DA Skin Research Articles

LIVER CONTROLS ALLOIMMUNE RESPONSE FOLLOWING DONOR SPLEEN CELLS INJECTION I

273 Introduction: The tolerance of allografts after donor-specific injection (DSI) by intraportal or intravenously have been reported, therefore liver considered as an one of the immune-modulate organs. It is unknown, however, whether the underlying mechanisms after DSI involve the liver alone or a combination of other tissue/organ(s). In this study, we developed the new model to examine the relationship between a liver and extrahepatic tissue/organ(s) in the alloimmune responses induced by antigen inoculation. Methods: WS (RT 1k) rats were sensitized with 5×107 DA (RT 1a) spleen cells which were injected intravenously. One, two or three days after DSI, a total hepatectomy was performed and the liver was transplanted into another WS rat (sensitized-liver-grafted; SLG), the hepatectomized WS rat underwent liver transplantation from a naive WS rat (sensitized-liver-removed; SLR). In SLG, the alloantigens accumulated in liver on just time transplanted liver after DSI; in SLR, in the extrahepatic tissue/organ(s). Following these treatments, the animals were grafted with DA skin or heart (Tx) on 10 days after antigen administration. The immune responses following these treatments were evaluated in terms of allografts survival, Th1/Th2 cytokine profile and perforin mRNA in various organs, T cell response (allogeneic MLR before just Tx and delated-type hypersensitivity (DTH) response after Tx) and B cell response (complement-dependent cytotoxicity (CDC) titer and donor-specific immunogloburin after DSI and Tx). Results: SLG animals on one day vigorously rejected skin or heart allografts (5.5±0.2 days; vs other groups, p<0.005, 4.2±0.3 days; vs other groups, p<0.005) compared with non-treated controls (8.5±0.2, 11.6±0.5). In contrast, SLR animals rejected allografts in a significantly delayed fashion (16.5±1.1; vs other groups, p<0.005, 27.7±2.2, vs other groups, p<0.005). On the other hand, SLG and SLR animals transplanted liver on other days rejected in a manner similar to non-treated controls. When skin grafts from LEW (RT 1l) rats were used as a third party, SLG and SLR animals rejected in a manner similar to non-treated controls; when LEW rats were used as a source of spleen cells, these animals rejected DA skin grafts in a same manner. SLG animals on one day associated with a significant higher IFN-γ and perforin mRNA upregulation, CDC titer and DTH response after cardiac transplantation, SLR animals associated with the decrease of them. Before cardiac transplantation, Th1 cytokine levels in liver of SLG and in spleen of SLR on one day highly upregulated rapidly and decline thereafter, mixed lymphocyte proliferative reaction significantly increased in SLG (11412.4±2898.8 cpm; vs other groups, p<0.005) than SLR (87691.2±1308.2) and non-treated controls (87069.0±1944.5). There was no significant difference about the point of peak switched from a IgM to a IgG response in each group. Conclusion: Our results in vivo and vitro on one day after DSI clearly indicate that two compartments of the immune system (liver and other organs) behave differently to alloantigen, suggested early Th1 reaction in liver or spleen after DSI correlate with T cell clonal activation or suppression but not B cell before cardiac transplantation. The liver may play an important role to determine unresponsiveness or rejection after DSI.

Fulminant second-set allograft rejection and endoscopic findings following small bowel transplantation in the rat

In the presensitized recipient who has been exposed to donor antigens, second-set rejection takes the form of severe hyperacute graft rejection. Second-set allograft rejection was studied following small bowel transplantation in the rat. Heterotopic intestinal grafting was performed from DA (RT1a) donors to PVG (RT1c) recipients 4 weeks after DA skin sensitization. The endoscopic images and histological specimens were compared with those of syngeneic and first-set rejected grafts. Endoscopically, diffuse erosions of the graft were detected from day 1. Mucosal necrosis progressed rapidly, and was accompanied by massive bleeding on days 3-5. These findings were similar to the course of severe necrotizing hemorrhagic enteritis. Histologically, interstitial edema and hemorrhage with massive infiltrations of neutrophils were manifested from day 1. Mesenteric vessels were completely occluded by thrombi on days 3-5. The grafted intestine had became totally necrotic by day 5. Microscopic findings strongly suggested that destructive graft necrosis was due to vascular damage caused by humoral factors. All the presensitized rats (n = 11) died showing systemic septic signs by day 11 after small bowel transplantation. We conclude that lethal hyperacute rejection occurred in presensitized recipients, even when the graft was transplanted heterotopically. Endoscopic evaluation is beneficial for the early diagnosis of graft rejection. Immediate graft removal should be mandatory as a rescue treatment in second-set rejection of the small intestine.

