The effects of cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in the rat

We have previously reported 5-30-fold increases in the expression of class I and class II major histocompatibility complex (MHC) antigens in rejecting heart and kidney allografts in the DA-to-PVG rat strain combination. We examine here the effects of immunosuppression with cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in this strain combination. Immunohistological studies and quantitative absorption analyses using monoclonal antibodies and assay systems specific for donor class I and class II MHC antigens were used throughout. Heart allografts in cyclosporine-treated rats were examined on day 3,5,7,9,11, and 14 after transplantation, and kidney allografts in cyclosporine-treated rats were examined at day 7. In addition, untreated heart and kidney isografts were studied at days 1,3,5, and 7 after grafting. Immunohistological studies on frozen sections showed that cyclosporine-treated heart and kidney allografts showed no induction of class II MHC antigens, in contrast to untreated heart and kidney allografts. Class I MHC antigen induction did occur in spite of cyclosporine-therapy, but at levels lower than those seen in untreated allografts. Moreover, the pattern and degree of class I induction in the cyclosporine-treated allografts resembled very closely those seen in isografts, and so this induction was, in all probability, a consequence of the transplantation procedure rather than of specific immune responses. We also noted, in the cyclosporine-treated heart allografts, that all donor interstitial dendritic cells had disappeared and been replaced by recipient interstitial dendritic cells by the end of the second week after grafting. In addition, there was no reduction in the class II antigen content of kidney allografts treated for 7 days with cyclosporine. The absence of class II antigen induction in allografts where rejection is effectively suppressed with cyclosporine might be of clinical value in the differential diagnosis between rejection and cyclosporine toxicity in renal transplantation, and between active and inactive cellular infiltrates in heart transplantation.     

Active enhancement of rat kidney allografts. Effect of pretreatment with prednisolone and donor-specific antigen.

(Lewis x Brown Norway) F1 hybrid rat kidney allografts were transplanted to bilaterally nephrectomized Lewis recipients pretreated in various ways. The mean survival time of untreated controls was 16.1 +/- 1.7 days. All rats pretreated with 1.67 g/kg of semi-soluble Brown Norway spleen extract and 5 mg/kg of prednisolone on days 15, 8, and 1 before transplantation survived indefinitely. Pretreatment with semi-soluble or soluble extract alone prolonged survival modestly (36.5 +/- 13.6 and 30.8 +/- 5.6 days, respectively), but the former induced indefinite survival in two of eight animals. Prednisolone on its own failed to bring about prolongation of survival and the combined use of soluble extract and prednisolone did not reveal a synergistic effect. Cytotoxic antibody titres in animals showing indefinite survival were very low, and there was no correlation between antibody titres and prolonged survival. It is assumed that the pretreatment with semi-soluble extract and prednisolone inhibited the formation of cytotoxic antibodies as well as cell-mediated immunity, and encouraged the formation of enhancing antibodies. To study the cellular and humoral reactivity of five prolonged survived kidney recipients, 1st and 2nd donor-specific skin grafts were carried out. The humoral and cell-mediated responses were somewhat delayed in these recipients but otherwise normal except for the absence of the second-set phenomenon.     

Absence of donor MHC antigen expression ameliorates chronic kidney allograft rejection

BACKGROUND: In previous studies, we have demonstrated that a subset of mouse kidney allografts has prolonged survival without any immunosuppressive treatment. Chronic rejection (CR) develops in these long surviving grafts. The pathologic features of CR in this model are similar to CR in human kidney grafts. METHODS: To explore the role of donor major histocompatibility complex (MHC) antigens in the development of CR, we performed vascularized kidney transplants using kidneys from donor mice that lack expression of both MHC class I and II antigens (MHC-/-). RESULTS: Survival was significantly improved in recipients of MHC-/- allografts. This enhanced survival was associated with higher glomerular filtration rate (GFR) in MHC-/- allografts (4.92 +/- 0.54 cc/min/kg) compared to controls (2.19 +/- 0.63 cc/min/kg; P = 0.004). The typical histologic features of CR were markedly reduced in MHC-/- allografts. Semiquantitative histopathological scores for MHC-/- grafts (13.3 +/- 2.1) were significantly lower than in control allografts (19.0 +/- 1.0; P = 0.04). Along with this improvement in structural abnormalities, significantly fewer CD4+ T (38.3 cells/mm(2) vs. 75.0 cells/mm(2); P = 0.008), CD8+ T cells (38.7 vs. 96 cells/mm(2), respectively; P = 0.008) and macrophages (60 vs. 134 cells/mm(2), respectively; P = 0.04) infiltrated MHC-/- allografts compared to controls. The levels of intragraft cytokine mRNA expression also were reduced in MHC-/- allografts compared to control allografts. Finally, serum alloantibodies were virtually undetectable in recipients of MHC-/- kidney allografts. CONCLUSIONS: Cell surface expression of donor MHC antigens promotes the development of CR. Donor antigen expression promotes the accumulation of infiltrating cells in the graft and the development of donor specific alloantibodies. Abrogation of these responses is associated with improved graft survival and reduced CR in MHC-/- grafts.     

