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 ) 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.
1. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation
in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med
2000; 343: 230.
2. Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation
. N Engl J Med
2006; 355: 1318.
3. Poggioli R, Faradji RN, Ponte G, et al. Quality of life after islet transplantation
. Am J Transplant
2006; 6: 371.
4. Toso C, Shapiro AM, Bowker S, et al. Quality of life after islet transplant: Impact of the number of islet infusions and metabolic outcome. Transplantation
2007; 84: 664.
5. Kaufman DB, Platt JL, Rabe FL, et al. Differential roles of Mac-1+ cells, and CD4+ and CD8+ T lymphocytes in primary nonfunction and classic rejection of islet allografts. J Exp Med
1990; 172: 291.
6. Bottino R, Fernandez LA, Ricordi C, et al. Transplantation of allogeneic islets of Langerhans in the rat liver. Effects of macrophage depletion on graft survival and microenvironment activation. Diabetes
1998; 47: 316.
7. Piemonti L, Leone BE, Nano R, et al. Human pancreatic islets produce and secrete MCP-1: Relevance in human islet transplantation
2002; 51: 55.
8. Andres A, Toso C, Morel P, et al. Macrophage depletion prolongs discordant but not concordant islet xenograft survival. Transplantation
2005; 79: 543.
9. Omer A, Keegan M, Czismadia E, et al. Macrophage depletion improves survival of porcine neonatal pancreatic cell clusters contained in alginate macrocapsules transplanted into rats. Xenotransplantation
2003; 10: 240.
10. Rossi L, Serafini S, Antonelli A, et al. Macrophage depletion induced by clodronate
-loaded erythrocytes. J Drug Target
2005; 13: 99.
11. Rossi L, Brandi G, Schiavano GF, et al. Heterodimer-loaded erythrocytes as bioreactors for slow delivery of the antiviral drug azidothymidine and the antimycobacterial drug ethambutol. AIDS Res Hum Retroviruses
1999; 15: 345.
12. Fraternale A, Rossi L, Magnani M. Encapsulation, metabolism and release of 2-fluoro-ara-AMP from human erythrocytes. Biochim Biophys Acta
1996; 1291: 149.
13. Rossi L, Castro M, D'Orio F, et al. Low doses of dexamethasone constantly delivered by autologous erythrocytes slow the progression of lung disease in cystic fibrosis patients. Blood Cell Mol Dis
2004a; 33: 57.
14. Annese V, Latiano A, Rossi L, et al. Erythrocytes-mediated delivery of dexamethasone in steroid-dependent IBD patients-a pilot uncontrolled study. Am J Gastroenterol
2005; 100: 1370.
15. Magnani M, Rossi L, Fraternale A, et al. Erythrocyte-mediated delivery of drugs, peptides and modified oligonucleotides. Gene Ther
2002a; 9: 749.
16. Rossi L, Serafini S, Cenerini L, et al. Erythrocyte-mediated delivery of dexamethasone in patients with chronic obstructive pulmonary disease. Biotechnol Appl Biochem
2001a; 33: 85.
17. Magnani M, Rossi L, Brandi G, et al. Targeting antiretroviral nucleoside analogues in phosphorylated form to macrophages
: In vitro
and in vivo
studies. Proc Natl Acad Sci
1992; 89: 6477.
18. Magnani M, Rossi L, Fraternale A, et al. Targeting antiviral nucleotide analogues to macrophages
. J Leukocyte Biol
1997; 62: 133.
19. Rossi L, Serafini S, Cappellacci L, et al. Erythrocyte-mediated delivery of a new homodinucleotide active against human immunodeficiency virus and herpes simplex virus. J Antimicrob Chemother
2001b; 47: 819.
20. Melzi R, Battaglia M, Draghici E, et al. Relevance of the hyperglycemia on the timing of functional loss of allogeneic islet transplants: Implication for mouse model. Transplantation
2007; 83: 167.
21. Ozasa T, Newton MR, Dallman MJ, et al. Cytokine gene expression in pancreatic islet grafts in the rat. Transplantation
1997; 64: 1152.
22. Molano RD, Pileggi A, Berney T, et al. Prolonged islet allograft survival in diabetic NOD mice by targeting CD45RB and CD154. Diabetes
2003; 52: 957.
23. Yasunami Y, Kojo S, Kitamura H, et al. Vα14 NKT cell-triggered IFN- γ production by Gr-1+CD11b+ cells mediates early graft loss of syngeneic transplanted islets. J Exp Med
2005; 202: 913.
24. Marselli L, Dotta F, Piro S, et al. Th2 cytokines have a partial, direct protective effect on the function and survival of isolated human islets exposed to combined proinflammatory and Th1 cytokines. J Clin Endocrinol Metab
2001; 86: 4974.