-endocrine pancreas (Ilieva et al. 1999). In addition, non-specific inflammation at the grafted site, which may be partly related to damage to islets during isolation, could play a role on the initial fate of transplanted islets (Vargas et al. 1998, Berney et al. 2001). An increased expression of inflammatory cytokines may participate in this non-specific inflammation, and induce the functional stunning or destruction of transplanted islets (Ozasa et al. 1997, Rabinovitch 1998, Berney et al. 2001, Gysemans et al. 2003).
The effects of pro-inflammatory cytokine interleukin-1ß (IL-1ß) in islet cells are particularly relevant (Mandrup-Poulsen 1996). IL-1ß has been conclusively shown to impair glucose-stimulated insulin production in mouse, rat, and human islets (Sandler et al. 1987, Eizirik 1991, Giannoukakis et al. 2000), and to increase ß-cell death (Delaney et al. 1997, Hoorens et al. 2001, Steer et al. 2006). In vitro observations indicate that the cytotoxic effect of IL-1ß in islet cells involves the induction of nitric oxide synthase (iNOS) and the production of nitric oxide (NO; Hoorens et al. 2001, Steer et al. 2006). In islet transplantation, it has been suggested that IL-1ß may contribute to early graft dysfunction, but the analysis of IL-1ß gene expression has been mostly limited to islets transplanted to non obese diabetic (NOD) mice (Gysemans et al. 2000, 2003). Moreover, direct evidence of the presence of the cytokine in islet grafts has not yet been provided, and the question of the cellular origin of cytokines in the graft has not been investigated. In this study, we provide evidence of the expression of both IL-1ß and iNOS genes and proteins in the initial days after syngeneic islet transplantation.
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Materials and Methods |
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Animals Male Lewis rats (B&K Universal, UK), aged 7–10 weeks, were used as donors and recipients of transplantation. The recipients were made diabetic by a single i.p. injection of streptozotocin (STZ; Streptozotocin, Sigma Immunochemicals) 65 mg/kg body weight, freshly dissolved in citrate buffer (pH 4.5). Diabetes was confirmed by the presence of hyperglycemia, weight loss, and polyuria and only those rats with blood glucose higher than 20 mmol/l on a minimum of two consecutive measurements were included in the study. Blood glucose was determined between 0900 and 1100 h in non-fasting conditions, unless stated otherwise. Blood was obtained from the snipped tail and glucose was measured with a portable glucose meter (Glucocard, A. Menarini Diagnostics, Barcelona, Spain). Animals were kept under conventional conditions in climatized rooms and fed with tap water and standard pelleted food available ad libitum.
Insulin treatment Twelve to fourteen days after STZ injection, when diabetes was well established in injected rats, the animals were started on insulin in order to maintain blood glucose as close as possible to the normal range on transplantation day and on subsequent days. This experimental approach was used to mimic human islet transplantation into diabetic patients, and to avoid the potentially confounding effects of hyperglycemia. Insulin was given as one s.c. implant of sustained insulin release (Linplant, Linshin Canada, Inc., Scarborough, ON, Canada; Merino et al. 1997). Insulin-treated groups were transplanted after 7 days on insulin and a minimum of 2 days of normoglycemia, and the insulin implants were removed the day of graft harvesting.
Islet isolation and transplantation Islets were isolated by collagenase (Collagenase P, Boehringer Mannheim Biochemicals, Mannheim, Germany) digestion of the pancreas as previously described (Montaña et al. 1993). All isolations were performed with the same batch of collagenase to avoid differences in endotoxin activity among isolations (Vargas et al. 1998). Isolated islets were hand-picked under a stereomicroscope two or three times until a population of pure islets was obtained. Islets > 75 and <250 µm in diameter were counted into groups of 500 islets, and transplanted under the left kidney capsule of the recipient on the day of isolation. The islets were transplanted to normoglycemic, insulin-treated, STZ-diabetic rats, or to normoglycemic non-STZ-injected rats. After transplantation, the capsulotomy was cauterized with a disposable low-temperature cautery pen (AB Medica, S.A, Barcelona, Spain), and the lumbar incision was sutured.
Graft harvesting The kidney was exposed, and the kidney capsule surrounding the graft was incised and removed with the graft. The grafts were harvested 1, 3, or 7 days after transplantation and processed for RNA extraction and gene expression studies. For immunohistochemical analysis, grafts harvested on days 1 and 3 after transplantation were immersed in 4% paraformaldehyde-PBS, and processed for paraffin embedding.
