1 Department of Pediatrics, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania
2 Division of Immunogenetics, Diabetes Institute, Rangos Research Center, Childrens Hospital of Pittsburgh, Pittsburgh, Pennsylvania
3 Department of Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania
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ABSTRACT |
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To become more successful, allotransplantation of human islets requires the availability of less toxic antirejection protocols and larger quantities of usable islets (1). In addition, the islet isolation process remains inefficient, often yielding an insufficient mass of functional islet cells to achieve adequate function, even in the absence of immune-mediated rejection.
Many factors contribute to islet loss and insufficient ß-cell metabolic function after transplantation (25). New data have shown that islet function is already undermined by stressful events before transplantation (6,7). These findings implicate the effects of organ explantation, cold storage time, and the isolation procedures themselves as potential threats to islets. The nonphysiological biophysical and biochemical ambient conditions that are present during organ harvesting and isolation require abrupt metabolic adaptation by islets, which may result in functional impairment and eventually cell death. Despite efforts to optimize the conditions of pancreas preservation ex vivo (810) and the islet isolation process to improve islet yield, only a significantly limited part of the islet pancreatic content survives the process of isolation and subsequent culture.
Although the cascade of events during pancreatic cell isolation that may cause ß-cell dysfunction and death is not fully characterized, recent lines of research in rodents (11,12) and humans (13) have indicated that oxidative stress plays a major role in triggering the death of islets and surrounding exocrine tissue. Other reports have demonstrated that oxidative stress is strongly connected to the adverse effects of chronic hyperglycemia on insulin biosynthesis by islet ß-cells (1416).
It has been widely reported that islet ß-cells are highly susceptible to oxidative stress because of their reduced levels of endogenous antioxidants (1719). Under extreme conditions of stress, the islet antioxidant defenses may become overwhelmed, leading to a state of redox imbalance and the production of reactive oxygen species (ROS). One potential ROS-dependent target molecule is the nuclear transcriptional factor nuclear factor-B (NF-
B). It is now known that NF-
B is a key transcription factor involved in regulating proinflammatory cytokines, chemokines, adhesion molecules, and inflammatory enzymes. Blockage of NF-
B, by administration of an NF-
B decoy or use of antisense oligonucleotide treatment, protects ß-cells from the effect of interleukin (IL)-1ßinduced NO production (20,21). Furthermore, it has recently been demonstrated that the native enzyme manganese superoxide dismutase delivered to mouse islets by gene therapy approaches proved beneficial in improving islet cell survival after transplantation (22).
Stress induced during pancreas harvesting and cold storage contributes to lowering the quality of the islets. The additional effect of isolation stress on the generation of islet damage has not been fully investigated. In this study, we focused on the response of islets to isolation stress. We hypothesized that a number of cellular mechanisms occur at this stage that can lead to adverse effects on the final product and negatively influence islet survival. Our aim was to characterize the events that are linked to isolation and possibly identify ways to reverse their consequences. We found that NF-B activation as well as the generation of apoptosis and necrosis, involving the poly(ADP-ribose) polymerase (PARP) pathway (23), occur during pancreatic islet isolation and persist throughout the culture. One interesting observation was that even under standard culture conditions, isolated islets produced and released cytokines and chemokines such as IL-6, IL-8, and macrophage chemoattractant protein 1 (MCP-1), molecules involved in cellular response to stress. The response of islets to stress could be further amplified by applying an in vitro model of chemical stress that reproduces the events occurring during the isolation process, ultimately causing islet dysfunction.
Antioxidative treatment with the antioxidant probe AEOL10150 (MnTDE; manganese [III] 5,10,15,20-tetrakis [1,3,-diethyl-2imidazoyl] manganese-porphyrin pentachoride [TDE-2,5-IP]) effectively reduced the levels of NF-B in treated islets. This phenomenon correlated with reduced production of select cytokines and chemokines and decreased activation of apoptotic and necrotic pathways, allowing for enhanced protection of islets. Our data demonstrated that isolation-induced stress causes islet responses that result in reduced functional capacity. Damage can be prevented, to a large extent, by antioxidant treatment that targets early cellular activation.
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RESEARCH DESIGN AND METHODS |
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Antioxidant treatment.