A role of anti-"LD" antibody in induction of active enhancement in rat heart allografts.

Heart allografts from S-D to Wistar rats pretreated with 5 x 10(7) spleen cells taken from donor strain 1 or 2 weeks before, survived indefinitely. Sera taken from the pretreated rats showed high lymphocytotoxic titers and obvious blocking activity against LMC of immunized Wistar spleen cells. Even though the sera were exhaustively absorbed with RBC, this blocking activity was not impaired, although CDC was reduced. The active prolongation of graft survival, therefore, correlates with the presence of anti-LD antibody.     

Effect of azathioprine and prednisolone on passive enhancement of rat renal allografts.

Passive enhancement provides only partial suppression of rejection in the (DA X Lewis)F1 to Lewis renal allograft model. Suboptimal (8 mg/kg/day) and supraoptimal (30 mg/kg/day) doses of azathioprine administered with enhancing serum failed to suppress the rejection reaction in enhanced animals. Similarly, suboptimal (4 mg/kg/day) and optimal (16 mg/kg/day) doses of methylprednisolone were ineffective. However, the onset of rejection in enhanced animals was delayed by the use of both azathioprine (30 mg/kg/day) and methylprednisolone (16 mg/kg/day). The survival times of enhanced animals treated with azathioprine were significantly shorter than those of animals treated with enhancing serum alone, suggesting that this agent may prevent the development of autoenhancement. Although suboptimal doses of antilymphocyte serum suppress rejection in this enhancement model, the dose requirements of conventional immunosuppressive agents appear to be maximal rather than minimal.     

Infiltration patterns of macrophages and lymphocytes in chronically rejecting rat kidney allografts

The migration of circulating leukocytes to sites of inflammation or antigen is based, at least in part, on the activities of adhesion molecules. In the context of organ transplantation, some of these have been shown to be upregulated during acute allograft rejection. As their role during chronic rejection has not been examined, we have used an established rat model to compare sequentially the presence of host cells within the grafts, as defined immunohistologically, with patterns of in vitro leukocyte binding and their dependence upon particular adhesion molecules. Various donor populations of peripheral blood lymphocytes (PBL), lymph node lymphocytes (LNL), and splenic monocytes were interacted with snap-frozen sections of allografted, isografted, and native kidneys at serial intervals up to 24 weeks after transplantation. Monocyte binding in the allografts rose at 8 weeks and peaked at 12 weeks, a period preceding the maximum numbers of macrophages noted immunohistologically in the chronically rejecting grafts at 16 weeks. Lymphocyte binding and infiltration patterns were similar, remaining stable throughout the follow-up period and consistently greater than those noted in isografts. In vitro binding of the monocytes was inhibited by mAbs against ICAM-1, LFA-1, CD18, and MAC-1; MAC-1 did not influence lymphocyte binding, although the other mAbs were effective. We conclude that adhesion molecules are responsible, at least in part, for patterns of cell populations infiltrating chronically rejecting renal allografts.     

The alloantibody response and active enhancement of some rat renal allografts.

Lewis rats that were given injections of 10(6) to 10(8) (Lewis X Brown Norway) F1 hybrid bone marrow cells produce predominantly, if not exclusively, 19S lymphocytotoxic antibodies. A number of Lewis rats that received transplants of perfused renal allografts from bone marrow donors at, or near, the peak of IgM response survival for well over 200 days with good renal function and no histological evidence of chronic rejection. All long-surviving rats had detectable lymphocytotoxic antibodies up to 120 days after allografting; late enhancing antibodies had the restricted specificity possibly identical or similar to anti-I region antisera. All rats bearing prolonged renal allografts were unable to accept donor-specific skin grafts or to respond with specific lymphocytotoxic antibodies following skin grafting. The possible involvement of non-complement-fixing 19S alloantibodies in active enhancement of rat renal allografts is discussed.     