Gene expression studies
RNA isolation and cDNA synthesis Total RNA was extracted from six groups of 500 freshly isolated islets and from days 1, 3, and 7 islet grafts using RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. Isolated islets and islet grafts were immersed in 350 µl guanidine isothiocyanate-containing buffer plus 3.5 µl ß-mercaptoethanol. Samples were homogenized and lysed by mechanical disruption with repeated passing through a Pasteur pipette. The lysate was centrifuged and the non-lysed kidney capsule was discarded. Total RNA was eluted in diethyl pyrocarbonate-treated water and quantified at 260 nm (DU 640 Spectrophotometer, Beckman, Fullerton, CA, USA).
To perform cDNA synthesis, total RNA was treated with RQ1 RNase-free DNase (Promega) at 37 °C for 30 min and predenatured at 60 °C for 10 min with 500 ng random primers (Promega). cDNA synthesis was carried out on total RNA using Superscript reverse transcriptase (400U; Invitrogen) in a 40 µl reaction at 42 °C for 1 h in a Gene Amp PCR System 9600 (Perkin-Elmer, Norwalk, CT, CA, USA).
PCR
PCR amplification was performed on 2–10 µl cDNA in a 50 µl reaction, containing 0.2 µM of each primer, 200 µM dNTPs, and 1 U Taq DNA polymerase recombinant (Invitrogen). The sequences of the specific oligonucleotide primer pairs used and the conditions for the PCR are given in Table 1
. Samples were amplified by cycles of denaturation at 94 °C for 30 s, annealing at different temperatures depending on the gene for 30 s, and elongation at 72 °C for 45 s in a Gene Amp PCR System 9600. PCR cycles were 28 for cyclophilin and 34 for IL-1ß and iNOS. The PCR products were electrophoresed on 2% agarose gels, transferred to nylon membranes, and hybridized using oligonucleotide probes. The sequences of the oligonucleotide probes used and the temperature conditions of the hybridization are given in Table 2. Oligonucleotide probes were radiolabeled with 32P-dATP, using T4 polynucleotide kinase. The hybridization procedure for oligonucleotide probes included prehybridization for 1 h in buffer containing 0.5 M phosphate buffer (pH 7.2), 10 mM EDTA, and 7% SDS, and overnight hybridization in the same buffer adding 20 pmol specific labeled probe. Post-hybridization washes were performed with 2xSSC/0.1% SDS at 37 °C and membranes were opposed to X-ray film.
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Determination of kidney-specific gene
Islet grafts harvested with the kidney capsule can be contaminated with kidney cortex. To exclude the presence of kidney cortical cells in our RNA samples, we amplified the kidney-specific gene novel kidney transporter (NKT), a gene product related to the organic ion transporter family (Vasir et al. 2001). The sequences of the specific oligonucleotide primer pairs used and the conditions for the PCR are given in Table 1
. RT-PCR was performed as previously described, and amplified through 40 cycles.
Immunohistochemical studies
Apoptosis detection Three micrometer graft sections were double stained by immunoperoxidase for apoptotic nuclei using the TUNEL technique (In situ Cell Death Detection Kit; ApopTag, Intergen, Oxford, UK), and by alkaline phosphatase for the endocrine non-ß-cells of the islets, as described (Biarnes et al. 2002). ß-cells and apoptotic nuclei were identified and counted using an Olympus BH-2 microscope connected to a video camera with a color monitor. When assessing apoptotic nuclei, we excluded regions with necrosis. ß-Cell apoptosis was expressed as percentage of TUNEL positive ß-cells. A minimum of 1000 cell nuclei were counted per graft; the sections were systematically sampled, all endocrine nuclei were counted, and when needed a second section was included.
ß-Cell apoptosis was also determined in 9 groups of 100 fresh islets isolated on different days and in 5 pancreata from control Lewis rats. The sections of isolated islets and pancreata were double stained and counted as described for grafts.
CD68, IL-1ß, and iNOS immunohistochemistry To identify the presence of macrophages, we stained CD68-expressing cells (monocyte/macrophage lineage marker) in freshly isolated islets and islet grafts. Rat spleen sections were used as a positive control. Endogenous peroxidases were blocked with 1% hydrogen peroxidase solution and antigen retrieval was performed by incubation in citrate buffer. Then, the sections were blocked with 5% horse serum (Biological Industries, Beit Haemek, Israel), and incubated overnight at 4 °C with mouse anti-rat CD68 antibody (final dilution 1:100; Serotec, Oxford, Oxon, UK). Immunostaining for CD68 was performed using an Immuno Pure ABC Peroxidase Mouse IgG Staining Kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Peroxidase reaction was visualized with 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide. Bright-field microscope was used to visualize the CD68-staining sections.
