Heterogeneity in the Evolution and Mechanisms of the Lesions of Kidney Allograft Rejection in Mice

The natural history and pathogenesis of the pathologic lesions that define rejection of kidney transplants have not been well characterized. We studied the evolution of the pathology of rejection in mouse kidney allografts, using four strain combinations across full major histocompatibility complex (MHC) plus nonMHC disparities, to permit more general conclusions. Interstitial infiltrate, MHC induction, and venulitis appeared by day 5, peaked at day 7-10, then stabilized or regressed by day 21. In contrast, tubulitis, arteritis, and glomerulitis were absent or mild at days 5 and 7, but progressed through day 21, indicating separate regulation and homeostatic control of these lesions. Edema, hemorrhage, and necrosis also increased through day 21. All lesions were T-dependent, failing to develop in T-cell-deficient hosts. Allografts into immunoglobulin-deficient hosts manifested typical infiltration, MHC induction, and tubulitis at days 7 and 21, indicating that these lesions are alloantibody-independent. However at day 21 kidneys rejecting in immunoglobulin-deficient hosts showed decreased edema, arteritis, venulitis, and necrosis. Thus the three groups of lesions are: T-cell-mediated interstitial infiltration, MHC induction, and venulitis, which develops rapidly then stabilizes; slower but progressive T-cell-mediated tubulitis and arteritis; and late antibody-mediated endothelial injury, which contributes to late edema, arteritis, and venulitis.     

IFN-γ is an Absolute Requirement for Spontaneous Acceptance of Liver Allografts

Experimental liver allografts undergo spontaneous acceptance despite undergoing rejection during the first few weeks post transplant. We explored the role of interferon-gamma (IFN-gamma) in the spontaneous acceptance of mouse liver allografts. Strain of mouse (CBA) liver allografts transplanted into normal BALB/c mice developed histologic changes typical of rejection that spontaneously regressed, permitting long-term survival of these allografts similar to that of syngeneic grafts. In contrast, CBA liver allografts in IFN-gamma-deficient hosts manifested not only infiltration but also hemorrhage and necrosis, with no survival beyond 14 days. Despite differences in survival, local expression of cytotoxic T-cell genes in the transplant was not increased in IFN-gamma-deficient hosts, but livers in interferon-gamma-deficient mice (GKO) hosts displayed much less induction of major histocompatibility complex (MHC) class I and II expression. To determine whether the difference in survival was secondary to the direct effects of IFN-gamma on the liver, we transplanted livers from IFN-gamma-receptor-deficient mice into normal hosts. Liver allografts lacking IFN-gamma receptors also developed hemorrhage and necrosis with minimal induction of MHC expression. Thus IFN-gamma mediates a direct effect on rejecting liver allografts that reduces hemorrhage and necrosis, induces MHC expression, and is absolutely required for spontaneous acceptance.     

Lesions of T-Cell-Mediated Kidney Allograft Rejection in Mice Do Not Require Perforin or Granzymes A and B

Organ allograft rejection is strongly associated with the presence of alloreactive cytotoxic T cells but the role of cytotoxicity in the pathologic lesions is unclear. Previous studies showed that the principal lesions of kidney rejection - interstitial infiltration, tubulitis, and endothelial arteritis - are T-cell-dependent and antibody-independent. We studied the role of cytotoxic granule components perforin and granzymes A and B in the evolution of the T-cell-mediated lesions of mouse kidney transplant rejection. By real-time RT-PCR, allografts rejecting in wild-type hosts at days 5, 7, 21, and 42 showed massively elevated and persistent expression of perforin and granzymes A and B, but evolution of tubulitis and arteritis did not correlate with increasing granzyme or perforin expression. Allografts transplanted into hosts with disrupted genes for perforin or granzymes A and B showed no change in tubulitis, arteritis, or MHC induction. Thus the development of the histologic lesions diagnostic of T-cell-mediated kidney transplant rejection are associated with but not mediated by perforin or granzyme A or B. Together with previous graft survival studies, these results indicate that the granule-associated cytotoxic mechanisms of T cells are not the effectors of T-cell-mediated allograft rejection.     