AEOL10150 (MnTDE; Incara Pharmaceuticals, Research Triangle Park, NC) was resuspended in HBSS at a concentration of 2 mmol/l. MnTDE was added as a supplement to culture or isolation medium at a final concentration of 34 µmol/l. Exposure of islet cells to MnTDE was carried out for a minimum of 30 min. In each experiment, treatment and control conditions were compared within the same donor tissue.
Islet culture.
The islet preparations were cultured in bacteriological Petri dishes at 37°C in an atmosphere of 5% CO2 in humidified air in CMRL-1066 (Gibco-BRL) medium supplemented with 10% FCS, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mmol/l L-glutamine (Life Technologies, Grand Island, NY) (26). The islets were cultured for a maximum of 7 days, with the medium being changed every second day. For studies involving islet survival, insulin release, and cytokine-chemokine production, the islets were hand picked using a dissecting microscope. In summary, groups of 50 hand-picked islets (diameter 150250 µm) were randomly assigned to control and experimental groups. Each batch was subcultured in 35 x 10-mm Falcon dishes at a concentration of 25 islets/ml for 60 h. In the treated groups of islets, MnTDE was added to the medium at a concentration of 34 µmol/l. To determine mass, islets were counted by two independent investigators at the beginning and end of the observation period. Variability of <1% was determined between counts. Samples of culture supernatant were collected and stored at 80°C for basal insulin release and cytokine and chemokine measurements.
Conditioned medium and chemical challenge.
To subject the islets to stress that mimics the chemical effects occurring during digestion, we exposed the islets to conditioned medium as previously described (27). Groups of 100 cell aggregates (islets and acinar tissue) were hand picked and incubated at 37°C with a HBSS solution containing Liberase (1.4 mg/ml) for 1 h (27) in the presence or absence of catalytic antioxidants. Cells were washed with fresh culture medium several times to eliminate the Liberase residues and subsequently cultured for 24 h. The culture supernatant collected 24 h after enzyme exposure was subsequently used as conditioned medium. As control medium for these experiments, 100 aggregates were incubated in HBSS without Liberase.
Hand-picked islets of similar size (150250 µm) were cultured in groups of 50 in CMRL-1066 medium, supplemented with conditioned or control culture medium (ratio 1:1 with fresh, untreated culture medium) with and without MnTDE. After 60 h of culture, the supernatant was collected from all groups, the islets were counted, and cell attachment and other morphological differences were evaluated. Insulin concentration in culture supernatant was measured using an enzyme-linked immunosorbent assay (ALPCO, Windham, NH); cytokines (IL-1ß and -6 and tumor necrosis factor- [TNF-
]) and chemokines (IL-8 and MCP-1) were quantified by Beadlyte Human Multi-Chemokine System 1 and Beadlyte Human Multi-Cytokine Beadmaster kits.
NF-B electrophoretic-mobility shift assay of human islets.
Human pancreatic cells collected during the isolation procedure as well as cultured and conditioned mediumtreated islets (islet-enriched fractions containing 80% islets/whole tissue) were subjected to NF-
B electrophoretic-mobility shift assay (EMSA). For nuclear and cytoplasmic protein extraction, the protocol was adapted from Tse et al. (28). The DNA-binding oligonucleotide and its corresponding complementary strand were used in these studies: 5'-AGTTGAGGGGACTTTCCCAGGC-3' (NF-
B consensus). For supershift analysis, after a 20-min incubation of the oligonucleotide and the nuclear protein extract, 1 µg of antibody against p50 (C-19; Cell Signaling, Beverly, MA) was added and the incubation was continued for an additional 15 min before electrophoresis.
Immunofluorescence and confocal microscopy.
Sections of pancreatic tissue and purified, hand-picked cultured islets were fixed in 2% paraformaldehyde in Histo-Gel (Richard-Allan Scientific, Kalamazoo, MI) and Affigel/Bluegel (BioRad, Hercules, CA) and frozen.