To identify the presence of IL-1ß and iNOS proteins and the contribution of macrophages to their expression, the sections were incubated overnight at 4 °C with goat anti-rat IL-1ß antibody (final dilution 1:30; R&D, Mckinley Place, MN, USA), or goat anti-mouse and rat NOS2 (iNOS) antibody (final dilution 1:10; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and with mouse anti-rat CD68 antibody (final dilution 1:100; Serotec) primary antibodies. To minimize autofluorescence, the sections were stained with Sudan Black B (Sigma) for 30 min. The sections were then incubated with donkey fluoroscein isothiocyanate (FITC)-conjugated monoclonal anti-goat secondary antibody (final dilution 1:400; Abcam, Cambridge, UK) and with rabbit anti-mouse AlexaFluor 546-conjugated secondary antibody (final dilution 1:400; Dako, Glostrup, Denmark). Incubation with DRAQ5 (Biostatus Limited, Leicestershire, UK) was used to dye cell nuclei with a low infrared fluorochrome. A Leica TC6-SL Spectral confocal microscope was used to visualize the fluorescence and images were processed with Leica Confocal Software, version 2.5 (Leica Microsystems Heidelberg GmbH, Mannheim, Germany).
Islets treated with 50 ng/ml tumour necrosis factor (TNF)-
+ 750 U/ml interferon (IFN)-
+ 10 µg/ml lipo-polysaccharide (LPS) for 4 h were used as positive control for IL-1ß-expressing cells. Islets treated with 50 U/ml IL-1ß for 24 h were used as positive control for iNOS-expressing cells.
Statistical analysis
Results were expressed as mean and S.E.M. (mean ± S.E.M.). Differences between means were evaluated by one-way ANOVA. The Fisher’s protected least significant difference (PLSD) method was used to determine specific differences between means when determined as significant by ANOVA main effects analysis. A P value of <0.05 was considered significant.
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Results |
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STZ-treated rats developed severe hyperglycemia with polyuria and weight loss shortly after STZ injection. Hyperglycemia was corrected with insulin treatment, and after transplantation the animals were even moderately hypoglycemic (Table 3). Severe hyperglycemia (> 20 mmol/l) recurred in all rats when the graft and insulin implants were harvested. Transplantation did not modify blood glucose in non-diabetic, non-STZ-injected rats.
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IL-1ß mRNA was detected in islets after isolation in five out of six samples from different isolation procedures, and was upregulated after transplantation. IL-1ß mRNA was ninefold increased on day 1 after transplantation, it was still sevenfold increased on day 3 when compared with fresh islets, and declined towards pretransplantation levels on day 7 (Fig. 1). iNOS mRNA showed a similar pattern of expression as IL-1ß: on days 1 and 3 after transplantation it was 14- and 4-fold higher respectively, than in freshly isolated islets. IL-1ß and iNOS mRNA levels in individual islet grafts were significantly and positively correlated (r = 0.56, P<0.05). To exclude the presence of kidney cortex contaminating the islet graft RNA samples, we examined the expression of NKT mRNA. No expression of NKT mRNA was detected in 7-day-islet grafts (data not shown).
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). ß-Cell death
ß-Cell apoptosis was similar in freshly isolated islets immediately after islet isolation (0.09 ± 0.04%) and control pancreases (0.04 ± 0.01%), but was significantly increased in day 3 grafts (0.30 ± 0.01%, ANOVA P<0.001, Fisher PLSD, P<0.01 between transplanted islets, and pancreas and isolated islets; Fig. 2). In addition, extensive areas of necrosis, similar to those previously reported in syngeneic mice islet grafts (Biarnes et al. 2002) were identified.
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In islet grafts, macrophages were abundant in the periphery of the islet tissue and in the necrotic areas (Fig. 3). In contrast, few CD68-positive cells were detected within the islet tissue.
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To determine the role of macrophages in IL-1ß and iNOS expressions, we double stained the graft sections with anti-CD68 antibody, and anti-IL-1ß or anti-iNOS antibodies. The double-stained sections were analyzed by confocal microscopy (Fig. 4). IL-1ß expression was detected almost exclusively in CD68-positive cells that accounted for ~95% of IL-1ß-expressing cells on days 1 and 3 after transplantation. Ten percent of CD68-positive cells expressed IL-1ß on day 1 and 14% on day 3. The rare IL-1ß-positive/CD68-negative cells (~5% of all IL-1ß-positive cells) were all found within the islets.