Induction of tolerance to heart transplants by simultaneous cotransplantation of donor kidneys may depend on a radiation-sensitive renal-cell population

BACKGROUND: To determine the mechanism by which cotransplantation of a donor kidney and heart allograft induces tolerance to both organs in miniature swine, we examined the renal elements responsible for tolerance induction. METHODS: Recipients received 12 days of cyclosporine, and transplants were performed across a major histocompatibility complex (MHC) class I mismatch. Group 1 animals received heart transplants (n=5); group 2 animals received heart and kidney allografts with no other manipulation (n=4); group 3 animals received heart transplants and donor-specific renal parenchymal cells (n=4); group 4 animals received heart and kidney allografts from lethally irradiated donors (n=7); group 5 animals received irradiated hearts and nonirradiated kidneys (n=2); group 6 animals received nonirradiated hearts and peripheral blood leukocytes from swine MHC matched to recipients and becoming tolerant to donor antigen (n=2); group 7 animals received nonirradiated hearts and donor-specific peripheral blood monocyte cells (PBMC) (n=2). RESULTS: Animals in group 1 developed vasculopathy and fulminant rejection by day 55. Animals in group 2 never developed vascular lesions. Parenchymal kidney cell infusion (group 3) did not prolong cardiac survival. Animals in group 4 developed arteriopathy by postoperative day (POD) 28. Group 5 recipients accepted allografts without vascular lesions. Adoptive transfer of leukocytes from tolerant swine (group 6) prolonged cardiac graft survival as much as 123 days, whereas donor PBMC infusion (group 7) did not affect cardiac survival or development of arteriopathy. CONCLUSIONS: Radiosensitive elements in kidney allograft may be responsible for tolerance induction and prevention of chronic vascular lesions in recipients of simultaneous heart and kidney allografts.     

Altered intragraft immune responses and improved renal function in MHC class II-deficient mouse kidney allografts

BACKGROUND: During renal allograft rejection, expression of MHC class II antigens is up-regulated on the parenchymal cells of the kidney. This up-regulation of MHC class II proteins may stimulate the intragraft alloimmune response by promoting their recognition by recipient CD4+ T cells. In previous studies, absence of donor MHC class II antigens did not affect skin graft survival, but resulted in prolonged survival of cardiac allografts. METHODS: To further explore the role of MHC class II antigens in kidney graft rejection, we performed vascularized kidney transplants using donor kidneys from A(beta)b-deficient mice that lack MHC class II expression. RESULTS: At 4 weeks after transplant, GFR was substantially depressed in control allografts (2.18+/-0.46 ml/min/kg) compared to nonrejecting isografts (7.98+/-1.62 ml/min/kg; P<0.01), but significantly higher in class II- allografts (4.38+/-0.60 ml/min/kg; P<0.05). Despite the improvement in renal function, class II- allograft demonstrated histologic features of acute rejection, not unlike control allografts. However, morphometric analysis at 1 week after transplantation demonstrated significantly fewer CD4+ T cells infiltrating class II- allografts (12.8+/-1.2 cells/mm2) compared to controls (25.5+/-2.6 cells/mm2; P=0.0007). Finally, the intragraft profile of cytokines was altered in class II- allografts, with significantly reduced expression of Th2 cytokine mRNA compared to controls. CONCLUSIONS: These results support a role of MHC class II antigens in the kidney regulating immune cells within the graft. Further, effector pathways triggered by class II antigens promote renal injury during rejection.     

Cytokine and T cell receptor gene expression at the site of allograft rejection.

Intragraft cytokine and T cell receptor gene expression was analyzed in rejecting renal allografts by polymerase chain reaction (PCR). Message for IL-1 beta, IL-6, and TNF-alpha was detected in nephrectomy tissue with pathological evidence of acute or chronic rejection. Similarly, mRNA for both IL-6 and TNF-alpha was present in renal biopsies from acute rejecting kidneys. IL-2R, IL-4, and IL-5 mRNA was present in both rejecting and rejected kidney allografts, indicating that these cytokines may play a role in ongoing renal allograft rejection. Conversely, IL-2, IL-7, and IFN-gamma message was detected infrequently. In order to address the diversity of T cells in rejecting kidneys, we have analyzed the clonality of the TcR present within the allograft tissue. Rearranged TcR genes were identified in all allografts examined (n = 16) indicating the presence of T cells bearing the alpha/beta TcR. We have determined that there is a heterogeneous infiltration of T cells in the rejected allograft with TcR representing x = 7.47 +/- 2.4 families rearranged in samples obtained from nephrectomies, whereas x = 5.33 +/- 0.58 families were detected in samples obtained from biopsy tissue. These data indicate that (1) cytokines are produced locally which may contribute to graft cell destruction, (2) the heterogeneity of intragraft T cells during kidney allograft rejection may exist because nonspecific lymphocytes have been recruited to the site by locally produced cytokines or because T cells are responding to multiple epitopes or multiple donor antigens. Detection of intragraft cytokines and TcR may prove useful in elucidating the mechanism of rejection and therefore lead to improved immunosuppression.     

