A dominant role for non-MHC gene effects in susceptibility to cyclosporin A (CsA)-induced autoimmunity.

Lethally irradiated LEW rats reconstituted with syngeneic bone marrow and given CsA for a 4-week period develop a graft-versus-host-like disease upon withdrawal of CsA. This T cell-mediated autoimmune disease is referred to as CsA-induced autoimmunity (CsA-AI). CsA-AI-susceptible LEW rats and resistant BN rats differ greatly in the composition of their peripheral T cell compartment. To dissect the role of MHC and non-MHC genes in the development of peripheral T cell subsets in combination with susceptibility to CsA-AI the respective MHC congenic strains (LEW-1N and BN-1L) were examined for their T cell subsets and for their ability to develop CsA-AI. In this study we show that the Th1/Th2-like cell ratio as well as susceptibility to CsA-AI are under control of the non-MHC genes. This suggests that the Th1/Th2-like cell ratio is a critical determinant for development of CsA-AI. Alternatively, resistance can be attributed to lack of target organ susceptibility due to the absence of the target autoantigen in resistant rat strains. This interpretation is rejected, since both BN as well as BN-1L rats consistently develop the characteristic macroscopic and microscopic signs of CsA-AI upon adoptive transfer with autoreactive LEW-1N and LEW T cells, respectively. Therefore, it can be concluded that the non-MHC genes encode for immune deviation and thereby determine susceptibility or resistance to CsA-AI.

Beta 2-microglobulin-dependent T cells are not necessary for alloantigen-induced Th2 responses after neonatal induction of lymphoid chimerism in mice.

We have analyzed the requirement for beta 2-microglobulin (beta 2m)-dependent T cells in the generation of allogeneic Th2 responses in vivo. A neonatal injection of semiallogeneic cells in BALB/c mice induces a state of chimerism that promotes the differentiation of donor-specific CD4+ T cells toward the Th2 phenotype. Polyclonal T-B cell interactions occur in this model between host Th2 and donor B cells, resulting in the production of IgE Abs. IgE production and Th2-priming are critically dependent upon the early production of IL-4. Our data in the present paper demonstrate that: 1) IgE synthesis and the up-regulation of MHC class II and CD23 molecules on B cells are independent of beta 2m expression in the host, 2) no difference in the induction of CD4 alloreactive Th2 cells could be observed between beta 2m-/- and their wild-type control littermates when Th2-priming was measured in adult mice, and 3) the Th2 response and IgE production is induced in the complete absence of beta 2m-dependent T cells both in the host and in the inoculum. Therefore, using a variety of assays, we could not demonstrate diminished responses in mice with a disrupted beta 2m gene in this model of Th2-mediated allogeneic interaction, indicating that beta 2m-dependent NK1.1+ and CD8+ T cells are not required for the generation of alloreactive Th2 responses in vivo.

Malignant neoplasms in cyclosporin A-induced autoimmunity (CyA-AI).

Lethally X-irradiated LEW rats reconstituted with syngeneic bone marrow and given low-dose Cyclosporine A (CyA) for 5 weeks develop, after withdrawal of CyA, symptoms of disease resembling graft-vs.-host disease (GVHD) as seen after allogeneic bone-marrow transplantation. Symptoms of disease may include acute dermatitis and chronic disease resembling scleroderma. Since anti-class II MHC cytotoxic lymphocytes are generated in this model, it has been proposed as an anti-tumor regimen in humans. We now report that LEW rats treated according to this protocol may, after cessation of CyA administration, paradoxically develop malignant neoplasms. Of 48 experimental animals, 31 developed rapidly progressive subcutaneous and/or intracutaneous tumors commencing at 6 weeks, 13 weeks and 6 months after cessation of CyA. Tumors were of mesenchymal origin, usually high-grade sarcoma, adenocarcinoma or both mesenchymal and epithelial tumors. Such tumor incidence exceeded the incidence of tumor growth in X-irradiated controls, and in rats subjected to thymectomy prior to X-irradiation and CyA administration. CyA by itself induced no tumors. Our results show that total body X-irradiation is required for tumor development but that the presence of CyA-induced autoimmune disease increases the incidence significantly.

Macrophage infiltration at the neuromuscular junction does not contribute to AChR loss and age-related resistance to EAMG.

