The contribution of terminal complement components to acute and hyperacute allograft rejection in the rat

Acute rejection and antibody-mediated hyperacute allograft rejection are affected by activation of the complement cascade. Split products of early complement components influence the localization, activation, and effector functions of platelets, granulocytes, monocytes, and lymphocytes, while the formation of membrane attack complex (C5b-C9) can lead to rapid cell destruction. Therefore, we compared acute and Ab-mediated hyperacute allograft rejection in a recently described model of C6 deficient PVG (C-) (RT1c) rats and their normal counterpart PVG (C+) (RT1c) rats. Cardiac allografts from fully MHC disparate ACI donors were heterotopically grafted into naive and skin graft sensitized PVG (C-) and PVG (C+) rats. ACI cardiac allografts were rejected acutely (8.3 +/- 2 days; n = 7) by naive PVG (C+) recipients, but survived significantly longer in PVG (C-) recipients (22 +/- 10 days; n = 10). Presensitized PVG (C+) rats rejected ACI cardiac allografts hyperacutely in 6.1 +/- 2.4 hr (n = 5). In contrast, ACI cardiac allografts transplanted into presensitized (PVG (C-) rats had markedly longer survival of 91 +/- 14 hr (n = 5). The alloantibody responses of naive PVG (C+) and PVG (C-) recipients 7 days after cardiac allografting, and of presensitized PVG (C+) and PVG (C-) recipients at time of cardiac allografting were not significantly different as measured by flow cytometry against ACI lymphocytes. Immunofluorescence demonstrated deposition of IgM, IgG and C3 in ACI allografts in PVG (C-) as well as in PVG (C+) recipients. Deposition of C6 was only found in grafts rejected by PVG (C+). The significantly longer survival of ACI cardiac allografts in C6-deficient PVG (C-) rats indicates that the membrane attack complex contributes to acute as well as antibody-mediated hyperacute allograft rejection.     

Flow cytometric analysis of proliferative activity of pleomorphic adenoma of salivary gland

BACKGROUND: In a previous study, it was shown that a spontaneously tolerated DA (RT1a) liver allograft in a PVG (RT1c) recipient was able to induce tolerance of a DA small bowel graft performed 17 days later in spite of infiltration of the intestinal grafts by mononuclear cells. AIMS: To compare the phenotype of graft infiltrating cells in rejecting and tolerated small bowel grafts in order to elucidate the mechanism(s) which block the graft infiltrating cells from mediating rejection. METHODS: Multiparameter immunofluorescence was used to compare the phenotype and state of activation of donor and recipient cells isolated from intestinal grafts rejected or tolerated after liver transplantation. RESULTS: Three differences were found. Firstly, there was a more rapid replacement of lamina propria (LP) cells by recipient lymphocytes in tolerated than in rejected grafts. Secondly, the proportion of LP recipient CD8alphabeta+ lymphocytes bearing the high affinity receptor for interleukin 2 was significantly less in tolerated grafts (1.1%, range 0-2%) than in rejected grafts (21.3%, range 9-26%). Finally, tolerated grafts contained significantly less NK lymphocytes (NKR-P1+) and macrophages than rejected intestinal allografts. CONCLUSIONS: These observations make it possible to delineate clear cut differences in the phenotype of cells infiltrating rejecting versus tolerated grafts. Furthermore, the data suggest that liver transplantation induces tolerance of intestinal grafts by hampering the activation of recipient TcRalphabeta+ CD8alphabeta+ T cells and subsequently the recruitment of non-specific effector cells.     

Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection

In a rat model of corneal transplantation, Fischer 344 (RT1(lv1)) rats received orthotopic corneal isografts or Wistar-Furth (RT1(u)) donor allografts. Rejection was observed in 25 of 26 allograft recipients, at a median time of 18 days, with all isografts surviving > 100 days. Flow cytometric analysis of aqueous humour identified cellular infiltration of the aqueous at the time of allograft rejection, in contrast to the acellular aqueous found in isografts at corresponding times following transplantation. A higher proportion of CD8+ than CD4+ cells was found at days 1-3 following rejection, whereas there was a higher proportion of CD4+ cells at days 5-8. No changes in peripheral blood T cell subsets were found at the time of rejection. Immunohistochemical analysis of cells infiltrating recipient iris and grafted cornea undertaken at days 1-2, 4 and 7-10 following onset of rejection, demonstrated inflammatory cells in the graft epithelium, stroma and aggregated on the endothelium. Large numbers of macrophages, T cells (CD4+ > CD8+ at all time points), natural killer (NK) cells and neutrophils were detected in graft tissue at days 1-2 and 4, diminishing after that time. Most infiltrating cells expressed MHC class II antigen, and a smaller number expressed IL-2R. Expression of the co-stimulatory marker B7 was identified in a few cells at day 4 in the region of the graft-host wound. The immune response in graft rejection was characterized at day 4 also by expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells of iris and corneal vessels, demonstration of interferon-gamma on mononuclear cells in the peripheral (recipient) cornea, and tumour necrosis factor-alpha on aggregated mononuclear cells on the graft, but not recipient, endothelium. Only sparse cellular infiltrates were found in isograft controls, with inflammation located at the graft-host wound. These findings suggest that inflammatory cells reach a corneal allograft by two routes--from vessels in the peripheral recipient cornea, and from vessels in the recipient iris via the aqueous humour. Different aqueous and intragraft T cell subset proportions were seen early in rejection, although a preponderance of CD4+ cells was found in both aqueous and graft at later times.     

RT1-U: identification of a novel, active, class Ib alloantigen of the rat MHC

In common with other mammalian species, the laboratory rat (Rattus norvegicus) expresses MHC class I molecules that have been categorized as either classical (class Ia) or nonclassical (class Ib). This distinction separates the class Ia molecules that play a conventional role in peptide Ag presentation to CD8 T cells from the others, whose function is unconventional or undefined. The class Ia molecules are encoded by the RT1-A region of the rat MHC, while the RT1-C/E/M region encodes up to 60 other class I genes or gene fragments, a number of which are known to be expressed (or to be expressible). Here we report upon novel MHC class Ib genes of the rat that we have expression cloned using new monoclonal alloantibodies and which we term RT1-U. The products detected by these Abs were readily identifiable by two-dimensional analysis of immunoprecipitates and were shown to be distinct from the class Ia products. Cellular studies of these molecules indicate that they function efficiently as targets for cytotoxic killing by appropriately raised polyclonal alloreactive CTL populations. The sequences of these class Ib genes group together in phylogenetic analysis, suggesting a unique locus or family. The combined serological, CTL, and sequence data all indicate that these products are genetically polymorphic.     

Induction of allograft nonresponsiveness after intrathymic inoculation with donor class I allopeptides. I. Correlation of graft survival with antidonor IgG antibody subclasses

We have recently demonstrated that cardiac allograft rejection in the PVG.R8-to-PVG.1U rat strain combination involves the recognition of a isolated class I (RT1.Aa) molecules as peptides in the context of the recipient MHC molecules. Three synthetic peptides (P1, P2, and P3) corresponding to the alpha-helices of the RT1.Aa molecule served as T-cell epitopes for graft rejection. In this study, we demonstrate that two of these peptides (P2 and P3) are sufficient to induce immune nonresponsiveness (median survival time >237 days) to cardiac allografts when presented to the recipient immune system in the thymus 7 days before transplantation. This effect was time dependent, as intrathymic inoculation 60 days before transplantation did not prolong graft survival (median survival time=12 days). Previous studies have demonstrated a critical role for alloantibody responses in mediating graft rejection in this rat strain combination. We, therefore, studied the role alloantibody responses may play in the observed immune nonresponsiveness. The titers of alloantibody in serum samples harvested from graft recipients at different times after transplantation were measured. We used recipient primary aortic endothelial cells genetically manipulated to express the donor RT1.Aa molecule as targets in an enzyme-linked immunosorbent assay. High titers of anti-RT1.Aa IgM antibody were detected in unmanipulated controls at the time of graft rejection. The IgM antibody switched to high IgG titers in intrathymically inoculated rats with accelerated or delayed rejection. Graft rejection in intrathymically manipulated recipients that had achieved a transient state of immunological nonresponsiveness correlated with higher titers of the IgG2b alloantibody. In marked contrast, the long-term graft survivors expressed undetectable or low levels of the IgG2b antibody and moderate to high levels of the IgG1 and IgG2a subclasses. These data suggest that the IgG2b alloantibody may contribute to the rejection reaction, whereas IgG1 and IgG2a may be involved in active enhancement of graft survival.     

