Accumulating evidence over the past few years has suggested that angiotensin II might be produced by and act directly on immune cells. For example, Gomez and associates reported that rat leukocytes express angiotensinogen mRNA and synthesize angiotensinogen protein (28
). Moreover, angiotensin peptides are released by rat alveolar macrophages (39
), mouse lymphocytes, and granuloma cells in murine schistosomiasis (14
). Addition of angiotensinogen or angiotensin I peptides to immune cell preparations can augment the release of angiotensin II (14
), implying that these cells possess the requisite enzymatic machinery to perform the complete conversion of angiotensinogen substrate to active angiotensin II. Although some studies suggest that these pathways may be independent of renin and/or ACE (41
), ACE activity and mRNA expression have been documented in macrophages and T cells (42
), and ACE expression may be upregulated in inflammatory conditions (42
). Enhanced expression of ACE in inflammatory cells has been linked to diseases such as sarcoidosis (42
), although its pathophysiological role has not been defined. Local accumulation of ACE has also been identified in atherosclerotic lesions, particularly in regions of inflammatory cell infiltration (48
). In these settings, it has been suggested that local expression and accumulation of ACE may contribute to enhanced production of angiotensin II in tissues.
The potential for angiotensin II to directly modulate inflammatory cell functions was suggested by the observation that human mononuclear leukocytes express specific binding sites for angiotensin II (27
). Tsutsumi and associates subsequently demonstrated large numbers of binding sites for angiotensin II in rat spleen and found that these sites were primarily AT1
). This is consistent with our receptor autoradiography findings of diffuse AT1
-specific binding in mouse spleen, depicted in Figure a. Our studies further show that these are predominantly AT1A
receptors and that AT1A
receptors are expressed in a variety of splenocyte populations including T cells, macrophages, and B cells.
Effects of angiotensin II to modulate T-cell functions have also been suggested by previous studies. In a preliminary report, Vance and Kelly reported that proliferation of a nephritogenic T-cell clone was significantly augmented by the addition of angiotensin II and that this effect seemed to be mediated by AT1
). In contrast, Simon and associates reported inhibition of PHA-induced proliferation by angiotensin II (50
). Thus, although the data are conflicting, there are several reports suggesting that angiotensin II modulates immune functions and that this peptide may have direct effects on T-cell proliferation. In the experiments described here, we find that exposure of splenocytes to angiotensin II, with no other exogenous stimulus, is sufficient to trigger proliferation. This response is blocked by specific AT1
receptor antagonists and it is absent in cells from Agtr1a–/–
mice, indicating that these effects are mediated by AT1A
To determine whether the actions of angiotensin II to stimulate lymphocyte proliferation play a role in modulating immune responses, we used the MLR as a model of the cellular alloimmune response. The MLR is designed to mimic the conditions that might occur in a transplanted organ when recipient immune cells are activated by recognition of foreign MHC antigens expressed on the donor tissue. When the responder cell population came from Agtr1a–/– mice that lack AT1A receptors, we found that proliferative responses in MLR were significantly reduced, by approximately 50%. Proliferation was also reduced when purified T cells from Agtr1a–/– mice were used as responders. Because AT1 receptors are also normally expressed on antigen-presenting cells, we tested whether the response would be affected if AT1A receptors were absent from stimulator cells that provide the antigenic stimulus to trigger the MLR. In this case, proliferation was similar to that induced by wild-type stimulators. Taken together, these data suggest that reduced proliferation of AT1A receptor–deficient cells in MLR is due to the absence of direct effects of angiotensin II on responder cell populations, including T cells.
We have extensively analyzed the lymphoid organs of Agtr1a–/– mice by size, cell numbers, and cell constituents and have found no discernible differences compared with wild-type animals (see Table ). Nonetheless, to ensure that the differences in MLR were not due to a subtle developmental defect caused by the absence of AT1A receptors, we also performed MLR experiments in wild-type cells in the presence of an ACE inhibitor or an AT1 receptor antagonist. Both inhibition of angiotensin II production and blockade of AT1 receptors produced a dose-dependent inhibition of the proliferative response. The level of inhibition achieved by pharmacologic blockade, approximately 50% of normal, was similar to that seen in the genetic experiments. The similarity between the effects of ACE inhibition and AT1 receptor blockade suggests that virtually all the effects of angiotensin II to modulate T-cell proliferation are mediated by AT1 receptors. These experiments also suggest that adequate components of the RAS are present in cultured splenocytes to produce angiotensin II in sufficient quantities to induce cellular effects. Therefore, these studies identify a “tissue RAS” in lymphoid organs that contributes to the regulation of cellular immune responses.
