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We examined the production of cytokine message in allogeneic and syngeneic rat pancreatic islet grafts using specific primers and polymerase chain reaction. Freshly isolated islet preparations contained transcripts for interleukin (IL)-1alpha, IL-6, IL-10, and interferon-gamma (IFN-gamma) but not for IL-2. IL-1alpha in allogeneic grafts showed increased and consistently high expression from 1 to 7 days after transplantation, but the level in syngeneic grafts fell quickly to pretransplant levels. IL-2 and IFN-gamma transcripts were found in allogeneic grafts at 1, 3, 5, and 7 days after transplantation with a peak at day 5, but these cytokines were almost absent from syngeneic grafts. The peak of IL-6 expression was 1 day after transplantation in both syngeneic and allogeneic grafts, and then the level fell quickly. IL-10 was produced at approximately the same high level at all time points in both syngeneic and allogeneic grafts. The results show that freshly isolated islet preparations contain IL-1alpha, IL-6, IL-10, and IFN-gamma transcripts at the time of transplantation. The initial production of cytokines in islet grafts, especially IL-1, may explain phenomena such as graft nonfunction, rapid rejection, and lack of response to immunosuppression.

The small bowel has a unique amount of closely associated lymphoid tissue in the form of mesenteric lymph nodes (MLNs) and Peyer's patches (PPs). It is rather unclear how this may affect the immune response to transplants involving small bowel. It is clear, however, that host-derived leukocytes infiltrate this lymphoid tissue very rapidly after transplantation of small bowel, which suggests the possibility of an early immune response within this compartment. To investigate this possibility, we analyzed, using a semiquantitative reverse transcriptase-polymerase chain reaction, the level of cytokine transcripts within isolated MLNs and PPs for the first 7 days after small bowel transplantation. Heterotopic small bowel (n=32) transplants were performed using the following rat strain combinations: syngeneic Lewis (Lew)-->Lew (n=8), blood group D Agouti (DA)-->DA (n=8), allogeneic Lew-->DA (n=8), and allogeneic DA-->Lew (n=8). Two rats from each group were killed at 1, 3, 5, and 7 days after transplantation. RNA was prepared separately from PPs and MLNs before analysis of transcripts for interleukin (IL) 2, IL-4, IL-10, IL-6, IL-1alpha, and interferon (IFN) gamma. No increase in transcripts for IL-2 or IL-10 was observed in either PPs or MLNs of syngeneic grafts. A small rise in IL-6, IL-1alpha, and IFN-gamma transcripts was seen in MLNs and IFN-gamma transcripts in PPs of syngeneic grafts. In contrast, in allografts an extremely early increase in cytokine transcripts was observed; all cytokine transcripts tested were elevated within the first 24 hr after transplantation. Indeed, the peak response of both IL-2 and IL-10 occurred within 1 to 3 days after grafting. This early immune response in the lymphoid tissue may not be controlled by immunosuppression delivered only at the time of transplantation, and therefore may be responsible for the difficulty in achieving adequate immunosuppression in small bowel transplantation.

Background: Pretransplant blood transfusion has a well-known beneficial effect on posttransplant graft survival. Recently, it has been proposed that the clinical benefit of transfusion is due to HLA-DR antigen sharing between the blood donor(s) and the recipient. Immunological studies have suggested that this might result from a functional deletion of donor-reactive cytotoxic T lymphocytes., Methods: We investigated frequencies of alloreactive lymphocyte precursors with cytotoxic or interleukin-2-producing helper function by limiting dilution analysis in 10 renal dialysis patients before and after transfusion with fresh, allogeneic whole blood. Five patients received blood transfusions from donors matched for one HLA haplotype (or one HLA-B-DR antigen) and the other five patients received blood from fully HLA-mismatched donors., Results: Contrary to some previous reports, frequency analysis of cytotoxic T lymphocyte precursors revealed no significant differences between the two treatment groups in terms of development of blood donor-specific hyporesponsiveness after transfusion. Split-well analysis of cytotoxic T lymphocyte precursors reactive with single-mismatched HLA antigens demonstrated that the effects of transfusion on alloreactive specificity are complex and may vary depending on the particular antigens mismatched between the recipient and blood donor. Analysis of donor-specific helper T lymphocyte precursor frequencies revealed a significant decrease of interleukin-2-producing cells 3 months after transfusion in the total patient population. This effect was most prominent in the recipients of HLA-mismatched blood, but it also exhibited some degree of nonspecificity, as frequencies of third-party reactive helper T lymphocyte precursors were also significantly reduced., Conclusions: Our overall results suggest that the degree of HLA matching between blood donor and recipient does not greatly influence the effect of blood transfusion on the T lymphocyte allorepertoire. The apparent induced down-regulation of helper T lymphocyte activity may play a role in the reported immunosuppressive effects of allogeneic blood transfusion.

Understanding of the events preceding acute cellular rejection of kidney transplants would be useful in the development of immunosuppressive strategies to prevent rejection. Information about these events in humans has been scarce, because of the lack of early, serial, biopsy samples. We took daily fine needle aspirates from kidney allografts for the first 10 days after transplant. Samples were analyzed by morphological cytology of graft-infiltrating cells, and reverse transcriptase-polymerase chain reaction for detection of interleukin (IL)-2, IL-4, IL-6, IL-10, and gamma-interferon gene expression. During the first 4 days, all of the grafts developed a low-grade monocyte-rich mononuclear cell infiltrate, accompanied by IL-10 gene expression. Thereafter, the infiltrates either remained stable or intensified. Of the 13 grafts with dense infiltrates, seven developed graft dysfunction. The remaining six did not, despite significant interstitial infiltrates. Both rejecting and nonrejecting dense infiltrates were associated with a biphasic pattern of IL-2 and gamma-interferon gene expression, preceding and accompanying lymphocytic graft infiltration. Grafts that did not develop dense infiltrates had no detectable IL-2 or gamma-interferon gene expression and did not suffer cellular rejection during the study period. The development of both rejecting and nonrejecting infiltrates was strongly associated with DR mismatches between donor and recipient. IL-2 and gamma-interferon gene expression are necessary, but not sufficient, for the development of acute cellular rejection in the first 10 days of kidney transplantation, and are more closely associated with the period leading up to rejection than with the period of graft dysfunction.