On histocompatibility barriers, Th1 to Th2 immune deviation, and the nature of the allograft responses

In the present study, we have sought to determine the basis for the frequent failure of Th1 to Th2 immune deviation to blunt the severity of allograft rejection, as such immune deviation has proven highly effective in the treatment of several T cell-dependent autoimmune states. Our study demonstrates that treating islet allograft recipient mice with anti-IL-12 mAb is highly effective in producing Th1 to Th2 immune deviation in several model systems (i.e., fully MHC, partially MHC, or multiple minor Ag barriers). Nevertheless, anti-IL-12 failed to prolong the engraftment of fully MHC-mismatched islet allografts. However, anti-IL-12-treated recipients carrying MHC-matched but multiple minor Ag-mismatched allografts experienced prolonged engraftment; allograft tolerance was frequently achieved in the DBA/2J (H-2d) to BALB/c (H-2d) strain combination. In another model, in which the host response was dominated by CD4+ T cells responding to donor allopeptides presented upon host APCs in the context of self MHC class II molecules, anti-IL-12 treatment proved to be extremely potent. Thus, Th1 to Th2 immune deviation produces prolonged engraftment as compared with recipients of MHC-mismatched allografts when rejection is dependent upon indirectly presented allogeneic peptides and a reduced mass of responding alloreactive T cells.     

Transient contralateral hypoglossal nerve palsy following third molar surgery under day-case general anaesthesia: a case report and review of the literature

We have noticed that bone marrow transplanted in a vascularized limb graft providing a continuous supply of donor BMC may prolong the survival time of skin graft from the same donor. The question arises whether the raised microchimerism plays a role in the prolonged survival of skin allograft. The aim of the study was to follow the development of microchimerism after allogeneic vascularized bone marrow transplantation (VBMTx) concomitantly with the rejection processes of transplanted skin. The BN rats served as donors and LEW rats as recipients of VBMTx and free skin flap allograft. Hind limb was transplanted followed by a full-thickness skin graft on the dorsum. Cellular microchimerism was investigated in recipients of VBMTx and skin grafts in blood, spleen, mesenteric lymph node and bone marrow with monoclonal antibody OX27 directed against MHC class I polymorthic RTI on BN cells and quantitatively analysed in FACStar. In VBMTx group free skin flap survived 70 days after weaning of CsA. Intravenous infusion of BMC in suspension equivalent to that grafted in hind limb did not prolong skin graft survival after cessation of CsA therapy. Donor-derived cells could be detected in VBMTx recipients as long 70 days after wearing of CsA but not in recipients of i.v. suspension BMC grafting.     

