Chandra Nataraj

Receptors for prostaglandin E2 that regulate cellular immune responses in the mouse

Production of prostaglandin E(2) (PGE(2)) is enhanced during inflammation, and this lipid mediator can dramatically modulate immune responses. There are four receptors for PGE(2) (EP1-EP4) with unique patterns of expression and different coupling to intracellular signaling pathways. To identify the EP receptors that regulate cellular immune responses, we used mouse lines in which the genes encoding each of the four EP receptors were disrupted by gene targeting. Using the mixed lymphocyte response (MLR) as a model cellular immune response, we confirmed that PGE(2) has potent antiproliferative effects on wild-type responder cells. The absence of either the EP1 or EP3 receptors did not alter the inhibitory response to PGE(2) in the MLR. In contrast, when responder cells lacked the EP2 receptor, PGE(2) had little effect on proliferation. Modest resistance to PGE(2) was also observed in EP4-/- responder cells. Reconstitution experiments suggest that EP2 receptors primarily inhibit the MLR through direct actions on T cells. Furthermore, PGE(2) modulates macrophage function by activating the EP4 receptor and thereby inhibiting cytokine release. Thus, PGE(2) regulates cellular immune responses through distinct EP receptors on different immune cell populations: EP2 receptors directly inhibit T cell proliferation while EP2 and EP4 receptors regulate antigen presenting cells functions.

Angiotensin II regulates cellular immune responses through a calcineurin-dependent pathway

The renin-angiotensin system (RAS) is a key regulator of vascular tone and blood pressure. In addition, angiotensin II also has a number of cellular effects that may contribute to disease pathogenesis. Using Agtr1a(-/-) mice, which lack AT(1A) receptors for angiotensin II, we have identified a novel function of the RAS to modulate the immune system. We find that angiotensin II, acting through type 1 (AT(1)) receptors on immune cells, triggers the proliferation of splenic lymphocytes. These actions contribute to the vigor of cellular alloimmune responses. Within lymphoid organs, sufficient components of the RAS are present to activate AT(1) receptors during an immune response, promoting cell growth. These actions require activation of calcineurin phosphatase. In an in vivo model of cardiac transplantation, the absence of AT(1) signaling accentuates the immunosuppressive effects of the calcineurin inhibitor cyclosporine. We conclude that inhibition of AT(1) receptor signaling should be useful as an anti-inflammatory and immunosuppressive therapy. Furthermore, the actions of the RAS to promote lymphocyte activation may contribute to inflammation that characterizes a number of diseases of the heart and the vascular system.

A Role for Fractalkine and Its Receptor (CX3CR1) in Cardiac Allograft Rejection

The hallmark of acute allograft rejection is infiltration of the inflamed graft by circulating leukocytes. We studied the role of fractalkine (FKN) and its receptor, CX3CR1, in allograft rejection. FKN expression was negligible in nonrejecting cardiac isografts but was significantly enhanced in rejecting allografts. At early time points, FKN expression was particularly prominent on vascular tissues and endothelium. As rejection progressed, FKN expression was further increased, with prominent anti-FKN staining seen around vessels and on cardiac myocytes. To determine the capacity of FKN on endothelial cells to promote leukocyte adhesion, we performed adhesion assays with PBMC and monolayers of TNF-α-activated murine endothelial cells under low-shear conditions. Treatment with either anti-FKN or anti-CX3CR1-blocking Ab significantly inhibited PBMC binding, indicating that a large proportion of leukocyte binding to murine endothelium occurs via the FKN and CX3CR1 adhesion receptors. To determine the functional significance of FKN in rejection, we treated cardiac allograft recipients with daily injections of anti-CX3CR1 Ab. Treatment with the anti-CX3CR1 Ab significantly prolonged allograft survival from 7 ± 1 to 49 ± 30 days (p < 0.0008). These studies identify a critical role for FKN in the pathogenesis of acute rejection and suggest that FKN may be a useful therapeutic target in rejection.