ABSENCE OF TOLERANCE TO NONINHERITED MATERNAL TRANSPLANTATION ANTIGENS EXPRESSED ON SKIN AND CORNEA1

Tolerance to noninherited maternal antigens (NIMAs) of the MHC was investigated in a rat model involving both skin and corneal transplants. Recipient animals were obtained by backcrossing F1 hybrids to parental strain animals. In one group of experiments, crosses were (DA[RT1a] x LEW[RT1(1)]) female-to-LEW male and, in a second group, (DAxPVG[RT1c]) female-to-PVG male. Homozygous backcross offspring (RT1(1/1) or RT1c/c) were putatively tolerant to DA NIMAs if the mother was a hybrid animal, having been exposed to these antigens in utero. The equivalent offspring of hybrid fathers, i.e., LEW female x (DAxLEW) male or PVG female x (DAxPVG) male, served as putatively nontolerant controls. Hemagglutinating antibody levels were measured against the class I RT1Aa antigen on days 7 and 14 after up to three consecutive subcutaneous DA strain skin grafts. Significantly lower titers were found in the putatively tolerant group 7 days after the first skin graft in the RT1a-to-RT1(1) combination (P < 0.05). Levels were not significantly different at any other time point, or at any time point in the RT1a-to-RT1c combination. Tolerance to a corneal graft was not demonstrated in either the strongly rejecting RT1a-to-RT1(1) combination or weakly rejecting RT1a-to-RT1c, whether or not animals were presensitized to RT1a antigens with DA skin grafts. We conclude that tolerance to NIMAs is unimportant in this clinical rat model of transplantation.

Rejection of skin allografts by indirect allorecognition of donor class I major histocompatibility complex peptides.

LEW (RT1l) rats were immunized with peptides corresponding to the alpha helical region of the alpha 1 domain (peptide 1), the beta sheet of the alpha 2 domain (peptide 2), and the alpha helical region of the alpha 2 domain (peptide 3) of the RT1-Aav1 classical class I molecule of the DA (RT1av1) strain. The immunizations were without carriers, and the objective was to prime to indirect allorecognition without influencing direct recognition of the RT1-Aav1 molecule. The LEW rats mounted strong primary and secondary antibody responses to peptides 1 and 3, but only weak secondary responses to peptide 2. None of the antipeptide antibodies crossreacted with intact RT1-Aav1 class I molecules. The immunization also resulted in LEW antigen-presenting cell-dependent, CD4+ T cell proliferative responses, which were very strong against peptide 1 and weakest against peptide 2. LEW rats immunized with peptides 1 or 3, but most effectively with both peptides 1 and 3 together, showed accelerated rejection of DA skin allografts. This effect was not observed in LEW rats immunized with peptide 2. In response to the DA skin allograft, the peptide-immunized LEW rats showed markedly accelerated kinetics of antibody production to the intact RT1-Aav1 molecule. These data demonstrate that indirect allorecognition can play an important role in allograft rejection and have important implications for understanding allograft rejection and its regulation.

Stimulation of CD4+ T lymphocytes by allogeneic MHC peptides presented on autologous antigen-presenting cells. Evidence of the indirect pathway of allorecognition in some strain combinations.

A preliminary analysis of the alloantibody response to free, unconjugated class I and class II MHC peptides in several rat and mouse strains was performed, to screen for an effective interaction between the allogeneic MHC peptides and recipient MHC molecules. The PVG rat strain was noted to produce very strong, MHC-restricted, primary and secondary responses to a synthetic peptide derived from the alpha helical region of the alpha 2 domain of an RT1.C/E class I MHC molecule of the DA strain. In vitro proliferation studies demonstrated that CD4+ but not CD8+ T cells of the PVG strain responded in a recipient APC-dependent manner to the peptide, whereas the BN strain (which showed no antibody response to this peptide) gave no T cell proliferation. Immunization of PVG rats with the peptide did not influence the rejection of DA skin allografts. The relevance of these studies to the possible mechanisms of allograft rejection by an indirect pathway are discussed.

Allorecognition of isolated, denatured chains of class I and class II major histocompatibility complex molecules. Evidence for an important role for indirect allorecognition in transplantation.