Renal allograft rejection: protection of renal epithelium from natural killer cells by cytokine-induced up-regulation of class I major histocompatibility antigens

The potential of natural killer (NK) cells to contribute to renal allograft rejection was modelled by mixing NK cells with cultured renal epithelial cells. It was found that the renal cells were readily lysed by cytokine-activated NK cells. Renal cells which were previously stimulated by culture with either interferon-gamma (IFN-gamma) or supernatant from mixed leucocyte cultures (MLC) were relatively resistant to such lysis; stimulation with tumour necrosis factor-alpha (TNF-alpha) had no effect. None of these cytokine preparations had any effect on the lysis of renal cells by either specific cytotoxic T lymphocytes or the antibody-dependent cell-mediated cytotoxic mechanism. The expression of class I major histocompatibility complex (MHC) antigens was up-regulated by stimulation of renal cells with either IFN-gamma or MLC supernatant; treatment with TNF-alpha had no effect on the expression of these antigens. Protection from NK cell-mediated lysis appeared to correlate with the expression of class I MHC antigens by the renal cells. Artificial removal of these MHC antigens by treatment with citric acid significantly increased the susceptibility of cytokine-stimulated renal cells to lysis by activated NK cells. This increase was not caused by enhanced binding of NK cells to acid-treated renal cell targets. These results suggest that high levels of class I MHC antigen expression block NK cell triggering after engagement with renal epithelial cells. It is concluded that cytokines present within the renal microenvironment during rejection protect graft cells from lysis by NK cells by causing local upregulation of the expression of class I MHC molecules.     

Tolerance induction ameliorates allograft vasculopathy in rat aortic transplants. Influence of Fas-mediated apoptosis

Based on successful induction of donor-specific unresponsiveness by alloantigenic stimulation in several animal models of acute rejection, we hypothesized that similar immune manipulations would also inhibit the evolution of chronic rejection and transplant vasculopathy. To induce immune tolerance, DA rats received a PVG heart allograft and were immunosuppressed with cyclosporine for 30 d. At day 100 the animals were challenged with a PVG aortic allograft after either 1 or 18 h of cold ischemia. 8 wk after the aortic transplantation, the grafts were investigated for morphological changes, infiltrating cells, apoptosis, and Fas-Fas ligand expression. Control allografts showed advanced transplant arteriosclerosis, whereas tolerance-induced aortic allografts displayed reduced neointimal formation, less medial atrophy, fewer apoptotic cells, and fewer Fas- and FasL-expressing cells. Prolonged ischemic storage time did not profoundly alter the morphological changes of the allografts. Fas expression was found in T cells, macrophages, vascular smooth muscle cells, and endothelial cells, whereas FasL was expressed mainly by T cells and macrophages. FasL mRNA expression was evident throughout the entire allograft wall. In conclusion, induction of allospecific tolerance can effectively prevent transplant arteriosclerosis. Cold ischemia damage does not abrogate the beneficial effect of tolerance, but creates a separate identity of mainly endothelial lesions. Furthermore, Fas-mediated apoptosis appears to be involved in the pathological lesions seen in chronic rejection.     

Immunogenicity of rat hepatocytes in vivo: effect of cholestasis-induced changes in major histocompatibility complex expression

Hepatocytes normally express few major histocompatibility complex (MHC) class I and no MHC class II molecules, a phenomenon which could explain their low immunogenicity. However, in pathological situations, such as allograft rejection and cholestasis, hepatocytes strongly express MHC class I molecules and their immunogenicity could be different. The aim of this study was to assess the role of MHC expression on the immunogenicity of hepatocytes in vivo. Hepatocytes were obtained from normal and cholestatic DA rats by whole-liver perfusion with EDTA. Cholestasis was induced by ligation-section of the common bile duct. MHC expression on hepatocytes was assessed by cytofluorimetry after labelling with monoclonal antibodies against MHC class I and class II antigens. The percentage of hepatocytes expressing MHC class I was 9.8 +/- 2.2% in normal rats and 77.2 +/- 3.3% in cholestatic rats (P = 2 x 10(-4)); MHC class II expression was present on 1 +/- 0.5% of normal hepatocytes and 0.4% +/- 0.1% of cholestatic hepatocytes (P > 0.05). Lewis rats received a DA or Wistar-Furth heart allograft 7 days after intravenous injection of 2 x 10(7) hepatocytes from normal or cholestatic DA rats. The DA heart allograft was rejected in 6.3 +/- 0.4 days in Lewis controls, 8.8 +/- 1.1 days (N.S.) in Lewis recipients that received normal DA hepatocytes and 17.6 +/- 3.0 days (P = 2 x 10(-4)) in Lewis recipients that received hepatocytes from cholestatic DA rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Qa-1b molecule binds to a large subpopulation of murine NK cells