Cold ischemia augments allogeneic-mediated injury in rat kidney allografts

BACKGROUND: Some clinical studies demonstrate that kidney grafts with prolonged cold ischemia experience early acute rejection more often than those with minimal ischemia. The mechanism, however, is putative. Therefore, the aim of this study was to unravel the impact of ischemia on the immune response in rat kidney allografts compared with that in isografts. METHODS: To induce ischemic injury, donor kidneys were preserved for 24 hours in 4 degrees C University of Wisconsin solution before transplantation. No immunosuppression was administered. The histomorphology according to the BANFF criteria for acute rejection and infiltrating cells were assessed at days 1, 2, 3, 4, 6, and 8 post-transplantation. RESULTS: In allografts, exposure of the kidney to ischemia led to a significantly earlier onset of interstitial cell infiltration and tubulitis compared with nonischemic allografts. The BANFF score of interstitial cell infiltration was 1 +/- 0 vs. 0.25 +/- 0.29 at day 3 and 2 +/- 0 vs. 1.25 +/- 0.25 at day 4. In contrast, in isografts, the effect of ischemia on the histology was not significant. From day 6, the histologic differences between ischemic and nonischemic grafts disappeared. Ischemia led to a more intense expression of P-selectin (day 1), intercellular adhesion molecule-1 (ICAM-1; day 2), and major histocompatibility complex (MHC) class II on endothelium and proximal tubular cells (day 2) in both allografts and isografts. Concurrently with the up-regulated ICAM-1 and MHC expression, significantly more CD4(+) cells and macrophages infiltrated the ischemic allografts at days 2 and 3 and the ischemic isografts at day 4. Importantly, the influx of these cells after ischemia was significantly greater in allografts than in isografts. CONCLUSIONS: Cold ischemia augments allogeneic-mediated cell infiltration in rat kidney allografts. The earlier onset of acute rejection in 24-hour cold preserved allografts may be prevented by better preservation or treatment using tailored immunosuppression.     

Effects of tolerance induction on the actions of interferon-gamma on porcine cardiac allografts

It is well known that interferon-gamma (IFN-gamma) not only plays a critical role in antigen-dependent but also in antigen-independent tissue injury; however, it is not clear how tolerance induction affects the actions of IFN-gamma in the transplant setting. To address this question, we compared the effects of IFN-gamma on porcine recipients of near-syngeneic, rejecting, and tolerant heart transplants. IFN-gamma was infused continuously into the left anterior descending artery of hearts transplanted into 3 groups of major histocompatibility complex (MHC) inbred miniature swine, each treated with a 12-day course of cyclosporine A (CyA). Group 1 recipients received a MHC class I disparate heart, group 2 recipients received a near-syngeneic heart, and group 3 recipients were cotransplanted with a MHC class I disparate heart and kidney, which uniformly induces tolerance to both grafts. An additional group of animals was not transplanted but received intracoronary IFN-gamma infusion into their native hearts. IFN-gamma perfusion not only accelerated the acute rejection of MHC class I disparate hearts (mean survival time = 19 +/- 7.21 vs 38 +/- 8.19 days, P = .025), but caused near-syngeneic heart transplants, which otherwise survive indefinitely, to reject within 35 days (n = 3). In contrast, IFN-gamma perfusion had no demonstrable effects on interstitial rejection, the development of vascular lesions, or graft survival in tolerant heart plus kidney allograft recipients (n = 4) or in autologous hearts (n = 2). These results suggest that tolerance induction mitigates the damaging effects of IFN-gamma itself and that the beneficial effects of tolerance induction on acute and chronic rejection may extend to antigen-independent factors like ischemia/reperfusion injury.     

