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Prolonged Islet Allograft Survival in Diabetic Mice Upon Macrophage Depletion by Clodronate-Loaded Erythrocytes

Rossi, Luigia1,6; Migliavacca, Barbara2; Pierigé, Francesca1; Serafini, Sonja1; Sanvito, Francesca3; Olivieri, Stefano3; Nano, Rita4; Antonioli, Barbara4; Magnani, Mauro1; Bertuzzi, Federico4,5

doi: 10.1097/TP.0b013e31816360f3
Brief Reports

Early impairment of islet function and graft loss strongly limit the success of allogenic islet transplantation in insulin-dependent diabetes. Macrophages play a key role in this process thus the depletion of these cells may strongly affect islet survival. In this study, we have evaluated the effect of the depletion of macrophages in mouse allograft rejection using a new approach based on a single infusion of red blood cells loaded with the synthetic analogue of pyrophosphate clodronate. Graft survival was 19.4±0.89 and 20±2 days in the two control groups treated with physiological solution and unloaded erythrocytes, respectively; 25±1.9 days in the group treated with free-clodronate and 35±6 days in the erythrocytes-loaded group. Our results indicate clodronate selectively targeted to the macrophagic cells by a single administration of engineered erythrocytes can significantly prolong islet graft survival and open new therapeutic strategies in islet transplantation.

1 Institute of Biological Chemistry, University of Urbino, Urbino, Italy.

2 The San Raffaele Telethon Institute for Gene Therapy, Milan, Italy.

3 Pathology Unit, San Raffaele Scientific Institute, Milan, Italy.

4 Unit of Cell Therapy for Type 1 Diabetes Mellitus, San Raffaele Scientific Institute, Milan, Italy.

5 Medicine Department, Mediterranean Institute for Transplantation and Advanced Specialized Therapies, Palermo, Italy.

This work was supported by the Fondo per gli Investimenti della Ricerca di Base (RBNE01TBTR) and the AIDS Project of the Italian National Institute of Health.

L.R. and B.M. contributed equally to this work

6 Address correspondence to: Luigia Rossi, Istituto di Chimica Biologica “G. Fornaini”, Università degli Studi di Urbino “Carlo Bo”, Via Saffi, 2–61029 Urbino, Italy.


Received 20 March 2007. Revision requested 20 October 2007.

Accepted 13 November 2007.

Insulin-dependent diabetes mellitus (type 1 diabetes) is a serious chronic disorder caused by the progressive loss of insulin-producing pancreatic β cells. Clinical trials have demonstrated that islet transplantation can result in remarkable improvement in the quality of life of patients (1–4). However, early impairment of islet function and graft loss greatly limit the success of allogenic islet transplantation. Nonspecific inflammatory events occurring at the transplant site immediately after grafting, involving the production of cytokines, free radicals, and sinusoidal endothelial cells activation, may contribute to islet cell damage (5, 6). It is known that macrophages play a key role in this inflammatory response; in fact, islets directly recruit macrophages into the site of transplant by the release of monocyte chemoattractant protein-1 (MCP-1), the most powerful chemoattractant chemokine for macrophages, in the absence of detectable infections or endotoxin contamination (7). As expected, it was shown in preclinical studies that depletion of macrophages represents a valid strategy to prolong survival and functioning of grafts after pancreas islet allo- and xenotransplantation (6, 8, 9).

Alternatively to free clodronate or clodronate loaded in liposomes, we have recently proposed using autologous erythrocytes (red blood cells [RBCs]) to target clodronate selectively to macrophages (10). Erythrocytes, in fact, possess some advantages compared to liposomes: they can be easily obtained in large numbers, are natural components of organisms, have long in vivo life span, and possess a greater ability to retain drugs. Furthermore, the morphological, immunological, and biochemical properties of carrier RBC are similar to those of native cells. In human studies, the use of erythrocytes as a drug delivery system has been extensively investigated in vitro (11, 12) and recently evaluated in vivo (13–16). Moreover, erythrocytes can be used as a drug-targeting system allowing the selective administration of substances to macrophages upon loaded-erythrocyte phagocytosis (17–19). Drug-loaded red cells in fact, can be modified to increase their phagocytosis by inducing band 3 clustering and opsonization by autologous immunoglobulin (Ig) Gs and complement (up to C3b). Macrophages recognize and phagocytize these drug-loaded red cells through the Fc and C3b receptors determining the specificity of the process. On the basis of these considerations, the ability of clodronate-loaded erythrocytes to prolong survival of allogenic islet graft in diabetic mice was evaluated.

C57BL/6 mice (Charles River SpA, Calco, Italy) were used as islet donors and Balb/C mice (Charles River SpA, Calco, Italy) both as blood donors and diabetic recipients. Diabetic animals were obtained through a single intravenous dose injection of streptozotocin (180 mg/Kg Biomol-DBA) and they were considered diabetic when fasting glycemia was >350 mg/dl in two consecutive measurements (normal values are in the 70–130 mg/dl range). Macrophage depletion in diabetic mice was achieved by means of single administrations of clodronate-loaded RBC. Clodronate (kindly provided by Roche Diagnostics GmbH, Mannheim, Germany) was loaded in murine erythrocytes to a final concentration of 7.51±0.3 μmoles/ml packed RBC by a procedure of hypotonic dialysis, isotonic resealing and reannealing, essentially as previously reported (10). Clodronate-loaded erythrocytes were then resuspended at 20% Ht in autologous plasma and 350 μl of suspension (corresponding at about 150 μg of clodronate) was intravenously (i.v.) injected into mice (=12 animals) 2 days before the islet transplantation. Simultaneously, another group of mice (n=12) received 150 μg of clodronate in 350 μl of physiological saline solution. As controls, both diabetic mice (n=12) receiving a suspension of unloaded RBC (i.e., erythrocytes submitted to the loading procedure but without clodronate addition) and diabetic mice (n=12) receiving only a saline physiological solution (because no other relevant reagent was present in solution in which clodronate was resuspended) were used.

