Background. Acute rejection of MHC class II-disparate bm12 skin grafts by C57BL/6 recipient mice is characterized by massive graft infiltration by eosinophils, together with increased intragraft amounts of IL-4 and IL-5 mRNA. IL-5 blockade prevents the intragraft eosinophil infiltration and prolongs the survival of skin allografts. As the differentiation of T cell precursors into Th2 cells is largely driven by IL-4, we investigated the role of IL-4 in MHC class II-disparate allograft rejection. Methods. We performed skin grafts from MHC class II incompatible bm12 mice into wild-type C57BL/6 mice (IL-4+/+) or C57BL/6 IL-4 deficient mice (IL-4−/−). Graft survival, in vitro T cell reactivity, and histology were compared. Results. We observed that 50% of IL-4−/− mice rapidly rejected their bm12 allograft, whereas the other 50% retained their graft 60 days after transplantation. Histological examination of bm12 allografts retained by IL-4−/− mice showed a normal appearance with no inflammatory infiltrate and no eosinophils. Among IL-4−/− mice that acutely rejected their bm12 skin graft, we observed a dense polymorphonuclear infiltrate. The depletion of neutrophils significantly prolonged bm12 graft survival. Conclusions. Eosinophil infiltrates, typical of MHC class II disparate acute skin graft rejection, are critically dependent on the availability of IL-4. IL-4−/− mice reject MHC class II disparate skin grafts by a pathway of rejection where neutrophils play a direct causal role.

Background. The incidence of new-onset posttransplant diabetes mellitus (PTDM) is increased in renal transplant patients treated with tacrolimus.Methods. We retrospectively analyzed fasting plasma glucose and HbA1c levels as well as the dose of glucose-lowering agents in 34 renal transplant patients converted from tacrolimus to cyclosporine (CsA) for PTDM. Diabetes was defined according to current guidelines as repeated fasting plasma glucose (FPG) levels >= 126 mg/dL.Results. At conversion, 11 patients received insulin, 5 received oral agents, and 18 had no glucose-lowering therapy. Fasting plasma glucose levels decreased from 146 64 mg/dL at conversion to 111 +/- 26 mg/dL at 3 months and 104 21 mg/dL at 12 months (P < .001). HbA1c levels decreased from 6.8 +/- 0.8% at conversion to 6.0 +/- 0.6% at 12 months (P = .001). Insulin was stopped in 3, the dose reduced in 7, and remained stable in 1 of the patients. The average daily insulin dose among these patients was reduced from 31 +/- 17 units at conversion to 13 +/- 12 units at 12 months (P < .05). There was no significant change in the number of patients treated with oral glucose-lowering agents. Diabetes reversed (fasting plasma glucose <= 125 mg/dL without glucose-lowering therapy) in 44% (95% confidence interval, 23% to 64%) of patients during the first year after conversion (P < .001). Graft function, blood pressure, and lipid levels remained stable after conversion but the proportion of patients receiving lipid-lowering therapy increased from 18% to 49% (P < .01).Conclusions. Conversion from tacrolimus to CsA for PTDM was associated with a marked improvement in glucose metabolism and frequent reversal of diabetes.

Because rejection of allografts is primarily caused by T and B lymphocyte responses to allogenic histocompatibility molecules, the role of innate immunity in organ transplant rejection is often overlooked. However, the very first damages to vascularized organ allografts are caused by ischemia-reperfusion, an inflammatory reaction involving activation of vascular endothelial cells and release of neutrophil chemoattractants. Herein, we review experimental observations suggesting that the early neutrophil influx in organ transplants favors T cell-mediated rejection.

C57BL/6 mice injected with the 145-2C11 anti-CD3 mAb and grafted with MHC class II disparate bm12 skin develop a chronic rejection characterized by interstitial dermal fibrosis, a marked eosinophil infiltrate, and an obliterative intimal vasculopathy. Because these changes occur in the absence of alloreactive antibodies, we examined the contribution of cytokines in their pathogenesis. Chronically rejected grafts showed a marked accumulation of both IL-4 and IL-5 mRNA. Mixed lymphocyte reaction experiments established that mice undergoing chronic rejection were primed for IL-4, IL-5, and IL-10 secretion. In vivo administration of anti-IL-4 mAb completely prevented allograft vasculopathy as well as graft eosinophil infiltration and dermal fibrosis. Injection of anti-IL-5 mAb or the use of IL-5-deficient mice as recipients also resulted in the lack of eosinophil infiltration or dermal fibrosis, but these mice did develop allograft vasculopathy. Administration of anti-IL-10 mAb did not influence any histologic parameter of chronic rejection. Thus, in this model, IL-4- and IL-5-mediated tissue allograft eosinophil infiltration is associated with interstitial fibrosis. IL-4, but not eosinophils, is also required for the development of obliterative graft arteriolopathy.