Aged rats resistant to acetylcholine receptor loss in passive transfer experimental autoimmune myasthenia gravis (EAMG) do not reveal infiltrating macrophages at the neuromuscular junction (NMJ) as observed in susceptible rats. It was investigated whether this age-related resistance is due to impaired macrophage function in these aged rats. Reconstitution of aged rats with bone marrow from young donors did not lead to macrophage infiltration, nor did it abolish resistance to EAMG. Subsequently, it was investigated whether macrophages are a primary cause of acetylcholine receptor (AChR) loss in EAMG or are attracted to the NMJ secondary to tissue damage. In lethally irradiated young susceptible rats infiltrating macrophages were absent from the NMJ. However, similar AChR losses were observed in irradiated and non-irradiated rats. These results suggest that macrophages do not contribute to acetylcholine receptor loss in the effector phase of passive transfer EAMG and that age related resistance to passive transfer EAMG is not primarily determined by the absence of infiltrating macrophages.

Multiple effects of cyclosporin A on the thymus in relation to T-cell development and autoimmunity.

The immunosuppressive reagent cyclosporin A paradoxically is able to generate autoimmunity. Cyclosporin A is known to interfere with the signal transduction upon T-cell receptor cross-linking, an event required not only for T-cell activation in the periphery but also for T-cell maturation and selection in the thymus. In this review the multiple effects of cyclosporin A on the thymus are explained in terms of a differential response of thymocyte subsets upon recognition of MHC-peptide complexes. This hypothesis holds the effects of cyclosporin A on the thymic stromal cells to be secondary to its effect on the thymocytes: generation of autoreactive cells is the result of direct interference with negative selection and is not due to the partial disappearance or alteration of thymic stromal cells and, depending on the concomitant reestablishment of the autoregulatory T-cell circuit in the periphery, autoimmunity may develop.

The lesions of cyclosporine-induced autoimmune disease can be equally well elicited by CD4 or CD8 effector T cells.

Lethally irradiated Lewis rats reconstituted with syngeneic bone marrow and given cyclosporine for 4 weeks develop a graft-versus-host-like disease upon withdrawal of CsA. Autoreactive T cells inducing this thymus-dependent autoimmune disease, termed CsA-AI, are demonstrable by adoptive transfer, provided regulatory cells in recipient rats are eliminated. Earlier studies have not unequivocally defined the effector T cells responsible for development of CsA-AI. Some of these studies suggest that both CD4 and CD8 T cells are required, while other studies indicate disease transfer by CD4 or CD8 T cells only. To further clarify this issue, it was necessary to study putative effector T cells in a well-defined setting. Hence, adoptive transfer studies were designed wherein the effect of the T cells of interest could be studied without being influenced by T cells of unwanted origin. Accordingly, recipient rats were thymectomized prior to irradiation, lymph node cells (LNC) from diseased donor rats were depleted of CD4 or CD8 cells before adoptive transfer, and recipients were treated in vivo with CD4- or CD8-depleting mAb. The results showed that CsA-AI developed after adoptive transfer with LNC depleted of either CD4 or CD8 cells. Analysis of PBL and of histologic specimens confirmed the absence of the depleted subset. In both instances, the typical MHC class II expression on keratinocytes and the presence of ED1+ macrophages were identical to the lesions in the primary donors, where both CD4 and CD8 T cells were present. Analysis of the T cell Receptor beta-chain variable region repertoires revealed that their expression patterns in LNC of diseased donors or recipients was comparable to that in normal thymus or LNC--hence, there was no restricted BV repertoire. Taken in toto, our observations indicate that CsA-AI involves both CD4 and CD8 T cells, and that these subsets can generate identical macroscopic and microscopic signs of disease.

Effect of in vivo rapamycin treatment on de novo T-cell development in relation to induction of autoimmune-like immunopathology in the rat.