Two days later, 300 handpicked islets (as previously described [20]) were transplanted under the kidney capsule and blood glucose levels monitored daily for the first week, then three times per week. All transplanted animals became normoglycemic after transplantation. Three consecutive values of glycemia >250 was the parameter followed to define graft rejection. At different days from transplantation (range 0–60), the percentage of islet graft survival was determined (Fig. 1). As shown, control animals became diabetic 19.4±0.9 days after transplantation, similar to our animal models maintained in specific pathogen free (SPF) facility. Similarly, graft survival was 20±2 days in mice treated with unloaded RBC. A significant increase in islet graft survival was observed in mice treated by free clodronate (25±1.9 days, P<0.001) confirming what was previously reported (18) about the ability of clodronate to decrease macrophagic cells, also when administered as a free drug.



Animals treated by clodronate-loaded RBC further delayed graft rejection (35±6 days, P<0.001 versus control group and P<0.001 versus free clodronate group; analysis of variance [ANOVA], Scheffè posthoc test), confirming again the increased ability of clodronate to deplete macrophages when administered through erythrocytes. One animal in this group remained normoglicemic even 60 days after transplantation. In this animal, the removal of islet graft by nephrectomy was followed by a subsequent increase of glycemic values up to 310 mg/dl, thus excluding a regeneration of native islets. Moreover, since transient alterations of the levels of selected cytokines and inflammatory mediators have been reported to occur both after intrahepatic and under kidney capsule islet transplantation (21, 22), the ability to modify these responses, by means pretreatments of recipients mice with free or loaded clodronate, was evaluated. Serum levels of interleukin (IL)-4 and interferon (INF)-γ were monitored for 1 month starting from 3 days after transplantation. These cytokines are produced by activated macrophages and were proposed as parameters for the inflammatory reaction towards the graft. In particular, IFN-γ is implicated in early graft loss in transplanted islets (23). IL-4 has a protective effect on islets exposed to proinflammatory cytokines and might have a role in graft acceptance (24). Considering the absolute values of IL-4 and INF-γ, data was not significantly different between the experimental groups (data not shown).

The ability of our strategy to deplete macrophages in diabetic mice has also been evaluated. To this end, two groups of diabetic mice (9 mice/group) received 150 μg clodronate, both as free drug and through RBC, 2 days before transplantation. As control, diabetic untreated mice were used. At different times after transplantation (3, 5, 10, and 30 days), 12 mice were randomly sacrificed and spleens harvested for histological examination. Formalin-fixed, paraffin-embedded 5-μm sections from murine spleen were incubated with the rabbit antihuman lysozyme 1:600 (DAKO), after antigen retrieval by water-bath using Tris-ethylenediamine tetraacetic acid of pH 8. Sections were sequentially incubated with biotin-conjugated goat antirabbit antibody and with peroxidase-conjugated streptavidin. The immunoreaction was revealed by horseradish peroxidase, using 3,3′diaminobenzidine as chromogen and the slides were slightly counterstained with Harris' hematoxylin. An additional immunochemistry analyses was performed to confirm data on macrophage staining. Formalin-fixed, paraffin-embedded 5-μm sections from murine spleen were incubated with the rat antimouse F4/80 antigen, which is expressed in a wide range of mature tissue macrophages (AbD Serotec MCA4997GA), after antigen retrieval by pronase 0.05%. Sections were sequentially incubated with biotin-conjugated rabbit antirat antibody (Vector) and with peroxidase-conjugated streptavidin (Jackson Laboratories). The immunoreaction was revealed as above described. Number of macrophages per high-power field was evaluated. Five fields per mouse were examined. Compared to control mice and to clodronate-treated mice, macrophage depletion in the group of clodronate-loaded RBC was more efficient and sustained compared to that by free clodronate (Fig. 2). Number of macrophages/field all together in the first 10 days after transplantation were significantly reduced in the clodronate-loaded RBC (311±66 versus 561±33, P=0.008; ANOVA, Scheffè post-hoc test) and not in free clodronate mice (415±47 versus 561±33, P=0.112).



Macrophage repopulation was complete 30 days after the treatment in both the experimental groups, but it was almost complete 5 days after the free-clodronate treatment group. This is expected because clodronate-loaded RBC are uptaken directly by macrophages. The reduced number of macrophages, particularly those activated, seems to explain the prolonged islet allograft survival in diabetic mice upon treatment with clodronate-loaded erythrocytes by the reduction of the inflammatory reaction towards islets in the site of implant. As expected, clodronate-loaded RBC appears to have simply an anti-inflammatory effect able to delay, but not prevent, islet rejection. Inflammation mediated by macrophages could impair islet survival and function by two different mechanisms: directly exerting toxic effects on transplanted islets or promoting efficient immune recognition of the graft (8). The values of our results consist in the identification of a new safe strategy to improve islet engraftment and to reduce the inflammation in the site of implant. This should also improve the control of rejection but immunosuppressive strategies are to be added.

In conclusion, our results show that clodronate, selectively targeted to the macrophagic cells by a single administration of engineered erythrocytes, is able to significantly prolong islet graft survival. This approach opens new perspectives for the definition of therapeutic strategies for islet allotransplant. In addition, the possibility of using erythrocytes as carrier of other drugs is now matter of further studies. This should be of particular value, especially for immunosuppressants, with the aim of reducing their dosage and concentration and therefore their toxicity.

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Islet transplantation; Inflammation; Clodronate; Macrophages

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