The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 11 5724-5728
Copyright © 2004 by The Endocrine Society
Tissue Factor and CCL2/Monocyte Chemoattractant Protein-1 Released by Human Islets Affect Islet Engraftment in Type 1 Diabetic Recipients
Federico Bertuzzi,
Simona Marzorati,
Paola Maffi,
Lorenzo Piemonti,
Raffaella Melzi,
Francesca de Taddeo,
Veronica Valtolina,
Armando D’Angelo,
Valerio di Carlo,
Ezio Bonifacio and
Antonio Secchi
Department of Medicine (F.B., S.M., P.M., R.M., F.D.T., V.V., A.S.), Unit of Immunology of Diabetes (L.P., E.B.), Coagulation Service and Thrombosis Research Unit (A.D.), and Surgical Department (V.D.C.), Vita-Salute University San Raffaele Scientific Institute, 20132 Milan, Italy
Address all correspondence and requests for reprints to: Federico Bertuzzi, Unit of Cell Therapy for Type 1 Diabetes, S. Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, Italy. E-mail: bertuzzi.federico{at}hsr.it.
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Abstract
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Islet survival in the early posttransplantation period is likely to be influenced by inflammatory events in and around islets. Twenty-seven human islet preparations were transplanted by 24 infusions into 14 patients with brittle type 1 diabetes under the Edmonton protocol. Patients were monitored for their coagulation [cross-linked fibrin degradation products (XDPs)] and liver function test [aspartate and alanine aminotransferase (AST and ALT)] as markers of early posttransplant complications, and these were correlated with in vitro islet number, purification, volume, monocyte-chemoattractant protein-1 (CCL2/MCP-1) and tissue factor (TF) islet release. Consistent with activation of coagulation pathways and hepatic damage, serum XDP values increased early after 11 infusions and transaminase after 13 of 24 infusions. TF and CCL2/MCP-1 were detected in supernatants of 21 and 22 islet preparations, respectively. Serum XDP peak values were correlated with TF/equivalent islets (EI) (r2=0.26, P = 0.001) and CCL2/MCP-1/EI (r2 = 0.42; P < 0.001); serum transaminase areas under the curve in the first week posttransplantation were correlated with CCL2/MCP-1/EI (r2 = 0.55; P < 0.001 for ALT and r2 = 0.51; P = 0.001 for AST) and TF/EI (r2 = 0.31; P = 0.002 for ALT, and r2 = 0.36; P = 0.002 for AST). These data suggest that reducing the islet proinflammatory state may be a means to reduce the early posttransplant complications and perhaps improve islet engraftment.
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Introduction
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ISLET TRANSPLANTATION IS a potential cure for patients with complicated type 1 diabetes; its success rate is progressively improved (1). The interest toward this transplant is principally based on the easy procedures, performed under local anesthesia and repeatable in the same recipient. Few procedure-related complications were reported, mainly consisting in portal vein thrombosis early after the tissue infusion (2, 3, 4) and recently also transiently elevated liver enzymes (2, 5). These phenomena may be considered part of the early postinfusion inflammation at the site of transplant, which threatens islet survival (6, 7) and increases the number of islets required to achieve insulin independence (1). The release of proinflammatory factors by islets may be an important factor in determining the extent of this inflammation. Islets were recently shown to produce and release tissue factor (TF), and it was suggested that this could activate the coagulatory cascade in islet recipients (8). It has also been demonstrated that islets may promote inflammation through their release of chemokines such as monocyte chemoattractant-1 (CCL2/MCP-1), one of the most powerful macrophage-attracting chemokines (9, 10). Here we examine whether the proinflammatory state of the islets as measured by TF and CCL2/MCP-1 release correlates with biochemical parameters of coagulation and liver function post-islet transplant.
