John W. Tawes

Intragraft cytokine expression and tolerance induction in rat renal allografts.

BACKGROUND Intragraft cytokine expression was evaluated in a model of renal transplantation. ACI and Lewis rats were used as donors and recipients, respectively, for heterotopic renal transplantation. METHODS Treated allograft rats (n=10) received a preoperative dose of rapamycin and cyclosporine, followed by 7 days of cyclosporine postoperatively. Isograft rats (n=5) and control allograft rats (n=4) received no immunosuppression. Sacrifice was performed after 120 days. Expression of interleukin (IL)-4, IL-10, and interferon-gamma (IFN-gamma) transcripts was determined with semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS All treated allograft rats had normal function with 50% histologic rejection. All isografts had normal function. IL-4 and IL-10 were in greater density in allografts with normal histology, whereas IFN-gamma was only seen in allografts with cellular rejection. No IL-10 was seen in isografts, but IL-4 was detected in 3/5 isografts. CONCLUSIONS We conclude that the lymphocyte populations elaboration of IL-4 and IL-10 is associated with tolerance, whereas the production of IFN-gamma and absence of IL-4 is associated with histology suggestive of acute cellular rejection.

Analysis of functional renal allograft tolerance with single-dose rapamycin based induction immunosuppression.

The induction of transplantation tolerance is one of the primary goals following solid organ transplantation. The combination of a single dose of rapamycin (RAPA) with a short course of cyclosporine (CsA) has been shown to induce transplantation tolerance in the nonfunctional rat heterotopic cardiac transplant model. The purpose of this study was to assess this effective induction protocol in a functional renal transplant model. Male ACI (RTl(a)) and Lewis (RT1(1)) rats were used as donor and recipients respectively. Allografts received a single RAPA dose of (1.5 mg/kg) combined with CsA (10 mg/kg) 12-14 hr prior to transplantation. CsA (5 mg/kg) was given daily on days +1 - +7. Untreated Lewis to Lewis isografts served as histological controls. Chimerism, assessed in recipient skin, and intragraft interleukin (IL) 10 expression was determined utilizing PCR and RT-PCR techniques respectively. Treated animals and isografts were sacrificed 120-130 days posttransplant for functional and histological evaluation. Allografts (n=9) were functionally tolerant with serum creatinine (0.77+/-0.1 vs. 0.88+/-0.1; P=0.275), blood urea nitrogen (37.6+/-4.6 vs. 23.3+/-1.9; P=0.123), and 24 hr protein excretion (27.0+/-4.4 vs. 17.9+/-5.2; P=0.131) similar to single kidney ACI controls. Histologically, 45% (4/9) allografts were indistinguishable from isografts with no evidence of rejection, and were considered immunologically tolerant. Donor/recipient chimerism was not detected. All immunologically tolerant allografts had evidence of intragraft IL-10 expression. Rejecting allografts and isografts did not express intragraft IL-10. This study confirms the efficacy of pre-engraftment single-dose RAPA combined with CsA in inducing true immunologic tolerance in this stringent functional renal transplant model. The expression of intragraft IL-10 in tolerant recipients suggests a Th-2 shift as the mechanism of tolerance in this model.

Intragraft cytokine expressionf in tolerant rat renal allografts with rapamycin and cyclosporin immunosuppression

The Th‐1/Th‐2 paradigm proposes clonal expansion of Th‐2 lymphocytes as the basis of tolerance towards allografts. Intragraft cytokine expression was evaluated in a highly stringent model of renal transplantation. ACI and Lewis rats were used as donors and recipients, respectively, for heterotopic renal transplantation. Group A (n=8) received a single dose of rapamycin and cyclosporin 12 h prior to engraftment, followed by 7 d of cyclosporin post‐operatively. Isografts (Group B, n=5) and control allografts (Group C, n=4) received no immunosuppression. Sacrifice was performed after 120 d. Intragraft expression of IL‐10, IL‐4, and IFN‐Γ was determined using qualitative reverse transcriptase‐polymerase chain reaction (RT‐PCR). All groups had functionally normal grafts at sacrifice, with 50% histological tolerance among Group A animals. No isografts showed evidence of cellular infiltrate, and all control allografts showed severe rejection. IL‐10 was only detected in the tolerant animals (p<0.001). Similarly, IL‐4 was detected predominantly in the tolerant allografts (p<0.05). IFN‐Γ was only isolated in rejected allografts, whether treated or untreated (p<0.001). We conclude that the expansion of Th‐2 cells is associated with tolerance, while the expansion of Th‐1 cells is associated with acute cellular rejection.

Immunobiology and long‐term graft function in a transplant heterotopic renal rat model

Abstract: Background: The Th1–Th2 paradigm proposes clonal expansion of Th2 lymphocytes as the basis of allograft tolerance. The Th2 cells have been found to be present in recipients with long‐term allograft survival. However, the presence of Th2 cells and tolerance is not a uniform finding. Previously we have shown that pre‐engraftment single dose rapamycin and a 7‐d course of cyclosporin induce transplantation tolerance to 120 d. In the present study, we investigated the immunobiology of grafts in a long‐term follow‐up (>350 d). Methods: Kidney allografts (n = 7), isografts (n = 5) and single nephrectomy (n = 3) groups were followed for 350 ± 87 d. Heterotopic kidney transplant was performed by the same surgeon in the allograft group (ACI‐Lewis) and the isograft group (Lewis‐Lewis). The left kidney was removed in the single nephrectomy group. The allograft group was treated with pre‐engraftment single dose rapamycin and a 7‐d course of cyclosporin. A kidney biopsy was performed at midpoint time for histological study and tissue was frozen for measuring intragraft cytokine expression (IL‐4, IL‐10) in all animals. Prior to biopsy, serum blood urea nitrogen (BUN) and creatinine (Cr) levels were studied. Serum BUN, Cr levels, plus 24‐h urinary protein (PRO) were measured prior to sacrifice. Randomly, four allograft rats received skin grafts (ACI, Lewis and Buffalo skin donors) after kidney biopsy. Skin grafts were studied for a mean of 6 weeks for signs of acceptance or rejection. Analysis of variance (ANOVA) with Tukeys test was used; p < 0.05 was considered statistically significant. Results: The mean follow up was 352 ± 87 d. BUN and Cr levels at biopsy time (mean 214 d) were not statistically different between the three groups (p = 0.19 and p = 0.66). At sacrifice (mean 352 d), BUN, Cr and PRO were statistically different between allograft and isograft groups (p = 0.013), and between allograft and single nephrectomy groups (p = 0.027). Functional and histological signs of graft loss occurred in three of seven (42.8%) of the allografts at 352 d. Using BANFF criteria, three allografts at biopsy time and seven allografts (100%) and four isografts (80%) at sacrifice time developed chronic histologic changes. Intragraft overexpression of IL‐4 and IL‐10 was seen at biopsy and sacrifice time in six of seven allografts and one of five isografts. All donor specific skin grafts (ACI‐Lewis) on allografts were accepted and third party (Buffalo) donor skin grafts were rejected in all animals (>95% skin necrosis). Conclusions: This highly stringent, functional, renal transplant model yields 100% normal renal function as compared with isografts at 120 d follow‐up. With the follow‐up extended to 350 d, 43% of the allografts loose function and develop a chronic allograft histology despite a demonstrated intragraft Th2 cytokine dominance and donor specific skin graft acceptance.