Scintigraphic imaging of MHC class II antigen induction in mouse kidney allografts: a new approach to noninvasive detection of early rejection

Mice with kidney transplants were investigated to determine whether early kidney allograft rejection could be detected by radioimmune scintigraphy targeting major histocompatibility complex (MHC) class II antigens induced on donor organ cells. Allografts from C3H/He (H2^k) donors were transplanted into BALB/c (H2^d) recipients. Each mouse was injected intravenously with 100 Ci of ¹²³I-labeled anti-MHC class II monoclonal antibody (mAb; Y17, anti-IE^k) 16 h before scintigraphy. After imaging, mice were sacrificed for tissue counting and histopathological examination. Radiotracer uptake in the nontreated allografts increased starting on the 3rd day after transplantation, peaked at around the 6th day, and then gradually decreased. Rejecting allografts with only focal perivascular mononuclear cell infiltration could be identified by scintigraphy. However, allografted mice without evidence of rejection and isografted mice did not show an increase in radiotracer uptake. Rejecting BALB/c kidney transplanted into C3H/He mice did not show an increase in Y17 mAb uptake, suggesting that class II antigens induced on donor kidneys are solely responsible for the mAb uptake in positive scintigrams of rejecting allografts. Five allografted mice were treated with anti-CD3 mAb and cyclosporin starting 3–9 days after transplantation. Radiotracer uptake decreased after 4 weeks of treatment and increased 2 weeks after the cessation of immunosuppressive treatment, reflecting suppression and recurrence of rejection, as determined by histological examination. These changes could be followed scintigraphically. We conclude that changes in class II antigen expression can be assessed by the ¹²³I-labeled anti-MHC class II antigen mAb and that it is a sensitive and noninvasive method for detecting kidney allograft rejection.     

ABO incompatibility in cadaver donor kidney allografts

We have found the ABO barrier does appear to remain intact in clinical kidney transplantation, as evidenced by a 4% 1-year graft survival in 25 cadaver donor, ABO-incompatible allografts. The one long-term surviving graft could have been a case of an A2 kidney and a B recipient, but this could not be determined. Although these grafts were the result of errors at a number of different points, most could have been avoided if a red cell crossmatch had been performed at the time of transplant. The fact that these transplants rarely occur should not overshadow the possibility that they could be avoided by the implementation of such a simple procedure.     

Survival of long nerve allografts following donor antigen pretreatment: a pilot study

In this study, the authors evaluated whether the pretransplant portal venous administration of UV-B irradiated donor alloantigen would induce tolerance to long peripheral nerve allografts in a swine model. They completed nerve allograft transplantation between four swine of separate lineages. Regeneration across the nerve allografts was followed for 10 months postoperatively. Sequential IN VITRO assays demonstrated the successful and prolonged suppression of the recipient immune response to donor antigen following antigen inoculation. Histomorphometric analysis demonstrated successful regeneration across the long nerve allografts in the pretreated recipients, but not across allografts in unimmunosuppressed recipients. A single pretransplant antigen delivery protocol has the potential to replace chronic medicinal immunosuppressant therapy and its associated morbidities. Furthermore, tolerance to long nerve allografts has immediate applicability to clinical requirements for bridging multiple, complex, long nerve gaps.     

Reduced immunogenicity of rat renal allografts after photochemical donor pretreatment and passive transfer of graft protection

Summary Photochemical pretreatment of the kidney donor (Sprague-Dawley rats/SD) with 8-methoxypsoralen (8-MOP) and ex vivo longwave ultraviolet (UVA) irradiation of the kidney graft (PUVA therapy) significantly prolonged survival in allogeneic recipients (BD IX rats). After more than 100 days 7 long-term surviving PUVA-pretreated SD kidneys were retransplanted into BD IX rats. Seven out of 7 secondary recipients survived for more than 100 days. Twenty BD IX recipients of normal SD kidneys were treated at the time of transplantation with serum (1 ml i.v.) and/or spleen lymphocytes (1x10⁷ i.v.) obtained from the PUVA-treated long-term survivors. A prolonged graft survival was achieved in 7 our of 20 rats, among them 4 out of 8 recipients of the serum-treated group. In conclusion, the long-term survival of PUVA-treated rat renal allografts is associated with a strong reduction of graft immunogenicity and the development of graft protecting humoral as well as cellular effectors.     

Indefinite survival of rat islet allografts following infusion of donor bone marrow without cytoablation

We have tested the effect of donor bone marrow cell (DBMC) infusion on the survival of pancreatic islet allografts in the rat, without the use of cytoablative recipient conditioning. Lewis and diabetic Brown Norway rats were used as donors and recipients, respectively. Donor islets were placed beneath the left renal capsule. Infusion of DBMC and temporary immunosuppression followed by delayed islet transplantation resulted in indefinite survival of all islet grafts (MST >180 days). Control animals demonstrated recurrent hyperglycemia (islet allografts rejection). Donor bone marrow derived cells were detected in the spleen and cervical lymph nodes of BN recipients of LEW bone marrow but not in the recipients of islet transplants alone. Second set full thickness skin grafts were performed in normal BN and in recipients of a previously successful ITX. Donor specific skin grafts were accepted in the animals that had received DBMC 40 days before the islet allograft, while animals receiving DBMC at the time of the islet allograft rejected the donor specific skin graft similarly to the controls. However, these animals did not reject a second set donor-specific islet transplant. The results indicate that radiation conditioning of the recipients was not necessary to induce microchimerism and graft acceptance in this rodent model of islet allotransplantation.