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Patients and Methods
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Human islet preparations were obtained after pancreas digestion according to the automated method and transplanted after 6–72 h of culture (10). Aliquots of 1000 equivalent islets (EI) were maintained in standard culture medium for 48 h (1000 IE/ml in CMRL plus 10% fetal calf serum, 1% antibiotics, and 1% glutamine) and characterized for purity, endotoxin content, viability (propidium iodide staining), function (insulin response to glucose by standard static incubation), and TF and CCL2/MCP-1 release. TF and CCL2/MCP-1 release were assayed by ELISA (American Diagnostic, Greenwich, CT, and Pierce Endogen, Rockford, IL, respectively) in 24 of the 27 islet preparations transplanted by 24 infusions into 14 patients. Transplantation was performed between 24 and 72 h after isolation. The islet preparation was suspended in 100 ml of Hanks’ solution (Hanks’ clinical grade, SALF, Bergamo, Italy) with 2% human albumin or transplant media (Mediatech, Herndon, VA) with 20% human albumin containing 35 U/kg recipient weight of heparin. Percutaneous trans-hepatic injection (under local anesthesia) was performed according to the protocol approved by the Local Ethical Committee. The maintenance of intraportal flow for a few minutes after islet infusion was evaluated by portography and ultrasound guidance. Patients were monitored for their coagulation [cross-linked fibrin degradation products (XDPs), prothrombin time, and activated partial thromboplastin time] and liver function test [aspartate and alanine aminotransferase (AST and ALT); total bilirubin and lactate dehydrogenase] 6–10 h after infusion and daily for the first week after the infusion. During the first 10 d after transplant, blood glucose was maintained between 4.4 and 7.0 mM by continuous insulin infusion. During follow-up, exogenous insulin was administered to maintain glycemic values within 4.4–9.9 mM (both in fasting and postprandial condition). In insulin-independent patients, insulin therapy was reintroduced when their glycemic values increased above 9.9 mM 2 d consecutively (11). Six patients were under statin treatment (atorvastatin 20 mg).
The immunosuppression therapy was based on the Edmonton protocol, which included daclizumab, sirolimus, and low-dosage tacrolimus (1). In the first week after transplantation, recipients were monitored for their coagulation, hepatic function, serum tacrolimus and syrolimus levels, and glucose homeostasis (glycemia and C-peptide values).
Data were correlated and compared using linear regression analysis; P < 0.05 was considered statistically significant. The patients gave informed consent for the study.
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Results
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Twenty-seven islet preparations were transplanted by 24 infusions [mean EI per infusion, 319,597 ± 80,274; mean EI/kg recipient body weight (BW) 5,391 ± 1,443] into 14 patients with brittle type 1 diabetes. All preparations were sterile; endotoxin content assessed in supernatants from eight preparations before transplantation were all less than 0.25 EU/ml. Islet TF and CCL2/MCP-1 release was available from 24 of these 27 islet preparations. We confirmed that human islet preparations release TF during 24 h of culture (0.24 ± 0.05 pg/EI, ranging from 0–0.81 pg/EI) and CCL2/MCP-1 (15.5 ± 3.6 pg/EI, ranging from 0.45–53.5 pg/EI). Detectable levels of TF and CCL2/MCP-1 were found in 21 and 22 islet preparations, respectively. A significant association was found between CCL2/MCP-1 and TF released by islets (P = 0.04; r2 = 0.18), as previously described (12). No relationships were observed between TF and CCL2/MCP-1 released by islets with in vitro islet parameters (propidium iodide staining for islet viability and insulin responsiveness to glucose; data not shown).
Procedure-related complications
Among transplanted patients, three of them showed a peritoneal hemorrhage after infusion. Indicative of activation of the coagulatory cascade, serum XDP values increased with a peak within 48 h posttransplant after 11 infusions (eight patients) (Fig. 1A
). No significant differences were observed between XDP changes after the first compared with changes after the second and the third islet infusions. No changes were observed in prothrombin time and activated partial thromboplastin time values. Ultrasound demonstrated a portal thrombosis of a small portal branch in one of these patients but not in the remainder.
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FIG. 1. Posttransplantation XDPs (A) and transaminase (B) (AST, continuous line; ALT, broken line) values in patients with type 1 diabetes that received islet allografts. Values are mean ± SE.