Rho Kinase Promotes Alloimmune Responses by Regulating the Proliferation and Structure of T Cells

Coordinated rearrangements of the actin-myosin cytoskeleton facilitate early and late events in T cell activation and signal transduction. As many important features of cell shape rearrangement involve small GTP-binding proteins, we examined the contribution of Rho kinase to the functions of mature T cells. Inhibitors of the Rho kinase pathway all had similar actions to inhibit the proliferation of primary lymphocyte cultures. Likewise, transfection of the human Jurkat T cell line with a dominant negative, kinase-defective mutant of Rho kinase diminished Jurkat cell proliferation. Furthermore, inhibition of Rho kinase substantially attenuated the program of cytokine gene expression that characterizes T cell activation, blocked actomyosin polymerization, and prevented aggregation of the TCR/CD3 complex colocalized with lipid rafts. These actions are relevant to immune responses in vivo, as treatment with a Rho kinase inhibitor considerably prolonged the survival of fully allogeneic heart transplants in mice and diminished intragraft expression of cytokine mRNAs. Thus, Rho GTPases acting through Rho kinase play a unique role in T cell activation during cellular immune responses by promoting structural rearrangements that are critical for T cell signaling.

Proinflammatory Actions of Thromboxane Receptors to Enhance Cellular Immune Responses

Metabolism of arachidonic acid by the cyclo-oxygenase (COX) pathway generates a family of prostanoid mediators. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting COX, thereby reducing prostanoid synthesis. The efficacy of these agents in reducing inflammation suggests a dominant proinflammatory role for the COX pathway. However, the actions of COX metabolites are complex, and certain prostanoids, such as PGE2, in some circumstances actually inhibit immune and inflammatory responses. In these studies, we examine the hypothesis that anti-inflammatory actions of NSAIDs may be due, in part, to inhibition of thromboxane A2 synthesis. To study the immunoregulatory actions of thromboxane A2, we used mice with a targeted disruption of the gene encoding the thromboxane-prostanoid (TP) receptor. Both mitogen-induced responses and cellular responses to alloantigen were substantially reduced in TP−/− spleen cells. Similar attenuation was observed with pharmacological inhibition of TP signaling in wild-type splenocytes, suggesting that reduced responsiveness was not due to subtle developmental abnormalities in the TP-deficient mice. The absence of TP receptors reduced immune-mediated tissue injury following cardiac transplant rejection, an in vivo model of intense inflammation. Taken together, these findings show that thromboxane augments cellular immune responses and inflammatory tissue injury. Specific inhibition of the TP receptor may provide a more precise approach to limit inflammation without some of the untoward effects associated with NSAIDs.

Rejection of kidney allografts by MHC class I-deficient mice

To evaluate the requirement for CD8+ T cells in kidney transplant rejection, we studied class I-deficient (class I-) mice that had received vascularized renal allografts. Because of the absence of MHC class I expression, these mice are grossly deficient in CD4-CD8+ alpha beta TCR+ cells. Despite the deficiency of CD8+ T cells in naive class I- mice, kidney allografts transplanted into class I- recipients developed significant reductions in renal blood flow and glomerular filtration rate to levels comparable to allograft controls. This functional deterioration was associated with histologic changes consistent with cellular rejection. There were no significant differences in the pattern, severity, or phenotypic character of the cellular infiltrate in allografts transplanted into class I- recipients compared to controls. In fact, substantial numbers of CD8+ T cells were present in these allografts, and the intensity and pattern of anti-CD8 staining was not different from controls. Virtually all of the CD8+ cells in the kidney grafts were class I- and CD4- and co-expressed CD8 alpha and beta chains; the majority were alpha beta TCR+. The CD8+ infiltrating cells were cytotoxic to donor targets but also exhibited activity against class I+ cells bearing self-MHC. Despite the marked CD8+ T cell infiltration of grafts, CD8+ T cells could not be detected by flow cytometry in freshly isolated splenocytes from the class I- recipients of allografts. High levels of circulating anti-class I antibodies were present in the serum of class I- recipients of kidney allografts, and these antibodies had unusual specificity in that they appeared to recognize framework epitopes of MHC class I. Thus, class I- mice readily reject kidney allografts. Although the number of CD8+ alloreactive precursors is substantially reduced in class mice, and their specificities are atypical, the pattern and character of the intra-graft CD8+ cellular response is not significantly altered. Thus, factors unrelated to precursor frequency determine the dimension of the intra-graft CD8+ response. Such factors might include cellular and/or biochemical properties of microenvironment within the graft.