Sections were cut onto gelatin-subbed slides, fixed in 2% paraformaldehyde, rinsed in PBS and 5% BSA, and blocked in 5% nonimmune goat serum for 45 min at room temperature. The primary antibodies used were rabbit anti-human IL-6 (#ab6672; Novus Biologicals, Littleton, CO; dilution 1:2,000), rabbit anti-human IL-8 (#ab7747-500; Novus Biologicals; dilution 1:100), mouse anti-human MCP-1 (#550416, BD Biosciences Pharmingen, San Diego, CA; dilution 1:500), and mouse anti-human insulin (#I-2018; Sigma; dilution1:3,000). Secondary antibodies used were goat anti-rabbit Cy3 (#111-165-144; Jackson ImmunoResearch, West Grove, PA; dilution 1:1,000) for IL-6 and IL-8, goat anti-mouse (#A-11026; Alexa 488; Molecular Probes, Eugene, OR; dilution 1:500) for insulin immunostaining, and goat anti-mouse Cy3-Fab (#115-167-003; Jackson ImmunoResearch; dilution 1:3,000) for MCP-1. Islets were viewed on an Olympus Fluoview BX61 confocal microscope (Melville, NY).
Western immunoblotting assays for poly(ADP-ribose) polymerase.
Snap-frozen pancreatic cells or islet-enriched (80% purity) preparations were lysed in cold radioimmunoprecipitation assay buffer (50 mmol/l Tris-HCl [pH 8.0], 150 mmol/l NaCl, 1.0% nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease and phosphatase inhibitors (1 mmol/l dithiothreitol, 1 mmol/l phenylmethylsulfonyl fluoride, 0.1 mmol/l sodium orthovanate, 1 µg/ml aprotinin, 1 µg/ml leupeptin). Lysed islets were cleared by centrifugation (10,000g, 10 min, 4°C). Protein content of the extract was measured using a bicinchoninic acid assay kit (Pierce Biotechnology, Rockford, IL). Then 40 µg of total protein content were electrophoresed on 15% SDS-PAGE, and the separated proteins were transferred onto an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was blocked with 5.0% nonfat dry milk in Tris-buffered saline with Tween (TBST; 0.15 mol/l NaCl, 10 mmol/l Tris-HCl, and 0.1% Tween 20 at pH 7.4) for 1 h at room temperature and then incubated with anti-PARP (Ab-2) antibodies (Oncogene, San Diego, CA; dilution 1:1,000) in blocking buffer for 2 h at room temperature. After being washed with TBST, the membrane was incubated with peroxidase-linked anti-goat IgG (Amersham-Pharmacia, Buckinghamshire, U.K. dilution 1:25,000) for 2 h. Detection was achieved using an enhanced chemiluminescence kit (Amersham-Pharmacia). Quantitation of Western blots by densitometry was performed with UN-SCAN-IT gel software (Silk Scientific, Orem, UT).
Statistical analysis.
Statistical analysis was carried out using the Students t test. P values < 0.05 were deemed statistically significant.
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RESULTS |
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Western blot analysis of PARP cleavage.
PARP degradation by caspase activity is considered a biochemical marker for apoptosis as well as necrosis (29,30). The same polyclonal antibody recognizes the 116-kDa intact PARP fragment as well as the 85- to 90-kDa proteolytic fragments that characterize apoptosis and minor cleavage products of 3550 kDa that are typical of necrosis. PARP cleavage was evaluated in pancreatic cell extracts during isolation (Fig. 2A) and in purified islets during culture (Fig. 2B), in the presence or absence of MnTDE treatment. The data showed that PARP degradation had already occurred during isolation, indicating that apoptosis and necrosis were ongoing. A comparison of the immunoblots of islets and pancreatic digests showed that a more pronounced cleavage of PARP was found in untreated control islets (those that exhibit higher levels of 85- to 90-kDa as well as 35- to 50-kDa PARP by-products) when compared with MnTDE-treated islets. Quantification of the differences in band density is reported in the bar graphs at the bottom of Fig. 2.
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Figure 3A shows that 1214% of the islets were lost during the 60-h observation period. The addition of antioxidants reduced the loss by half, to
7%. As shown in Fig. 3B, slightly higher, yet statistically significant, insulin concentrations were found in the culture medium of MnTDE-treated islets after the 60-h incubation, in comparison with control islets. The concentrations of insulin were normalized for the total islet number at each condition, which differed in relation to the treatment.
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Response of islets to artificially induced isolation stress.
To assess whether isolation stress is directly involved in the proinflammatory response of isolated islets, we designed an in vitro experiment aimed at mimicking such stress. One of the major events that characterizes the isolation procedure is the exposure of islets to proteolytic enzymes and by-products generated by pancreatic cells undergoing isolation. Conditioned medium was produced by exposing islets and acinar cells to isolation enzymes with and without catalytic antioxidants; after Liberase was washed out, it was used as culture supplement to different groups of islets.