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Serum AST and ALT values increased after 13 infusions (10 patients), suggesting liver dysfunction (Fig. 1B
). These patients also had elevated lactate dehydrogenase levels (data not shown) and no changes in total bilirubin. As previously described (5), significant differences were observed between transaminase changes after the first and after the second infusions [154 U/liter, ranging from 20–703 U/liter AST peak after the first infusion (n = 14), vs. 64 U/liter, ranging from 32–152 U/liter of second infusion (n = 7), P = 0.027; 158 U/liter, ranging from 19–495 U/liter ALT peak after the first infusion, vs. 63 U/liter, ranging from 24–156 U/liter ALT of second infusion, P = 0.027]. Further decreased changes in AST and ALT were observed after the third islet infusion in the same recipients [30 U/liter, ranging from 25–36 U/liter AST peak, and 33 U/liter, ranging from 25–41 U/liter ALT peak (n = 2, few cases for statistical analysis)]. Transaminase changes normalized in all recipients within 2 months after transplantation.
Serum peak XDP and transaminase area under the curve in the first week after transplantation were correlated with each other (r2 = 0.35 and P < 0.001 for ALT; r2 = 0.31 and P = 0.001 for AST).
Pathogenesis of XDP changes
The relationships between islet TF or CCL2/MCP-1 release and serum XDPs were examined for 21 infusions (no CCL2/MCP-1 and TF values were available for three infusions). Significant relationships were observed between serum XDP peak values and TF/EI (r2 = 0.26; P = 0.001) and CCL2/MCP-1/EI (r2 = 0.42; P < 0.001) (Fig. 2). The association remained significant also after the exclusion from the analysis of data of those recipients showing a peritoneal hemorrhage or a portal thrombosis observed by ultrasound examination. A correlation was observed also between XDP peak values and EI (r2 = 0.17; P = 0.043). No correlation was observed between XDPs and purity, the volume of preparations, or serum tacrolimus and syrolimus levels in recipients. Finally, no significant differences were observed in XDP values in patients with or without statin treatment.
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FIG. 2. Relationships between islet TF and CCL2/MCP-1 in vitro release and biochemical parameters of recipients. Significant relationships were observed between TF/EI and serum XDP peak values in the first 24 h after transplantation (A) and serum ALT area under the curve in the first week after transplantation (B). Significant relationships were also found between CCL2/MCP-1/EI and serum XDPs after transplant (C) and serum ALT area under the curve in the first week after transplantation (D).
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Pathogenesis of liver dysfunction
A significant relationship was found between serum transaminase area under the curve in the first week after transplantation and CCL2/MCP-1/EI (r2 = 0.55 and P < 0.001 for ALT; r2 = 0.51 and P = 0.001 for AST; Fig. 2) and TF/EI (r2 = 0.31 and P = 0.002 for ALT; r2 = 0.36 and P = 0.002 for AST; Fig. 2). The association remained significant also after the exclusion from the analysis of data of those recipients showing a peritoneal hemorrhage or a portal thrombosis observed by ultrasound examination. The correlation between CCL2/MCP-1/EI with transaminase remained highly significant, limiting the analyses to the first infusions (r = 0.72 and P = 0.004 for ALT; r = 0.80 and P = 0.001 for AST; n = 14) and nearly significant in the small number of recipients receiving second islet infusion (r = 0.83 and P = 0.042 for ALT; r = 0.77 and P = 0.072 for AST; n = 7).
No correlation was observed between transaminase changes with EI, purity, and volume of preparation or with serum tacrolimus and syrolimus levels in recipients.
In vivo graft function
Seven of 14 patients reached insulin independence (median transplanted EI/kg of recipient BW was 11,921, ranging from 13,950–3,571), another four patients showed a reduction in their insulin requirement to less than 50% (median transplanted EI/kg of recipient BW, 11,573, ranging from 14,647–5,230), and three patients showed detectable C-peptide levels with a reduction in their insulin requirement less than 50% (median transplanted EI/kg of recipient BW, 5,087, ranging from 6,667–3,859). No correlation was observed between graft function as measured by insulin requirement, C-peptide values, and TF and CCL2/MCP-1 released by islets or XDP and transaminase changes.