Classical RT1-A class I and RT1-B class II major histocompatibility complex (MHC) molecules were purified from DA (RT1avl) spleens, and the individual chains separated and purified by preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. LEW (RT1l) rats were immunized with the pure class I heavy chain, the RT1-B alpha chain and the RT1-B beta chain with the aim of priming to indirect allorecognition (i.e. after processing and presentation of DA MHC chains on LEW antigen-presenting cells) in the absence of any priming to direct allorecognition (i.e. to whole, undenatured, dimeric DA MHC molecules). LEW rats immunized with each of the three DA MHC chains produced alloantibodies to these chains, suggesting that indirect allorecognition did occur, because of the requirement for cognate recognition of B cells by T helper cells. This also demonstrated polymorphism of all three chains between the DA and LEW strains. The antibodies to the isolated, denatured MHC chains did not react to the whole MHC molecules on DA cells, with the possible exception of very weak reactions in some class I heavy chain-immunized rats. DA skin grafts placed on LEW recipients immunized with each of the DA MHC chains were rejected in an accelerated fashion. Following DA skin grafting, there was an accelerated production of antibodies to whole, undenatured class I MHC molecules, even in the LEW rats preimmunized with RT1-B alpha and RT1-B beta chains. These data suggest that indirect allorecognition can play an important role in the effector mechanisms of allograft rejection, and demonstrate T helper priming as one possible mechanism whereby this might be effective.

THE ROLE OF MHC AND NON-MHC ANTIGENS IN THE REJECTION OF INTRACEREBRAL ALLOGENEIC NEURAL GRAFTS

Embryonic DA retinal allografts that have survived for prolonged periods after having been transplanted into the brains of neonatal BN rats can be induced to reject following peripheral sensitization with a DA skin graft. The results show that histocompatibility antigens play the major role in the rejection of grafts placed in the CNS and that a disparity between the retinal and skin grafts for MHC antigens induces a more severe rejection response than does a non-MHC antigen disparity.

Strategies of monoclonal antibody therapy that induce permanent tolerance of organ transplants.

Treatment of normal rats with certain monoclonal anti-CD4 antibodies, beginning on the day of grafting, prevents heart graft rejection across a full MHC-haplotype mismatch. Adequate doses of antibody MRC OX-35, which is very potent in vivo, led to the induction of authentic specific tolerance of the transplants. In the very stringent grafting test of transplantation of DA skin to high-responder LOU recipients, adjunctive therapy with cyclosporine, in addition to the anti-CD4 antibodies, is required to induce tolerance. Since tolerance persists indefinitely after a 30-day course of treatment and appears to involve T cell-mediated suppression, this form of treatment is of potential clinical interest.

Serology of liver transplantation in the rat. I. Alloantibody responses and evidence for tolerance in a nonrejector combination.

The antibody response against class I and class II RT1 antigens has been studied in PVG rats grafted with DA liver. In this nonrejector combination, liver grafts survive permanently in all normal recipients and in about 50% of recipients presensitized by a DA skin graft, with concurrent induction of transplant tolerance for other DA organs and skin. Using a two-stage radioimmunoassay, the anti-class I (RT1Aa) levels in sera of normal PVG recipients of DA liver grafts were found to be low (maximal titer 1:50 serum dilution or less); after peaking at 2 weeks posttransplantation, they diminished to background levels by 6 weeks. The anti-RT1Aa response showed a close parallel to cell-mediated rejection events in the liver graft recipients. In contrast, anti-class II (RT1Ba/Da) responses reached much higher titers (over 1:1000), which were maintained for several weeks before declining after 4 months. Similar observations were made in presensitized recipients. The induction of tolerance in the alloantibody response was indicated by the inability of DA skin grafts to restimulate anti-RT1 antibody in liver recipients. The observations support the picture of "split tolerance" indicated by previous cellular studies in this combination.

Functional capacity of solid tissue transplants in the brain: evidence for immunological privilege.

The capacity of the mammalian brain to support the physiological function of allografts was assessed in parathyroidectomized Fischer strain rats bearing either isografts or immunogenic DA allografts of parathyroid glands implanted in their cerebral cortices. Established isografts and allografts survived indefinitely in the brain, maintaining normal serum calcium levels, with equal numbers of spontaneous failures (18-21%) in each group. Similarly, both MHC-compatible and incompatible skin allografts survived and were 'functional' at 40-50 days postgrafting as assessed by: continued formation of keratin; the presence of differentiated hair follicles and sebaceous glands; and frequent mitotic figures. No serum alloantibodies were induced by either MHC-incompatible parathyroid glands or skin in this site. However, both types of allografts were promptly rejected or failed to become established in the brains of specifically presensitized hosts. Furthermore, when Fischer hosts with long-established intracerebral DA parathyroid grafts received orthotopic DA skin grafts, their parathyroid grafts were rejected along with first-set rejection of the skin grafts. The tempo of this cellular immune response and the primary alloantibody response that accompanied it indicate that although the intracerebral grafts failed to induce detectable host sensitization or suppression, they remained susceptible to immune effectors. Thus, by using strongly immunogenic, adult tissues, we have established that the rat cerebral cortex is an immunologically privileged site, and the privilege is not dependent on lack of graft immunogenicity or alterations in host responsiveness. Furthermore, Ia+ (possible antigen-presenting) cells were rare in the cortical parenchyma sites used for transplantation though numerous in the choroid plexus of the ventricles and in certain areas of white matter. Therefore, privilege probably reflects deficient graft antigen presentation related to the paucity of Ia+ cells as well as to the brain's poor lymphatic drainage.