142 Minor transplantation antigens are constituted by the association of an MHC class I or class II molecule with a peptide derived from a protein which displays allotypic differences between the donor and the recipient. Previous experiments have shown that β2 microglobulin (β2m) peptides are constitutively present in the MHC class I and II molecules. These antigenic complexes are minor transplantation antigens in mice as indicated by the rejection of β2m-positive grafts by genetically β2m deficient mice. The aim of our study is to investigate the effector pathways responsible for the rejection induced by minor transplantation antigens. We performed skin grafts from wild-type C57BL/6 (β2m+/+) mice on C57BL/6 β2m deficient (β2m−/−) mice. 93% of β2m−/− mice (N=21) rejected their graft within 20 days after transplantation. β2m−/− mice depleted in vivo of CD4 (N=10) or CD8 (N=10) cells by mAb administration did not reject β2m+/+ skins, indicating the requirement for both CD4 and CD8 T cells in this process. CTL assays performed with T cells isolated from lymph nodes draining the rejected grafts showed that β2m−/− mice displayed significant cytotoxicity against β2m+/+ targets. Histological examination of rejected grafts showed tissue necrosis and infiltration by numerous eosinophils (number of eosinophils/0,0025 mm2: 36 vs 2 in syngeneic grafts, P<0,0001). This led us to search for the presence of Interleukin-5, the major eosinophil growth and differentiation factor. In vitro, lymph node T cells from β2m−/− sensitized mice produced large amounts of IL-5 when stimulated with β2m +/+ cells. Furthermore, IL-5 mRNA was detected by RT-PCR within rejected β2m+/+, but not in syngeneic skins. In conclusion, CD4 as well as CD8 T cells are required for the acute rejection induced by β2m-associated minor transplantation antigens. Both CTL activity and an IL-5/eosinophil pathway appear to be involved in the rejection process. Their respective roles are under current investigation.

CD4 T cells play a crucial role in the acute rejection of MHC class II-disparate skin allografts, mainly by Fas/Fas ligand-mediated cytotoxicity. Because recent observations indicate that eosinophils may be found within allografts rejected by CD4 T cells, we evaluated the role played by IL-5, the main eosinophil growth factor, and by eosinophils in the rejection of MHC class II-disparate skin grafts, C57BL/6 mice rapidly rejected MHC class II-disparate bm12 skin grafts. Rejected skins contained a dense, aggressive eosinophil infiltrate. Lymphocytes isolated from lymph nodes draining rejected bm12 skin were primed for IL-5 secretion, and IL-5 mRNA was present within rejected grafts. The IL-5/eosinophil pathway played an effector role in allograft destruction, because the rejection of bm12 skin was significantly delayed in IL-5-deficient mice as compared with wild-type animals. The role of the IL-5/eosinophil pathway was further investigated in MHC class II-disparate donor-recipient strains unable to establish Fas/Fas ligand interactions. Fas ligand-deficient gld/gld mice rejected bm12 skins, and bm12 mice rejected Fas-deficient lpr/lpr C57BL/6 skins, Neutralization of IL-5 prevented acute rejection in both combinations. We conclude that MHC class II-disparate skin allografts trigger an IL-5-dependent infiltration of eosinophils that is sufficient to result in acute graft destruction.

54 Skin grafts from MHC class II antigen disparate bm12 mice are acutely rejected by C57BL/6 mice. Cytotoxic CD4+ cells are known to be responsible for acute rejection in this setting through Fas/Fas-ligand (Fas-L) interactions. We performed skin grafts between bm12 mice and C57BL/6 Fas-L deficient (gld) mice in order to investigate the pattern of rejection in mice lacking CD4 cytotoxicity. The majority of gld mice acutely rejected bm12 grafts with a kinetic similar to that observed in wild-type mice (median survival time, Fas-L deficient vs wild-type mice: 15.6 vs 16.5 days, P=NS). In vitro CTL assays confirmed that CD4+ cells from gld animals, in contrast to wild type mice, could not mount a cytotoxic response against bm12 targets. On histology, bm12 grafts rejected by gld mice displayed a massive infiltrate by eosinophils. This led us to search for Interleukin-5, the major eosinophil growth and differentiation factor, in Fas-L deficient mice with acute rejection. T cells from bm12 sensitized gld mice secreted much higher levels of IL-5 than cells from naive animals in mixed lymphocyte reaction with donor alloantigens (median: 5063 pg/ml vs 572 pg/ml in naive animals, P<0.001). In addition, rejected grafts expressed IL-5 mRNA by RT-PCR, while no signal was present in syngeneic grafts. The effector role of IL-5 and eosinophils in the acute rejection process was demonstrated by a significant increase of skin graft survival in Fas-L deficient mice injected with a neutralizing anti-IL-5 mAb (% graft survival at day 35: 83% vs 27% in mice injected with a control mAb; P=0.027). The IL-5/eosinophil effector pathway also plays a role in the rejection of MHC class II incompatible skin grafts by recipients able to generate a normal CD4 cytotoxic activity. Indeed, the rejection of bm12 skin grafts was significantly delayed by IL-5 KO recipients as compared to wild-type animals (22.1 days vs 12.5 in wild-type recipients, p<0.001). In conclusion: 1) mice lacking alloreactive cytotoxic CD4+ cells reject MHC class II disparate allografts through an IL-5 and eosinophil dependent mechanism; 2) this process also contributes to acute rejection in normal mice.