Acute cell mediated graft rejection is frequently associated with an immune response dominated by cytokines like IL-2 and IFNgamma. While small bowel grafts are rejected acutely, there is little information on the type of immune response generated following transplantation and, in particular, whether the cytokine profile resembles that seen during the rejection of other solid organ grafts. In this paper we compare the expression of cytokines in isolated gut tissue following experimental small bowel transplantation with that in heart grafts. Heterotopic small bowel (n=32) and cardiac (n=32) transplants were performed using the following rat strain combinations: syngeneic Lewis (Lew) > Lew (n=8), blood group D Agouti (DA) > Lew (n=8) and allogeneic Lew > DA (n=8), DA > Lew (n=8). Two rats from each group were sacrificed at 1, 3, 5, or 7 days after transplantation. RNA was prepared separately from gut wall, after removing the Peyer's patches (PPs) and mesenteric lymph nodes (MLNs) and from heart. Cytokine (IL-1alpha, IL-2, IL-4, IL-6, IL-10 and IFNgamma) transcripts were analyzed using semiquantitative RT-PCR. Most notably, transcripts of only a single cytokine, IFNgamma, became progressively elevated with time in the rejecting small bowel grafts. This is in marked contrast to the findings presented here for rat cardiac grafts in which transcripts of all cytokines tested show an increase with rejection. This significant and steady increase in IFNgamma expression occurred before there was any clinical or histological evidence of rejection. These data demonstrate that the mechanisms of rejection in small bowel and other solid organ grafts are likely to be different. Further, the unique rise in IFNgamma expression in the gut wall may be a valuable and early indicator of graft rejection.

In a model of arterialized rat liver transplantation, the biological indicators of liver dysfunction and the phenotype and in vitro function of graft-infiltrating cells have been compared in rejected (DA-to-Lew) and spontaneously accepted (Lew-to-DA) grafts, 2-8 days after grafting. Recipients of rejected and nonrejected allografts had, during this time, similar loss in body weight and plasma levels of transaminase. The markers of cholestasis, however, increased from days 3 and 4 onward in the recipients of rejected grafts, but remained low and similar in the recipients of nonrejected allografts and those of syngeneic grafts. From days 2 to 6 the phenotype, IL-2 responsiveness, and donor-specific cytotoxic potential of the leukocytes infiltrating rejected and nonrejected allografts were comparable. On days 7 and 8, although the proportion of T cell subpopulations was identical in both combinations, activated CD4+ graft-infiltrating cells were reduced in the nonrejected grafts. Also at this time, donor-specific cytotoxic cells were no longer detected in nonrejected grafts, whereas activity had reached a peak in the rejected grafts. These results suggest that liver grafts in both the LEW-->DA (grafts not rejected) and DA-->LEW (grafts rejected) strain combinations undergo tissue damage, but that the type of damage differs between the two combinations. Specifically, cholestasis was only observed in grafts that would subsequently be rejected. The difference in graft damage occurred at a very early time point (3 or 4 days after grafting), at which time neither the intensity nor phenotype of the graft infiltrate, its IL-2 responsiveness, or its cytotoxic potential varied between the two combinations. Thus, a lack of immune reactivity, as assessed by these parameters, does appear not to be responsible for spontaneous acceptance of liver transplants in the LEW-->DA strain combination.

The induction of donor major histocompatibility complex (MHC) antigens on nonrejected and rejected rat renal allografts was compared at various times after transplantation in two strain combinations, DA-to-PVG and LEW-to-DA. Graft rejection was prevented by preoperative donor-specific blood transfusion (DST). Quantitative absorption analysis and immunohistology were performed using monoclonal antibodies specific for donor class I and class II MHC antigens. A significant increase in the expression of donor MHC antigens, both class I and class II, was demonstrated on nonrejected as well as rejected kidneys after transplantation. A kinetic analysis showed that induction of donor class I antigens was accelerated on the nonrejected grafts, and by day 5 the nonrejected kidneys showed increased expression of class I antigen when compared with the rejected grafts (a 37- vs. a 25-fold increase in expression). Increased expression of donor class I antigens persisted on the nonrejected grafts and was still detectable on long-term-surviving kidneys, 50 days after transplantation. The magnitude of class II antigen induction was similar on both rejected and nonrejected grafts (8-fold by 5 days after transplantation). Immunohistology demonstrated that class I and class II antigens were induced on identical structures in the kidney in both situations. In particular the vessel endothelia, which do not express class II antigens in normal kidney, become strongly positive in both rejected and nonrejected grafts 5 days after transplantation. Although renal allograft rejection is completely suppressed in rats given a single donor-specific blood transfusion before transplantation, graft survival cannot be explained by the lack of induction of donor MHC antigens. Donor MHC antigens are induced on these nonrejected kidney grafts, and therefore they could act as target molecules for the effector cells that mediate graft destruction. Thus the induction of donor MHC antigens on tissue allografts should not be considered as indicative of a rejection response resulting in graft destruction.