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Discussion |
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Similar to our previous results in mice islet grafts (Biarnes et al. 2002), we found that rat islet grafts show necrosis and increased apoptosis shortly after transplantation. Non-specific inflammation at the grafted site may contribute to this initial islet damage which results in massive loss of transplanted tissue (Biarnes et al. 2002), and can lead to primary non-graft function. Ozasa et al.(1997) found increased levels of IL-1, IL-2, IL-6, and IFN- transcripts in syngeneic and allogeneic islet grafts when compared with freshly isolated islets. However, they could not perform a quantitative comparison of the expression of transcripts on different days, and they did not analyze the presence of the relevant IL-1ß transcript. IL-1ß is a candidate which contributes to early graft inflammation in islet transplantation, but its expression has been investigated almost exclusively in autoimmune models in islet syngeneically transplanted to NOD mice (Gysemans et al. 2000, 2003). Cardozo et al.(2003) detected the expression of IL-1ß gene in Balb/c islet isografts 8 h after transplantation, but isolated, non-transplanted, islets were not studied and it could not be established whether IL-1ß expression was increased in islet grafts when compared with isolated islets. This is an important question, because previous studies have shown that the endotoxin activity present in collagenase preparations used in islet isolation elicits an inflammatory cytokine response in macrophages, and may be responsible for the induction of several pro-inflammatory cytokine genes in islets during the isolation process (Vargas et al. 1998, Berney et al. 2001). In this study, we have found clearly increased levels of IL-1ß mRNA in islet grafts on day 1 after transplantation when compared with isolated islets and to days 3 and 7 after transplantation, indicating that the expression of IL-1ß was enhanced by the transplantation process.
This is the first study that directly shows the presence of IL-1ß and iNOS proteins in recently transplanted islet grafts. IL-1ß is the major regulator of iNOS expression in ß-cells (Eizirik et al. 1992, Karlsen et al. 1995), and in rat islets, IL-1ß alone is sufficient to stimulate iNOS expression, and NO production. NO has a fundamental role in cytokine-induced ß-cell damage (Eizirik et al. 1992, Karlsen et al. 1995, Liu et al. 2000, Steer et al. 2006). Therefore, the expression of iNOS suggests that the presence of IL-1ß in the graft was biologically significant. The increased expression of IL-1ß and iNOS genes, along with the identification of IL-1ß and iNOS proteins in grafts, confirms the presence of an early inflammatory process in islet transplantation, and strongly suggests that they are important mediators of graft inflammation and islet damage in early islet transplantation. The relevance of this inflammatory process may go beyond the induction of direct islet damage in accordance with the proposed role of inflammation trigger of the immune response (Matzinger 2002). Therefore, in islet allotransplantation, the initial non-specific inflammation could enhance the subsequent immune rejection.
The question of the cellular origin of pro-inflammatory cytokines in islet grafts had not previously been investigated. We did not detect the kidney-specific gene NKT in graft homogenates, excluding the contamination from kidney tissue thus leaving the graft as the only source of IL-1ß and iNOS mRNAs. Using confocal microscopy, we found that macrophages were, by far, the main source of IL-1ß and iNOS in islet grafts. The role of macrophages in early graft failure has previously been suggested by the reports of improved graft survival with treatments that depleted or inactivated macrophages (Kaufman et al. 1990, 1994, Kenmochi et al. 1996, Bottino et al. 1998).
Other cell types, in addition to macrophages, had a less important contribution to IL-1ß and iNOS expressions in islet grafts, as indicated by 5% of IL-1ß-positive cells and 10–25% of iNOS-positive cells that were CD68-negative. IL-1ß expression by cells other than macrophages was found only within the islets. Transplanted islet preparations contain, in addition to the endocrine cells of the islets, exocrine tissue, capillary endothelial cells, macrophages, dendritic-like cells, fibroblasts, and other minor cell populations. Ductal and endothelial cells were a source of IL-1ß in human islets in response to LPS + TNF- + IFN-, although they represented a small number when compared with macrophages (Matsuda et al. 2005). In contrast, ß-cells were the major source of NO in rat and human islets in response to IL-1ß or IL-1ß + IFN- respectively (Corbett & McDaniel 1995, Arnush et al. 1998). We found that iNOS-positive/CD68-negative cells were similarly distributed inside and outside the islets. The intra-islet expression of iNOS by cells other than macrophages is important because it has been hypothesized that NO must be produced by ß-cells to induce ß-cell damage (Thomas et al. 2002). Nevertheless, the expression of iNOS by the abundant macrophages infiltrating the graft probably contributed also to ß-cell damage, similar to that described in other tissues (Bruns et al. 2000).
In summary, the increased IL-1ß and iNOS gene and protein expressions in syngeneic islet grafts confirm the presence of a non-specific inflammatory response in the initial days after islet transplantation. Macrophages were the main cellular source of IL-1ß and iNOS, confirming their essential role in early graft inflammation. Considering the well-established cytotoxicity of IL-1ß and NO, they probably contribute to the initial massive loss of transplanted islet cells and the phenomenon of early graft failure.
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Acknowledgements |
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References |
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Received in final form 21 September 2006
Accepted 11 October 2006
Made available online as an Accepted Preprint 27 October 2006
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P<0.05 among iNOS expression in day 1 after transplantation and all other groups.