The first injection of OKT3 in kidney transplant recipients activates the common pathway of coagulation. This may result in early thrombosis of graft vessels. To this day, the cells involved in this phenomenon have not been identified. The aim of this study was to investigate whether circulating monocytes participated in this OKT3-induced coagulopathy. The procoagulant activity (PCA) of circulating monocytes rose from (mean +/- SEM) 0.15 +/- 0.02 mU/mL to 0.40 +/- 0.05 mU/mL at 3 hours (P = .002) and 0.56 +/- 0.21 at 5 hours (P = .045) after the initial OKT3 injection. These monocytes displayed increased tissue factor expression at the same moments (mean flourescence intensity: 14 +/- 2 before OKT3 injection versus 54 +/- 14 at 3 hours, P = .008 and 34 +/- 7 at 5 hours, P = .01). Tissue factor mRNA was detected in blood by reverse transcriptase-polymerase chain reaction as early as 2 hours after OKT3 administration. The circulating monocytes also displayed a steady increase in membrane expression upregulation of ICAM-1, CD29, CD11b, and CD11c. In vitro experiments showed that OKT3 as well as 2 mitogenic, humanized anti-CD3 antibodies potently induced monocytic PCA whereas the 4 nonmitogenic anti-CD3 antibodies tested were over 1,000- fold less potent than OKT3. We conclude that (1) OKT3 induces in vivo tissue factor gene upregulation and membrane expression resulting in increased PCA of circulating monocytes; and (2) nonmitogenic anti-CD3 antibodies seem devoid of significant procoagulant properties.

In our experience the use of OKT3 as prophylaxis in renal transplantation has been associated with an increased incidence of both delayed graft function and thromboses of graft vessels. OKT3 nephrotoxicity might have been favored by restriction of perioperative fluid infusion to prevent pulmonary edema and by the use of very high dose (30 mg/kg) of methylprednisolone (mPDS) before the first OKT3 injection to reduce the release of cytokines. This led us to modify our perioperative management in three ways: (1) hydration status was optimalized; (2) the calcium-channel blocker diltiazem, considered beneficial for recovery of graft function, was administered on the day of transplantation; and (3) the dose of mPDS given before the first OKT3 injection was fixed at 8 mg/kg. Comparison of two consecutive series of patients (group 1, control patients, N = 172; group 2, managed as described above, N = 173) showed that: (1) the incidence of delayed graft function fell from 52% in group 1 to 22% in group 2 (P < 0.0001): (2) the incidence of pulmonary edema was not significantly increased in group 2 (3.5% vs. 1.7% in group 1, P = 0.5); and (3) the frequency of intragraft thrombosis fell from 7.6% in group 1 to 1.2% in group 2 (P = 0.0034). Multivariate analysis showed that the volemia/diltiazem program and avoidance of high mPDS dose were the most important factors responsible for the reduced occurrence of delayed graft function and graft vessels thrombosis, respectively. We conclude that a combined strategy of appropriate dosage of steroids before the first OKT3 injection, administration of a calcium-channel blocker and optimalization of volemia is safe and efficiently prevents against OKT3 nephrotoxic effects.

The use of OKT3 as prophylaxis in renal transplantation carries an increased risk of intragraft thrombosis, which is related to the systemic activation of the coagulation system that consistently occurs after the first dose of OKT3. As only a few patients develop thrombosis after OKT3 therapy, we searched for possible additional risk factor by comparing the demographic and clinical parameters of the 13 patients who developed thrombosis in our institution to those of 218 patients who did not. Multivariate analysis showed a relationship between the dose of methylprednisolone (mPDS) given before the first OKT3 injection and the risk of thrombosis: 6 out of 42 patients (14%) who received high (30 mg/kg) mPDS experienced a thrombotic event, as compared to 7 out of the 189 patients (3.7%) who received < or = 8 mg/kg of mPDS (P < 0.01). This led us to study the effects of mPDS on the procoagulant activity induced by OKT3 on peripheral blood mononuclear cells (PBMC) in vitro. The procoagulant activity of unstimulated PBMC (mean +/- SEM: 0.6 +/- 0.1 mU/ml) reached 3.0 +/- 0.7 mU/ml after OKT3 stimulation (P = 0.0062) and further increased to 7.4 +/- 2.0 mU/ml when PBMC were first preincubated overnight with mPDS before OKT3 stimulation (P = 0.018 as compared to OKT3 alone). This process involved the tissue factor/factor VII pathway, as shown by increased membrane expression of tissue factor on monocytes as well as by a marked reduction of the induced procoagulant activity when the clotting assay was performed with factor VII-deficient plasma.(ABSTRACT TRUNCATED AT 250 WORDS)