Downregulation of T cell receptor expression by CD8(+) lymphocytes in kidney allografts.

Allospecific CD8(+) T lymphocytes are an important component of the cellular response in allograft rejection. These cells recognize and engage MHC class I antigens, leading to allospecific cytolytic responses and graft rejection. In mouse kidney allografts that survive to 3 wk after transplantation, we noted that the majority of CD8(+) cells do not express surface alpha/beta T cell receptor alpha/beta(TCR), gamma/deltaTCR, or CD3. However, these CD8(+)TCR- cells did express surface markers characteristic of T cells, including Thy1.2, CD2, and CD5. In addition, the CD8(+)TCR- cells expressed mRNA for TCR Vbeta gene families, and nearly half stained positive for cytoplasmic Vbeta8 protein, suggesting that they are T cells that have downregulated alpha/betaTCR protein expression from their cell surfaces. When these surface TCR- cells were isolated from kidney allografts by flow cytometry and cultured in the presence of either allogeneic or syngeneic stimulators, nearly 100% of cells reacquired normal levels of alpha/betaTCR expression with disproportionate usage of Vbeta8 chains. After recovery of their surface TCR expression, the CD8(+)TCR- population demonstrated strong alloreactivity in culture. These results suggest that the substantial number of CD8(+)TCR- cells found in long-term surviving mouse kidney allografts are alpha/beta-T cells that have downregulated their cell surface expression of TCR. While in other systems this phenotype may identify cells that have engaged antigen, our results indicate that loss of TCR expression by CD8(+) kidney graft-infiltrating cells may not depend on antigen engagement and that elements in the microenvironment of the kidney graft play a key role in this process. Factors that modulate expression of TCR by graft-infiltrating lymphocytes may have an important role in regulating rejection responses.

The intragraft CD8+ T cell response in renal allograft rejection in the mouse.

To identify the role of donor class I alloantigens in regulating the CD8+ T cell response to a kidney allograft, we analyzed and compared the CD8+ infiltrate in kidney transplants from MHC class I-deficient (class I-) mouse donors and class I+ controls. One week after transplantation, there was a prominent CD8+ infiltrate in control allografts, whereas CD8+ T cells were virtually absent in grafts from class I- donors. In class I+ allografts, infiltrating CD8+ cells utilized a wide range of T cell receptor (TCR) Vbeta families and their Vbeta usage was similar to that of the systemic CD8+ population. However, there was a modest but significant overrepresentation of cells bearing Vbeta8 in the graft compared with the spleen due to an expansion of CD8+ Vbeta8.3+ cells. This could be detected as early as 1 week and became more pronounced by 3 weeks after transplantation. In 3-week allografts, only 52% of CD8+ cells expressed alphabetaTCR. Among T cells isolated from class I+ grafts, the CD8+ Vbeta8+ cells demonstrated allospecific responses that were numerically larger than responses of the CD8+ Vbeta8- population. In contrast to the early (1 week) time point, significant numbers of CD8+ cells could be isolated from class I- grafts by 3 weeks after transplantation and their Vbeta repertoire resembled that seen in controls. While increasing numbers of CD8+ Vbeta8+ were present in the class I- grafts at 3 weeks, this increase was not statistically significant. Thus, expression of class I alloantigens on a kidney graft plays an important role in regulating the rate of accumulation of CD8+ T cells in rejecting kidney grafts. However, the TCR Vbeta repertoire of the CD8+ T cell infiltrate is largely determined by factors that are independent of normal class I expression on the graft.