NF-B nuclear translocation.
Figure 4 shows NF-B translocation in isolated islet-enriched fractions treated with conditioned medium made with and without catalytic antioxidants. NF-
B binding to the consensus oligo was lower in islets incubated with conditioned medium made in the presence of MnTDE (Fig. 4, group 6) as compared with conditioned medium generated in the absence of MnTDE (Fig. 4, group 7). Two different islet preparations (from HP104 and HP107) are shown. Again, slightly different patterns were seen in the EMSAs; there were some nonspecific bands, depicted in Fig. 4 as nonspecific shift.
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Microenvironmental cytokines and chemokines.
After islets were incubated with conditioned medium, IL-6, MCP-1, and IL-8 concentrations increased (Fig. 5CE). Because the cytokine and chemokine content of the conditioned medium itself contributed only marginally to the measured concentration (data not shown), the rise was attributed to ex novo production and release. Supplementation of antioxidants resulted in significantly lower IL-6 and MCP-1 concentrations. IL-8 levels, however, as noted previously, peaked irrespective of catalytic antioxidant treatment. IL-1ß and TNF- concentrations were under the detection limit (data not shown).
Immunofluorescence analysis.
The presence of cytokines and chemokines in the culture medium of highly purified islets raised a question as to which cell types were responsible for the synthesis of the proinflammatory molecules. Immunofluorescence techniques were used to stain pancreatic tissue collected before isolation and samples of hand-picked islets with IL-6, IL-8, and MCP-1 antibodies. Figure 6A, D, and G illustrate islets exposed to conditioned medium containing insulin-positive cells as well as cytokine- and chemokine-positive cells. As shown, many MCP-1 and IL-6positive cells in isolated islets were also positive for insulin (Fig. 6A), whereas IL-8 stained nonß-cells. Figure 6 B, E, and H show islets exposed to conditioned medium made in the presence of MnTDE. Multiple sections from three different pancreatic organs were also analyzed before the isolation process was initiated. Figure 6C, F, and I show that insulin-positive cells before isolation did not react with any of the three cytokine- or chemokine-specific antibodies. In the intact pancreas, IL-6 and IL-8positive cells were not islet ß-cells.
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DISCUSSION |
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Other than the direct effects on islets, we have limited information on the modifications of acinar and other pancreatic nonislet cells during the process of isolation. Considering that the islet preparations used for clinical transplantation contain, in addition to islet ß-cells, considerable amounts of contaminant pancreatic nonislet cells, attention should also be devoted to studying the evolution of each cellular component of the islet graft and how different cells interplay in the islet microenvironment after isolation and transplantation.
Recent studies have shown that the stress evoked by isolation increases the expression of cytokine and chemokine genes in human islets (31). It has also been reported in rodent studies that pancreatic acinar cells can be involved in inflammatory responses, because exocrine tissue is capable of producing and releasing cytokines in response to isolation stress (12).
In this study, we provided evidence that cytokines and chemokines are produced and secreted by isolated human islets during culture and, to a higher extent, after elicited stress. The release of proinflammatory molecules in the microenvironment of isolated islets raises justified concerns, considering the wide clinical application of islet transplants, as the same islet ß-cells are known to be very sensitive to the detrimental effects of cytokines (3236). Beyond a direct toxic effect on ß-cells, it has also been reported that IL-6 interacts with a cascade of proinflammatory cytokines to induce hepatic production of serum amyloid-A, one of the major mediators of atherosclerosis (37). Chemokines such as MCP-1 and IL-8 may further jeopardize islet function after transplantation by enhancing recruitment and activation of host macrophages and leukocytes at the implant site. In the context of intraportal islet transplantations, these mechanisms may play a crucial role in the triggering of graft dysfunction and the eventual loss of islets. The clinical relevance of such events has been shown by a recent report demonstrating that human islet cells are able to attract monocytes and macrophages and that MCP-1 plays a relevant role in achieving long-term insulin independence in patients (38).
