Cyclosporine: A New Immunosuppressive Agent for ...

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Cyclosporine: A New Immunosuppressive Agent for Organ Transplantation DAVID J. COHEN, M.D.; ROLF LOERTSCHER, M.D.; MARIO F. RUBIN, M.D.; NICHOLAS L. TILNEY, M.D.; CHARLES B. CARPENTER, M.D.; and TERRY B. STROM, M.D.; Boston, Massachusetts Cyclosporine, a cyclic endecapeptide of fungal origin, has recently been released for use in clinical transplantation. Trials in kidney, heart, liver and bone marrow recipients were encouraging: 1-year graft survival rates were 7 0 % to 8 0 % for kidney and heart recipients, and 6 0 % to 6 5 % for liver allograft recipients. Cyclosporine is also effective in treating bone marrow recipients with acute graft-versushost disease. The drug selectively inhibits T-helper cell production of growth factors essential for B cell and cytotoxic T-cell differentiation and proliferation, while allowing expansion of suppressor T-cell populations. Drug absorption varies greatly, necessitating monitoring of drug level and individualization of therapy. Nephrotoxicity is the most frequent side effect of cyclosporine. An increased incidence of B-cell lymphomas seen when cyclosporine was used in conjunction with cytotoxic agents or anti-lymphocyte globulin has very rarely been observed when concomitant immunosuppression has been limited to low-dose corticosteroids. Lower initial doses of cyclosporine followed by more rapid tapering may reduce the incidence of nephrotoxicity without compromising improved graft outcome. IN THE EARLY 1960S, azathioprine was introduced for maintenance immunosuppression in clinical renal transplantation. Shortly thereafter, with the addition of highdose corticosteroid anti-rejection therapy and lower dose steroid adjunctive therapy, an acceptable regimen for use in cardiac, renal, and hepatic transplantation was established that has undergone little change until the present. The use of anti-lymphocyte globulin, either as anti-rejection therapy or as prophylactic immunosuppression immediately after engraftment, has remained a contentious issue. The salubrious effects of previous blood transfusions on kidney graft survival, recognized in the 1970s, led to their widespread use for pre-transplant conditioning. Immunosuppressive protocols have undergone a major change with the introduction of cyclosporine in renal, cardiac, hepatic, pancreatic, and bone marrow transplantation. This agent is a potent immunosuppressant, more effective than standard drug therapies in preventing rejection. A metabolite of the soil fungi Cylindrocarpum lucidurn Booth and Tolypocladium inHatum Gams, cyclosporine is a cyclic endecapeptide with a molecular weight of 1203. It includes several A'-methylated amino acids and one new amino acid (Figure 1). Cyclosporine exerts its • From the Departments of Medicine and Surgery. Laboratory of Immunogenetics and Transplantation. Brigham and Women's Hospital, and the Charles A. Dana Research Institute, the Harvard.Thorndike Laboratory. Department of Medicine. Beth Israel Hospital and Harvard Medical School; Boston. Massachusetts. Annals of Internal Medicine. 1984;101:667-682.

most well characterized effects on T-lymphocyte helper/ inducer subpopulations; B cell, macrophage, granulocyte and natural killer cell functions appear unaltered. The drug is not myelosuppressive, and in-vitro growth of bone marrow-derived myeloid, erythroid, and B-lymphoid cell lines remains unimpaired (1-8). Cyclosporine is not a panacea, however, as side effects, sometimes serious, are common, and immunologic graft loss still occurs, albeit at a reduced rate. Mechanism of Action