Elimination of donor CD47 protects against vascularized allograft rejection in mice

CD47 is a ubiquitously expressed transmembrane glycoprotein that plays a complex role in regulation of cell survival and function. We have previously shown that the interspecies incompatibility of CD47 plays an important role in triggering rejection of cellular xenografts by macrophages. However, the role of CD47 in solid organ transplantation remains undetermined. Here, we explored this question in mouse models of heart allotransplantation. We observed that the lack of CD47 in donor hearts had no deleterious effect on graft survival in syngeneic or single MHC class I‐mismatched recipients, in which both wild‐type (WT) and CD47 knockout (CD47 KO) mouse hearts survived long term with no sign of rejection. Paradoxically, elimination of donor CD47 was beneficial for graft survival in signal MHC class II‐ and class I‐ plus class II‐mismatched combinations, in which CD47 KO donor hearts showed significantly improved survival compared to WT donor hearts. Similarly, CD47 KO donor hearts were more resistant than WT hearts to humoral rejection in α1,3‐galactosyltransferase‐deficient mice. Moreover, a significant prolongation of WT allografts was observed in recipient mice treated with antibodies against a CD47 ligand thrombospondin‐1 (TSP1) or with TSP1 deficiency, indicating that TSP1‐CD47 signaling may stimulate vascularized allograft rejection. Thus, unlike cellular transplantation, donor CD47 expression may accelerate the rejection of vascularized allografts.     

Increased expression of MHC class II antigens in rejecting canine lung allografts

Expression of major histocompatibility complex class II antigens was investigated in the normal lungs and in lung allografts of mongrel dogs after single-lung transplantation. Cryostat sections were stained with an indirect immunoperoxidase technique that used B1F6 and 7.5.10.1 as anti-MHC class II monoclonal antibodies. In the normal lungs and native lungs of the recipient dogs after single-lung transplantation, only some cells of lymphoid tissue and macrophages/dendritic cells were MHC class II-positive. During acute rejection, increased infiltration with MHC class II-positive cells in perivascular, peribronchial, and interstitial areas and intraalveolar spaces was found in lung allografts. In addition, expression of MHC class II antigens was induced on the bronchial epithelium and vascular endothelium. Induced expression of MHC class II antigens on the bronchial epithelium and vascular endothelium in rejecting lung allografts was found as early as two days after single-lung transplantation. The intensity of MHC class II antigen expression on bronchial epithelium and vascular endothelium in graft lungs increased with the progression of rejection response and directly correlated with the bronchoalveolar lavage fluid (BALF) levels of biochemical markers, as tumor necrosis factor alpha, gamma-interferon (IFN-gamma), interleukin 2 (IL-2) and soluble interleukin 2 receptor (SIL-2R). Abnormal expression of MHC class II antigens on bronchial epithelium and vascular endothelium and abnormal elevation of BALF levels of the cytokines in lung allografts could be prevented by cyclosporine (CsA) treatment. Our results suggested that MHC class II antigen expression could be induced on the bronchial epithelium and vascular endothelium of canine lung allografts during acute rejection. This abnormal expression of MHC class II antigens on bronchial epithelium and vascular endothelium of graft lungs may serve as a specific index for diagnosis of lung allograft rejection when infection as an inducing factor can be excluded. Furthermore, bronchial epithelium and vascular endothelium of lung allografts have become MHC class II-positive, and are likely to be the targets for low-grade rejection, resulting in the development of bronchiolitis obliterans and occlusive vascular disease in lung allografts.     