Recent studies on human NK cells have demonstrated that the NK cell CD94/NKG2 receptors bind to the nonclassical MHC class I molecule HLA-E. A functional CD94/NKG2 complex has not yet been identified in rodents, but cDNA encoding rat and mouse CD94 and NKG2 have recently been cloned, suggesting that CD94/NKG2 receptors may exist in species other than man. The mouse nonclassical MHC class I molecule Qa-1 shares several features with HLA-E. This suggests that Qa-1 may be similarly recognized by murine NK cells. To study the ability of Qa-1 to bind to murine NK cells, we have produced a soluble tetrameric form of Qa-1b. In the present study, we demonstrate that Qa-1b tetramers distinctly bind to a large subset of fresh or IL-2-activated NK1.1+/CD3- splenocytes independently of the expression of Ly49 inhibitory receptors. Binding occurs whether NK cells have evolved in an MHC class I-expressing or in an MHC class I-deficient environment. Our data suggest the existence of a Qa-1-recognizing structure on a large subpopulation of murine NK cells that may be similar to the human CD94/NKG2 heterodimeric complex.     

Adoptively transferred CD4+ lymphocytes from CD8 -/- mice are sufficient to mediate the rejection of MHC class II or class I disparate skin grafts

Recent studies revealed that CD4+ cells initiate allograft rejection through direct recognition of allogeneic MHC class II Ags and indirect recognition of MHC peptides processed by self APCs. Both pathways were shown to help CD8+ cells that eventually lysed allogeneic MHC class I-presenting targets. There was little evidence, however, that CD4+ cells are sufficient for graft rejection. We studied skin graft rejection by CD8-deficient (CD8 -/-) mice. We showed that BALB/cJ(H-2d) CD8 -/- mice could reject allogeneic skin from C57BL/6J(H-2b) mice deficient in MHC class I or in MHC class II Ags. To understand the role of CD4+ cells in this process, we isolated them from CD8 -/- mice and transferred them to BALB/cJ nude mice that had been grafted with allogeneic skin (H-2b) from animals deficient in MHC class I or MHC class II. Nude mice injected with CD4+ cells rejected MHC class II and, albeit more slowly, MHC class I disparate skins. We showed in vitro evidence that CD4+ cells were not cytotoxic toward MHC class I or MHC class II disparate targets and that they recognized MHC class I allogeneic targets through indirect recognition. CD4+ cells produced Th1 cytokines, but not IL-4, following stimulation with allogeneic cells. Furthermore, intragraft TNF-alpha was elevated in skin grafted onto nude mice reconstituted with CD4+ cells compared with nonreconstituted mice. This suggests that MHC class II- or MHC class I-guided CD4+ cells alone are sufficient to induce rejection by the generation of cytokine-induced lesions.     

Frequency of ras mutations in liver neoplasms from B6C3F1 mice exposed to tetrafluoroethylene for two years

BACKGROUND: Cytotoxic T lymphocytes (CTLs) contribute to the rejection of transplanted tissues through two pathways: first, by direct recognition of foreign graft major histocompatibility complex (MHC) class I molecules; and second, by recognition of foreign graft-derived peptides presented by classical MHC class Ia molecules that are matched between graft and donor. However, a number of observations suggest that additional categories of CTL recognition patterns may exist, but they remain to be defined molecularly. METHODS: Previous studies showed that the murine nonclassical MHC molecule H2 M3 may be involved in allorecognition. We investigated whether other members of nonclassical MHC class Ib, namely Qa1 and Qa2, may be recognized. Alloreactive CTLs were generated from mice mismatched for non-MHC and/or MHC genetic backgrounds and tested using various target cells, including cells transfected with Qa1 or Qa2. Furthermore, candidate peptides were synthesized and used to generate CTLs specific for peptide presented by Qa1 or Qa2. RESULTS: The experiments demonstrate that allogeneic and xenogeneic peptides were recognized by CTLs when presented on shared nonclassical MHC class Ib Qa1 and Qa2 molecules. CONCLUSIONS: The results confirm that MHC class Ib molecules present peptides to CTLs. This potentially important alloreactivity pathway may be functional between most individuals because sharing of MHC class Ib alleles is frequent.     