Initial studies by Borel and coworkers (1, 9) showed a suppressive effect of cyclosporine on antibody production in a mouse model. This effect was subsequently shown in this and in other systems to be mediated by inhibition of the T-cell help required for B-cell activation (9, 10). The polyclonal B-cell response to Escherichia coli lipopolysaccharide—a direct stimulator of B cells—was not blunted by the presence of cyclosporine in the culture medium, nor was the activation of B cells by mitogenic T-independent antigens such as Epstein-Barr virus (1012). In fact, the growth of Epstein-Barr virus-infected B lymphoblastoid cell lines was favored by cyclosporine (13). Time course studies clearly showed that although administration of cyclosporine at the time of antigenic challenge suppressed antibody production, on-going antibody responses were not impaired (9). In fact, cyclosporine did not interrupt primary antibody responses unless introduced during the early steps of B-cell activation, before acquisition of responsiveness to B-cell growth factor (13). A direct effect of cyclosporine on B-cell function was discerned, however, in studies using anti-immunoglobulin as a surrogate to antigen. Here, cyclosporine interrupted an early, premitotic phase of B-cell activation (14). In contrast, direct inhibitory effects on T-cell responses were easily shown in several in-vitro systems (15-21). As with antibody production, suppression only occurred when the drug was present at initiation or in the early stages of test cultures. The effector functions of previously activated cytolytic or suppressor T-cells were not altered (15, 16). Similarly, pretreatment of T lymphocytes followed by removal of the drug before antigen or lectin exposure was without effect on subsequent responsiveness (17). The currently accepted model of the T-cell activation sequence requires expression and recognition of two types of signals: antigen or mitogen, and cell-derived "growth factors." (Figure 2). Antigen is presented by ©1984 American College of Physicians

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Figure 1. Chemical structure of cyclosporine.

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accessory cells to T-inducer cells, which are thereby triggered to release a factor (or factors) that target the macrophage. The macrophage is induced to release interleukin-1, previously termed lymphocyte activating factor. This monokine stimulates release of interleukin-2, previously termed T-cell growth factor, from antigen primed T-helper cells. Interleukin-2 acts on antigen-primed cytolytic T-cell precursors, other T-helper cells, and perhaps on T-suppressor cells, and is essential for their continued growth and proliferation (22). In the absence of interleukin-2, blastogenesis may occur, but cells are unable to enter the S-phase of their growth cycle (23). The manner in which cyclosporine interrupts this cascade of T-Tmacrophage interactions, thus preventing T-cell activation, differentiation and proliferation, has been studied in vitro by many workers. Cyclosporine was convincingly shown to impair interleukin-2 production by naive as well as activated T-helper populations in several different models. Hess and coworkers (17-19) extensively studied the effects of this agent on the human mixed lymphocyte culture. Suppression of proliferation and cytotoxic T-lymphocyte generation by cyclosporine in primary and secondary mixed lymphocyte cultures was associated with a dramatic inhibition of interleukin-2 production. Similar results were obtained in murine and guinea pig cultures (20-25). Bunjes and associates (25) showed that the conconavalin-A-induced release of interleukin-1 by some, but not all, macrophages was also impaired, presumably secondary to an effect on the T-cell help required for this function, because T-independent induction of macrophage interleukin- 1 release by lipopolysaccharide was resistant to the effects of cyclosporine. These results were confirmed by Palacios (21). Conflicting results have been noted in attempts to ascertain whether cells activated in cyclosporine-treated cultures acquire the ability to respond to interleukin-2 or whether the drug prevents expression of the interleukin-2 receptor. Larsson (20) found that the lectin-induced ex668