The differential effects of the Agtr1a
mutation on proliferation induced by anti-CD3 antibody compared with the nonspecific mitogen concanavalin A suggest that the actions of AT1A
receptors to modulate T-cell proliferation may be specific for stimulation through the TCR/CD3 complex. Engagement of the T-cell receptor with antigen in the setting of appropriate costimulation causes a brisk increase in intracellular calcium (51
). This leads to activation of calcineurin, a calcium- and calmodulin-dependent phosphatase (34
) that dephosphorylates NFATs (34
). Once these proteins are dephosphorylated by calcineurin, they translocate to the nucleus where they directly activate transcription of genes that promote T cell activation and proliferation (55
receptor stimulation is coupled to increased intracellular calcium levels in a number of systems (1
) including human PBMCs (56
). Accordingly, we considered the possibility that stimulation of AT1
receptors on lymphocytes leads to an increase in intracellular calcium concentration and that this AT1
-mediated calcium signal triggers calcineurin and NFAT activation. In support of this possibility, recent studies have demonstrated that AT1
receptor stimulation is sufficient to activate the calcineurin-NFAT pathway in cardiac myocytes and this activation leads to alterations of cell phenotype that can be inhibited by cyclosporine. In the heart, this pathway seems to be an important cause of myocardial hypertrophy (37
To examine the role of the calcineurin-NFAT pathway in the angiotensin II–mediated lymphocyte proliferation, we used cyclosporine, a specific inhibitor of calcineurin phosphatase (52
). Cyclosporine completely blocked the ability of angiotensin II to induce proliferation of cultured splenic lymphocytes, demonstrating that calcineurin is required for stimulation of lymphocyte proliferation by AT1
receptors. To determine whether this pathway is operative during an immune response in an intact animal, we studied the effects of the Agtr1a
mutation on cyclosporine responses in a mouse model of cardiac transplant rejection. In this model, donor and recipient are completely mismatched at the MHC locus, resulting in a very aggressive acute cellular rejection response to the allograft. Using these strain combinations, doses of cyclosporine of approximately 100 mg/kg per day are generally required to achieve prolongation of graft survival. In wild-type animals, doses of cyclosporine that were an order of magnitude below this therapeutic level had no effect on graft survival. In contrast, these small doses of cyclosporine caused a significant prolongation of graft survival in mice lacking AT1A
receptors on their lymphocytes. This result demonstrates the relationship between AT1
receptor signaling and calcineurin activation in an alloimmune response in vivo and suggests that these pathways cooperate to promote allograft injury in this model. Because AT1A
receptor expression is normal in the donor heart, these actions result from the absence of AT1A
receptor signaling in recipient tissues.
Although these transplant studies provide proof of the concept that AT1
receptor signaling on immune cells contributes to in vivo alloimmune responses, the effect in this powerful model of acute rejection is relatively modest. However, it is possible that the relative contribution of this pathway may be more significant in more indolent alloimmune responses, such as in chronic allograft rejection or in patients who are being treated with therapeutic doses of immunosuppressive agents. In this regard, a number of studies in animal models have demonstrated an important role for the renin-angiotensin system in the pathogenesis of chronic allograft rejection. For example, several groups have demonstrated that AT1
receptor blockade significantly ameliorates kidney injury in rat models of chronic renal allograft rejection and that these effects are not due to reduction in systemic blood pressure (22
). In a model of chronic rejection of cardiac allografts, AT1
receptor blockade significantly ameliorates intimal proliferation of coronary arteries, the pathological lesion that characterizes chronic rejection (20
). The efficacy of RAS antagonists in immunological diseases has largely been attributed to effects on vasculature and production of inflammatory mediators. Our studies suggest that blockade of direct actions of angiotensin II to promote the T-cell response may also contribute to these beneficial effects.
In summary, these studies identify potent effects of the RAS to modulate the immune system. Because inflammation is critical to the pathogenesis of diseases such as atherosclerosis (57
), the effects of angiotensin II on immune cell activation may contribute to the pathological effects of RAS dysregulation in cardiovascular disease. The enhanced sensitivity of AT1A
receptor–deficient mice to the immunosuppressive actions of cyclosporine suggests a rationale for the use of RAS antagonists in patients with organ transplants who are being treated with calcineurin inhibitors. In this setting, antagonism of the RAS may potentiate the immunosuppressive effects of calcineurin inhibition reducing the doses required to achieve adequate immunosuppression and therefore limiting dose-related toxicity.