Cyclosporine (CsA) and FK506 are structurally unrelated immunosuppressants, but function in similar ways. FK506 and rapamycin (RAPA), on the other hand, have structural similarities, but act by different mechanisms to yield immunosuppression. Besides their immunosuppressive action, CsA and FK506 are known to interfere with T-cell development. CsA treatment after lethal X-irradiation and syngeneic bone marrow transplantation results in autoimmune disease, which is referred to as CsA-induced autoimmunity. In this study, we examined the effect of RAPA on T-cell development by flow cytometry and immunohistochemistry in female Lewis and Brown Norway rats. Irradiation and syngeneic bone marrow transplantation were performed before a 4-week course of RAPA administration to determine de novo T-cell development in relation to possible autoimmune phenomena. RAPA interfered with the maturation of thymocytes to the CD4+CD8+ DP stage, which resulted in a relative increase in TCRalphabeta(-) immature thymocytes, localized in a rim along the outer cortex. The thymus of RAPA-treated animals had a thinner cortex, leading to stronger thymic atrophy. In the periphery, only a few T cells were observed at the end of RAPA treatment. In the Lewis rat, a normal CD4/CD8 T-cell ratio and an increased Th1/Th2 ratio was observed within the T-cell population. Six weeks after cessation of RAPA therapy, the T-cell compartment was restored to normal, with respect to number and phenotype. In Brown Norway rats, however, T-cell areas were barely detectable at the end of RAPA treatment. The CD4/CD8 T-cell ratio was decreased as a result of a lower number of CD4 T cells; the Th1/Th2 ratio was increased but Th2 remained higher. Similar to Lewis rats, the situation was almost normalized 6 weeks after cessation of RAPA administration. However, Brown Norway rats, in contrast to Lewis rats, showed T-cell infiltration and concomitant induction of MHC class II in the submandibular salivary gland, as well as insulitis, in the pancreas. Possible relationships to Sjogren's disease and diabetes remain to be established.

Susceptibility to clinically manifest cyclosporine A (CsA)-induced autoimmune disease is associated with interferon-gamma (IFN-gamma)-producing CD45RC+RT6- T helper cells.

Lethally irradiated Lewis (LEW) rats reconstituted with syngeneic bone marrow and given CsA for a 4-week period, develop, upon withdrawal of CsA, a graft-versus-host-like disease, so-called CsA-induced autoimmunity (CsA-AI). This T cell-mediated autoimmune disease is thymus-dependent; it is generally held that this disease is a consequence of aberrant T cell recovery brought about by CsA. In this study we determined mononuclear cell subsets phenotypically by tri-colour flow cytometry. A strong decrease in recent thymic emigrants (Thy1.1+, TCR alpha beta +) was observed as a consequence of CsA treatment, eventually resulting in decreased absolute peripheral T cell numbers. In these rats no altered CD4:CD8 T cell ratio was observed before onset of CsA-AI; CD4+ and CD8+ cells consisted predominantly of monocytes (CD4dim+, TCR alpha beta-) and natural killer cells (CD8+, TCR alpha beta-), respectively. LEW rats, x-irradiated, syngeneic bone marrow-reconstituted and treated with CsA, showed a marked and persistent, relative expansion of mature CD45RC+, RT6- Th cells. In contrast, Brown-Norway rats treated in a similar fashion, or LEW rats subjected to either CsA treatment or x-irradiation, did not show a comparable expansion of mature CD45RC+, RT6- Th cells, nor did these animals develop CsA-AI. The CD45RC+, RT6- Th cells produced IL-2, and moreover constituted the only Th subset producing IFN-gamma upon stimulation, and therefore were considered as Th1-like effector cells. These results are consistent with the view that a persistent preponderance of Th1 cells and not the mere presence of autoreactive cells determines whether or not clinically manifest CsA-AI will occur.

Cutaneous immunopathology of cyclosporin-A-induced autoimmunity in the rat.

Syngeneic bone marrow transplantation following lethal X-irradiation and subsequent administration of cyclosporin A (CsA) results after cessation of CsA treatment in an autoimmune disease which is thymus dependent and resembles graft-versus-host disease. The chronic dermal changes of this experimental autoimmune model have similarities with human scleroderma in terms of skin histopathology. In this study we evaluated the possible role of different effector leukocytes in the rat model of CsA-induced autoimmunity (CsA-AI) by examining the skin by immunohistology. In the acute phase both CD4+ and CD8+ TCR alpha beta + T-cells together with activated ED1+ macrophages and class II MHC-upregulated keratinocytes were seen in the epidermis; no selective use of TCR V beta was observed. Few TCR alpha beta + T-cells were seen in the dermis where CD4+ ED2+ macrophages were abundant. With the change from acute to chronic, scleroderma-like lesions the CD4+ T-cells disappeared from the epidermis and the TCR alpha beta + cells were now almost exclusively CD8+; both class II MHC-upregulated keratinocytes and macrophages persisted. Changes in TCR gamma delta + T-cells were not observed in the acute or chronic phase. As a possible effector mechanism CD4+ T-cells in the acute-phase of CsA-AI may cause the observed activation of macrophages and keratinocytes. Furthermore, CD4+ T-cells may be necessary for the homing of the CD8+ T-cells in the epidermis. Especially the activated keratinocytes are suspected of being the target cells which may perpetuate the ongoing autoimmune response into the chronic phase as established by CD8+ T-cells only.