pression of interleukin-2 responsiveness was inhibited in murine lymphocytes, such that even when cells were washed free of cyclosporine, they failed to proliferate in response to interleukin-2. Palacios (21) provided evidence to support this concept. These studies, however, did not discriminate between a decrease in interleukin-2 receptor expression and post-receptor defects. More recently, direct evidence showing that cyclosporine does not prevent expression of interleukin-2 receptors on activated T cells has been obtained by use of an anti-interleukin-2 receptor monoclonal antibody (26). Orosz and associates (27, 28) provided indirect confirmation with cloned murine T lymphocytes. Furthermore, addition of exogenous interleukin-2 to human allogeneic mixed lymphocyte cultures treated with cyclosporine restored proliferation, although those cultures still failed to generate cytolytic T cells (18, 19). We found full restoration of allogeneic responsiveness with the addition of lymphokines rich in interleukin-2 to cells cultured in the presence of cyclosporine (29, 30), as did Bunjes and associates (25). Although cyclosporine inhibited the release of various lymphokines, including cytotoxic differentiation factor (31), and gamma interferon (32, 33), macrophage responsiveness to lymphokine was not impaired (3). Coincident with the depression of T-helper/inducer cell function, inhibition of interleukin-2 production, and the subsequent abrogation of proliferation and cytotoxic T-cell development, there appeared to be relative sparing of T-suppressor subpopulations by cyclosporine. Studies by Hess and associates (34) showed the emergence of suppressor cells in primary human mixed lymphocyte cultures treated with cyclosporine, despite the virtual absence of proliferation. Similar results were seen in murine cultures (30, 35). The drug itself did not induce suppressor cells in the absence of antigen or lectin stimulation, and at sufficiently high doses inhibited suppressor cell development (17, 34). Doses of cyclosporine that markedly suppressed the induction of cytolytic T cells allowed the differentiation of potent alloantigen-activated

November 1984 • Annals of Internal Medicine • Volume 101 • Number 5

suppressor cells. With the increasing evidence that the generation of alloreactive T-suppressor cells depends on the availability of specific differentiation factors distinct from interleukin-1 and interleukin-2, it would appear that the environment created by cyclosporine uniquely favors production of T-suppressor cell-inducing lymphokines (36, 37). An analysis of the inhibitory effects of cyclosporine in guinea pig mixed lymphocyte culture showed that release of a genetically restricted suppressor factor by T cells was not impaired (38). Recently Mohagheghpour and associates (39) showed activation of suppressor-inducer T cells in cyclosporine-treated human cultures, whereas cytolytic T cell-inducer cell generation was inhibited. Considerable experience has accumulated with the use of cyclosporine in experimental transplantation, where remarkable successes have been achieved. Initial studies in a rat heart transplant model showed that indefinite allograft survival could be readily induced by short-term treatment with cyclosporine (40-42). A permanent state of unresponsiveness was established after 7 to 14 days of immunosuppression. Prolonged acceptance of cardiac allografts was also induced in pigs, for the first time, and primates, although continued drug administration was necessary (43-46). Results with renal transplant models were comparable: tolerance to fully histoincompatible kidneys was seen in rats and rabbits, whereas rejection followed discontinuation of drug treatment in the dog and in primates (47-60). Mixed but generally favorable results have also been reported with pancreatic islet cell and segmental pancreatic allografts in dogs, rats, and baboons, although very high doses of cyclosporine were frequently required, and rejection of pancreatic tissue was seen whereas simultaneously allografted kidneys or hearts remained unaffected (61-69). Studies on the immunosuppressive effects of cyclosporine have also examined lung (70), skin (71-73), liver (74), small intestine (75, 76), muscle (77), nerve (78), ovary (79), and cornea (80) transplant models. Deeg and coworkers (81) and Tutschka and coworkers (82) have reported facilitation of bone marrow transplant engraftment and induction of tolerance (graft to host) in the dog and rat, respectively. Tolerance induction was achieved in rats across major histocompatibility barriers, but not in any other species (81, 82). In the canine model, until recently, only minor histocompatibility differences could be overcome (82). The observation that a prolonged state of tolerance was created after only limited periods of drug treatment led investigators to probe its immunologic basis. Strong evidence has emerged showing that suppressor cells may be important in maintaining this unresponsive state. In cyclosporine-treated bone marrow chimeras, Tutschka and colleagues (84) have shown the presence of splenocytes capable of suppressing the donor T-cell responses to host antigens. Similarly, lymphocytes from cyclosporine-treated rats showing tolerance to cardiac allografts prevented the generation of cytotoxic T-cells in mixed lymphocyte culture. Cyclosporine treatment alone did not induce suppressor cells in unprimed animals but

Helper T Lymphocyte

Allogenelc Cell

Cytotoxic T Lymphocyte

MacrophageStimulating Lymphokine

Cytotoxic T Cell

Promote Differehtiation of B Lymphocytes (antibody secretion)

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