Donor Fas Is Not Necessary for T‐Cell‐Mediated Rejection of Mouse Kidney Allografts

It is important to resolve whether T‐cell‐mediated rejection (TCMR) is mediated by contact‐dependent cytotoxicity or by contact‐independent inflammatory mechanisms. We recently showed that the cytotoxic molecules perforin and granzymes A and B are not required for TCMR of mouse kidney transplants. Nevertheless, TCMR could still be mediated by cytotoxicity via Fas on donor cells engaging Fas ligand on host T cells. We examined whether the diagnostic TCMR lesions would be abrogated if donor Fas was absent, particularly in hosts deficient in perforin or granzymes A and B. Kidneys from Fas‐deficient donors transplanted into major histocompatibility complex (MHC)‐ mismatched hosts developed tubulitis and diffuse interstitial infiltration indistinguishable from wild‐type (WT) allografts, even in hosts deficient in perforin and granzymes A and B. Gene expression analysis revealed similar molecular disturbances in Fas‐deficient and WT allografts at day 21 transplanted into WT, perforin and granzyme A/B‐deficient hosts, indicating epithelial injury and dedifferentiation. Thus, donor Fas is not necessary for TCMR diagnostic lesions or molecular changes, even in the absence of perforin–granzyme mechanisms. We propose that in TCMR, interstitial effector T cells mediate parenchymal injury by inflammatory mechanisms that require neither the perforin–granzyme nor the Fas–Fas ligand cytotoxic mechanisms.     

Chronic antagonism of Mig inhibits cellular infiltration and promotes survival of class II MHC disparate skin allografts

BACKGROUND: The goal of the current study was to test the ability of monokine induced by IFN-gamma (Mig)-specific antibodies to inhibit long-term T cell infiltration into class II major histocompatibility complex (MHC) disparate skin allografts and to test cellular and molecular changes in the graft during the rejection observed following cessation of treatment. METHODS: C57BL/6 recipients of B6.H-2bm12 skin grafts were treated with normal rabbit serum (NRS) or rabbit Mig antiserum (Mig AS) every other day from day 7 until day 21 posttransplant and then weekly thereafter. Allografts were retrieved during the course of treatment and following cessation. Tissue sections were prepared and stained to compare infiltration by macrophages and CD4+ and CD8+ T cells and to assess collagen deposition in the grafts. RNA was prepared and tested by ribonuclease protection assay for intragraft levels of Mig, monocyte chemotactic protein-1 (MCP-1) and regulated on activation normal T-cell expressed and secreted (RANTES). RESULTS: T cell and macrophage infiltration into allografts was inhibited and graft survival maintained as long as Mig-specific antibodies were given. Following cessation of treatment, T cells and macrophages infiltrated the allografts. In contrast to the histology of acute rejection observed in allografts from NRS-treated recipients, the resulting rejection of the allografts from Mig AS-treated recipients was accompanied by dense collagen deposition and high level expression of Mig and RANTES. CONCLUSIONS: Mig directs T cell infiltration into B6.H-2bm12 skin allografts on C57BL/6 recipients. Delayed T cell and macrophage infiltration and rejection of the grafts following cessation of Mig AS treatment results in rejection that is histologically and molecularly distinct from acute rejection.     

A major role for host MHC class I antigen presentation for promoting islet allograft survival

The purpose of this study was to determine the role for CD8 T cells versus generalized MHC class I-restricted antigen presentation in islet allograft rejection and tolerance. Diabetic C57BI/6 (B6, H-2(b)) controls, C57BI/6 CD8-deficient (CD8 KO), or MHC class I-deficient C57BI/6 (beta 2m KO) recipients were grafted with allogeneic BALB/c (H-2(d)) islets. Islet allografts were acutely rejected in untreated B6, CD8 KO, and in beta 2m KO mice, indicating that neither CD8 T cells nor host MHC class I is required for allograft rejection. We then determined the efficacy of costimulation blockade in these same strains. Costimulation blockade with anti-CD154 therapy facilitated long-term islet allograft survival in both B6 and in CD8 KO recipients. However, anti-CD154 treated beta 2m KO recipients were completely refractory to anti-CD154 therapy; all treated animals acutely rejected islet allografts with or without therapy. Also, anti-NK1.1 treatment of wild-type B6 mice abrogated graft prolongation following anti-CD154 therapy. Taken together, results show a dramatic distinction between two forms of MHC class I-restricted pathways in allograft prolongation. Although anti-CD154-induced allograft survival was CD8 T-cell independent, an intact host MHC class I-restricted (beta 2m-dependent) pathway is nevertheless necessary for allograft survival. This pathway required NK1.1+ cells, implicating NK and/or NKT cells in promoting allograft prolongation in vivo.