Regulatory T cells in the control of autoimmunity: the essential role of transforming growth factor beta and interleukin 4 in the prevention of autoimmune thyroiditis in rats by peripheral CD4(+)CD45RC- cells and CD4(+)CD8(-) thymocytes

Previous studies have shown that induction of autoimmune diabetes by adult thymectomy and split dose irradiation of PVG.RT1(u) rats can be prevented by their reconstitution with peripheral CD4(+)CD45RC-TCR-alpha/beta+RT6(+) cells and CD4(+)CD8(-) thymocytes from normal syngeneic donors. These data provide evidence for the role of regulatory T cells in the prevention of a tissue-specific autoimmune disease but the mode of action of these cells has not been reported previously. In this study, autoimmune thyroiditis was induced in PVG.RT1(c) rats using a similar protocol of thymectomy and irradiation. Although a cell-mediated mechanism has been implicated in the pathogenesis of diabetes in PVG.RT1(u) rats, development of thyroiditis is independent of CD8(+) T cells and is characterized by high titers of immunoglobulin (Ig)G1 antithyroglobulin antibodies, indicating a major humoral component in the pathogenesis of disease. As with autoimmune diabetes in PVG. RT1(u) rats, development of thyroiditis was prevented by the transfer of CD4(+)CD45RC- and CD4(+)CD8(-) thymocytes from normal donors but not by CD4(+)CD45RC+ peripheral T cells. We now show that transforming growth factor (TGF)-beta and interleukin (IL)-4 both play essential roles in the mechanism of this protection since administration of monoclonal antibodies that block the biological activity of either of these cytokines abrogates the protective effect of the donor cells in the recipient rats. The prevention of both diabetes and thyroiditis by CD4(+)CD45RC- peripheral cells and CD4(+)CD8(-) thymocytes therefore does not support the view that the mechanism of regulation involves a switch from a T helper cell type 1 (Th1) to a Th2-like response, but rather relies upon a specific suppression of the autoimmune responses involving TGF-beta and IL-4. The observation that the same two cytokines were implicated in the protective mechanism, whether thymocytes or peripheral cells were used to prevent autoimmunity, strongly suggests that the regulatory cells from both sources act in the same way and that the thymocytes are programmed in the periphery for their protective role. The implications of this result with respect to immunological homeostasis are discussed.     

Effect of major histocompatibility complex expression on murine intestinal graft survival

BACKGROUND: Clinical intestinal transplantation has been plagued by frequent and severe graft rejection. It has been proposed that the major histocompatibility complex (MHC) antigens might play a critical role in this process owing to their extensive expression on enterocytes and mucosa-associated immune cells. METHODS: The present study examined the role of MHC antigens in intestinal graft rejection using MHC class I-deficient and MHC class II-deficient donors. RESULTS: Grafts with normal MHC expression were rejected by 9 days, whereas survival was prolonged to 14 days in the MHC class II-deficient grafts (P=NS) and to 20 days in the MHC I-deficient grafts (P<0.002). In all groups, early rejection was characterized by (1) increased crypt cell apoptosis, as detected by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique of in situ labeling; and (2) the increased expression of perforin and a CD8 phenotype in the graft-infiltrating cells. CONCLUSIONS: These data suggest that MHC antigens, CD8-positive T cells, and perforin-expressing cells contribute to intestinal graft rejection. Apoptosis of the progenitor epithelial crypt cells during early intestinal rejection may impair the gut's ability to regenerate and repair mucosal damage.