X-irradiation of the thymus is not required for the induction of cyclosporine-induced autoimmunity.

The thymus-dependent model of cyclosporine-induced autoimmunity (CsA-AI) in the Lewis rat requires a lethal total body X-irradiation and rescue with syngeneic or autologous bone marrow and cyclosporine (CsA) administration for at least 4 weeks; two to three weeks after cessation of CsA, the animals develop a graft-versus-host-like disease. The obligatory role of the thymus in the etiology of CsA-AI has been established unequivocally, but the way in which disease is thymus dependent is a topic of debate. In the present study we demonstrate that the model of CsA-AI requires the presence of a thymus for at least 2 weeks after total body irradiation and CsA administration, but that X-irradiation of the thymus itself is not necessary to bring about disease. Transplantation of neonatal thymus shows in addition that in the absence of X-irradiation of the thymus, CsA therapy is required to generate autoreactive cells, but that disease occurs only if peripheral autoregulatory cells are eliminated by X-irradiation.

Differential effects of X-irradiation and cyclosporin-A administration on the thymus with respect to the generation of cyclosporin-A-induced autoimmunity.

Cyclosporin A (CsA), a potent inhibitor of T-cell activation, has been shown to have several effects on thymocyte maturation, thymic stromal cells, and the generation of autoreactive T cells. In Lewis rats, the combination of lethal irradiation, syngeneic bone marrow transplantation, and a 4-week course of CsA administration causes the development of an autoimmune disease (CsA-AI) resembling allogeneic graft-versus-host disease. This occurs upon withdrawal of CsA, provided the thymus receives irradiation and is present during CsA treatment. In this study, the separate effects of irradiation or CsA treatment on thymic stromal cells and thymocytes, compared to the combinatory effects, were examined using immunohistochemistry and tricolor flow cytometric analysis. CsA treatment causes an involution of the thymic medulla and a strong reduction of the cell number of thymocytes and stromal cells residing in the medulla. However, within the remaining medullary area, changes in cell distribution and antigen density on these cells were not observed. Irradiation on the other hand causes a strong depletion of thymocytes. The thymocyte population is recovered within 2 weeks and a cortical and medullary region can be distinguished. CsA treatment in combination with irradiation results in a strongly inhibited recovery of the medulla during CsA treatment, whereas the cortex recovers to normal size and morphology. The presence of the medullary IDC and epithelial cells is reduced proportionally to the small size of the medulla. However, the distribution of these stromal cells is normal. During the CsA administration, the thymuses from irradiated and CsA-treated rats are very similar to thymuses from CsA-treated rats. In conclusion, no changes specific for irradiation plus CsA treatment have been observed. Regarding the distribution and size of medullary stromal cells and residing thymocytes, thymuses from irradiated and CsA-treated rats hardly differ from the thymuses from rats treated only with CsA. Therefore, irradiation seems essential in the generation of CsA-AI by eliminating suppressor-cell circuits in the periphery.

The effects of in vivo cyclosporin A administration on rat thymic dendritic cells.

Cyclosporin A (CsA) induces a graft-versus-host-like disease (GVHD) in lethally irradiated Lewis rats reconstituted with syngeneic bone marrow. The role of the thymus in the generation of disease has been unequivocally established. It has been suggested that the CsA-induced disappearance of thymic dendritic cells (DC) is responsible for the generation of the autoaggressive cells. In this study we quantify the loss of DC upon in vivo CsA administration in normal and bone marrow-reconstituted rats using an isolation technique. The phenotype of the DC is determined using MoAbs recognizing antigens which are expressed on thymic medullary DC. Furthermore, the functional aspects are assessed by determining the antigen presentation capacity. Short-term CsA exposure clearly affects the number of DC isolated from the thymus in a concentration-dependent manner. However, in all instances a substantial number of DC can be isolated from CsA-treated animals. These isolated DC exhibit an identical phenotype and function as DC isolated from control animals. Therefore, the partial deficiency of DC can not be held as essential for loss of tolerance.