The role of isolation stress appears to be central in eliciting a proinflammatory response by the isolated islets. Although a new design for less aggressive methods finalized to islet isolation is not yet available, novel approaches specifically aimed at preventing damage that occurs during such procedures should be identified and examined. The efficacy of blocking oxidative stress (and proinflammatory responses) as a way to enhance islet ß-cell survival has been supported by our groups work (13,22) as well as by that of others (11,3942). In particular, our group has shown that islets cultured in the presence of catalytic antioxidants exhibit higher survival after transplantation of marginal islet masses in mice (13). These encouraging results call for more experiments aimed at exploring possible clinical applications.
However, no clear information has been obtained on the mechanisms involved in the generation of the damage and, consequently, on possible ways to infer islet protection. Our data showed that NF-B nuclear translocation occurred in pancreatic cells during the isolation process and was maintained during culture. Moreover, catalytic antioxidants efficiently inhibited NF-
B DNA binding. This became particularly evident when pancreata were split and processed in parallel with and without antioxidants. At the isolation stage, apoptotic and necrotic pathways mediated by PARP activation were also detected, and, analogous to what was observed for NF-
B, were sensitive to protective antioxidant treatment. It is our assumption that blocking apoptotic and necrotic pathways at an early stage may have positive consequences for the long-term survival of the islets. The expression of NF-
B can be considered a triggering factor for the production of proinflammatory cytokines and chemokines. Their induction, in turn, contributes to islet dysfunction by cyclical amplification of the oxidative stress cascade involving more inflammatory mediators, resulting in a larger loss of ß-cell functional mass (17,43). We found that the proinflammatory cytokine IL-6 and the chemokines IL-8 and MCP-1 accumulated in the islet culture medium after isolation and reached higher concentrations after chemical stress. Reduced NF-
B translocation, consequent to antioxidative treatment, was correlated to reduced IL-6 and MCP-1 concentrations in the culture medium. IL-8 secretion, however, was not correlated to NF-
B activation, neither was it reduced by antioxidative treatment, alternatively suggesting that NF-
Bindependent mechanisms not affected by antioxidants regulate its production and secretion, as has also been suggested by others (12).
In this study we also addressed whether islets, and in particular ß-cells, contribute to the production of cytokines and chemokines in the microenvironment. Immunofluorescence staining of highly purified islets with IL-6, IL-8, MCP-1, and insulin-specific antibodies showed that islet nonß-cells reacted with IL-8 antibodies, whereas a number of ß-cells (insulin-positive cells) reacted with IL-6 and MCP-1 antibodies. These data confirmed and further expanded the finding that human islets can express genes for proinflammatory cytokines (31). Our results are therefore suggestive of a possible involvement of the ß-cells themselves in the production of IL-6 and MCP-1, whereas islet nonß-cells appear to be involved in the production of IL-8. However, this conclusion still needs further confirmation. Early blockage of oxidative stress appears to be a key treatment; however, many of the mechanisms linked to subsequent islet response are already amplified during the isolation, suggesting that efforts should be made to prevent their activation up front to achieve higher islet protection.
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ACKNOWLEDGMENTS |
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We are grateful to Amy Sands and Dr. Jing He for their excellent technical work, to Dr. Massimo Pietropaolo for critical review of the manuscript, and to Simon Watkins, PhD, Director of the Center for Biological Imaging at the University of Pittsburgh, for supervision of confocal analysis. We are also grateful to Incara Pharmaceuticals for the generous gift of the catalytic antioxidant AEOL10150.
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FOOTNOTES |
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Address correspondence and reprint requests to Jon D. Piganelli, PhD, Diabetes Institute, Childrens Hospital of Pittsburgh, Rangos Research Center, 3460 Fifth Ave., Pittsburgh, PA 15213. E-mail: jdp51{at}pitt.edu
Received for publication March 19, 2004 and accepted in revised form July 1, 2004
EMSA, electrophoretic-mobility shift assay; HBSS, Hanks balanced salt solution; HP, human pancreatic isolation; HTK, histidine tryptophan ketoglutarate; IL, interleukin; MCP-1, macrophage chemoattractant protein 1; MnTDE, manganese (III) 5,10,15,20-tetrakis (1,3,-diethyl-2imidazoyl) manganese-porphyrin pentachloride (TDE-2,5-IP); NF-B, nuclear factor-
B; PARP, poly(ADP-ribose) polymerase; ROS, reactive oxygen species; TBST, Tris-buffered saline with Tween; TNF-
, tumor necrosis factor-
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REFERENCES |
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