Prolongation of renal allograft survival in the rat by pretreatment with donor antigen and cyclosporin A.

Pretreatment of LEW rats with cyclosporin A (10 mg/kg/day orally for 14 days) plus 10(8) DA or (DA X LEW)F1 spleen lymphocytes on two occasions markedly prolonged the survival of subsequent DA renal allografts. The lymphocytotoxic antibody response was suppressed completely and renal biopsies showed a marked mononuclear cell infiltrate, but no other manifestations of rejection. Neither pretreatment with cyclosporin A alone nor pretreatment with splenic lymphocytes alone had any effect on the subsequent immune reaction. The immunosuppressive effect generated was dependent upon the dose of lymphocytes given, because pretreatment with 10(7) cells on two occasions plus cyclosporin A was relatively ineffective at prolonging renal allograft survival. Pretreatment with 10(8) (DA X LEW)F1 spleen cells plus cyclosporin A was also an effective combination, and ruled out the possibility that graft-versus-host (GVH) disease was responsible for the observed immunosuppression. This pretreatment protocol exhibited specificity in terms of rejection of a third-party (PVG) renal allograft, but not in terms of the lymphocytotoxic antibody response to this graft. Chimerism was not detected in long-term surviving animals using a 51Cr release assay. When pretreatment was performed in LEW rats with a DA skin allograft plus cyclosporin A, a subsequent DA renal allograft was rejected normally, in combination with the skin graft. These findings suggest that clonal deletion had not occurred in these pretreatment protocols, but that a shift in immunoregulatory mechanisms was responsible for the long survival. There are possible clinical implications for the use of pretreatment schedules using donor antigen and cyclosporin A.

The histological changes in immunologically tolerant lymphoid tissue.

SummaryThe histological features of the regional lymph node draining a skin homograft have been examined in normal and tolerant recipients. The response of this node was morphologically identical in normal and specifically tolerant hosts, the most important feature being the appearance of medullary cords packed with intensely pyroninophilic cells. Furthermore, a similar response was observed in the regional lymph nodes of (DA × Lewis)F1 hybrids bearing DA skin grafts of which they were “genetically tolerant”. It was concluded that the failure of graft rejection in tolerant rats could not be attributed to an absence of reactivity to the grafts on the part of the lymphoid tissue of the host.

Induction of tolerance in rats by treatment with antithymocyte serum and adult skin homografts

ACI rats bearing healthy DA skin homografts > 150 days after completion of a 35-day course of antithymocyte serum given at the time of grafting, accepted another DA skin graft for > 150 days. These animals promptly rejected third-party skin and transfer of the long-surviving homografts to new ACI hosts resulted in acute rejection, ruling out adaptation. Use of serum from hosts of long-accepted skin did not enhance survival of donor strain skin grafts in rats of the recipient strain. Specific unresponsiveness (tolerance) seems to have been achieved as the result of immunosuppression and adult skin as the only source of antigen.

Influence of the Ag-B locus on reactivity to skin homografts and tolerance responsiveness in rats.

SUMMARY The case with which rats of one strain can be rendered tolerant of skin grafts from another strain is correlated with their Ag-B genotypes and not with the median survival time of skin grafts exchanged between them. Thus, although adult Lewis recipients reject Ag-B-compatible Fischer skin grafts as promptly as they reject Ag-B-incompatible BN or DA skin grafts, Lewis recipients are much more easily rendered unresponsive of Fischer skin grafts than of BN or DA grafts. Where Ag-B compatibility prevails, tolerance is inducible with relatively low dosages (≤1 million) of cells originating from all components of the lymphohematopoietic tissue system, including the thymus. On the other hand, when donor and host differ at this locus not only are much higher numbers of cells required to induce tolerance, but there are wide disparities in the tolerance-conferring capacity of different cell types. Bone marrow is superior to spleen which, in turn, is better than lymph nodes; thymocytes are practically ineffective. Evidence is also presented which suggests that the facility with which tolerance can be abolished by transfer of isologous cells is a function of the dosage of the inoculum used to induce tolerance. The importance of the Ag-B locus in determining susceptibility to runt disease is also considered.