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Discussion
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The number of clinical trials aimed to explore the feasibility of islet transplantation as a cure of type 1 diabetes mellitus is increasing as a result of the success achieved by the Edmonton group (1). The insulin independence rate has improved, but the procedure is not completely free of complications. Some recipients show evidence of intravascular thrombosis or transient liver dysfunction with transaminase increased 10 times higher than normal range (2). Although these phenomena do not usually affect prognosis, their progression potentially could be fatal (13). The pathogenesis of these complications remains undefined as do strategies for their treatment. The evidence that human islets produced TF has suggested a possible key of interpretation of the thrombosis, even though a direct relationship between in vitro and in vivo parameters was not demonstrated (8).
We observed that in vivo markers of thrombosis and of hepatocellular necrosis directly correlated with TF and CCL2/MCP-1 released in vitro by islets. No relationship was observed with the volume of injected tissue, suggesting that the quality more than the quantity of transplanted tissue may be the determinant for islet engraftment. Our observations support the notion that pancreatic islets are able to produce many biologically active molecules and that these molecules may affect the clinical outcome of islet transplantation. The release of these molecules may be a consequence of an islet proinflammatory state triggered by the clinical condition of donors or by the stress during isolation procedures (10). Their release may also be regulated through similar pathways; CCL2/MCP-1, for example, can induce TF release in human aortic smooth muscle (14).
Changes of XDP and of transaminase values were correlated with each other, with some differences; liver dysfunction appeared delayed and more prolonged compared with XDP changes, and it is reduced in subsequent infusions in the same recipient. The limited transaminase changes after the second infusion might be related to the immunosuppression therapy, as previously suggested; a pretransplant immunosuppressive treatment might protect against hepatocyte injury around the newly transplanted islets (5). Also in islet transplantation recipients under standard immunosuppression therapy for a previous kidney graft, we never observed liver dysfunction (11, 15). Differently from transaminase, XDP increase is not significantly different between the first and the second infusion. An activation of the coagulatory cascade directly related to the transplanted IE per se might contribute to XDP changes independently from the inflammatory process and from the order of infusions.
No correlation was observed between transaminase or XDP changes and graft function. Furthermore, no correlation was observed between graft function and TF and CCL2/MCP-1 released by the islets. We previously reported a correlation between islet CCL2/MCP-1 release and long-term graft function in patients who received islets after kidney transplants under standard therapy with anti-lymphocyte globulin, cyclosporine, and mycophenolate mofetil (10). Several differences may explain the discrepancy. First, in the current series, the majority of patients received more than one islet preparation to reach insulin independence as a primary goal, and this makes it difficult to understand the clinical impact of single infusion. This contrasts with the islet-after-kidney protocol where the majority of patients received single infusions. Second, the immunosuppression therapy differed substantially between the two study cohorts, and some of the early posttransplant complications (i.e. elevations of serum transaminase) also differed under the two therapies.
There are, therefore, many determinants of early post-islet transplant complications, islet engraftment, and ß-cell function. Our observations suggest that the quality of the islets in terms of their proinflammatory state may determine the immediate local inflammatory response. They also suggest that specific therapies that modulate either the proinflammatory state of islets or the inflammatory response may be of clinical value. Control of potentially damaging reactions at the site of transplant should reduce the number of islets required to achieve insulin independence and also the incidence of clinical complications observed early after islet infusion and ultimately make islet transplantation a more attractive clinical option for patients with type 1 diabetes.
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Acknowledgments
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We are grateful to the "Associazione Cellule staminali, oltre il diabete" for having continuously supported our studies.
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Footnotes
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This work was supported by grants from the Juvenile Diabetes Research Foundation (5-2001-172, 1-2000-780, and JT-01) and from Telethon Italy (JT-01).
Abbreviations: ALT, Alanine aminotransferase; AST, aspartate aminotransferase; BW, body weight; CCL2/MCP-1, monocyte chemoattractant protein-1; EI, equivalent islets; TF, tissue factor; XDP, cross-linked fibrin degradation product.
Received April 12, 2004.
Accepted August 16, 2004.
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Abstract
Introduction
