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C Nataraj

Publications and source records attributed to C Nataraj.

17 recordsLinked to original sources
PubMed2001

Receptors for prostaglandin E(2) 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.

Animals↗
PubMed2000

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, CX(3)CR1, 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-alpha-activated murine endothelial cells under low-shear conditions. Treatment with either anti-FKN or anti-CX(3)CR1-blocking Ab significantly inhibited PBMC binding, indicating that a large proportion of leukocyte binding to murine endothelium occurs via the FKN and CX(3)CR1 adhesion receptors. To determine the functional significance of FKN in rejection, we treated cardiac allograft recipients with daily injections of anti-CX(3)CR1 Ab. Treatment with the anti-CX(3)CR1 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.

Animals↗
PubMed2000

Stimulation of thymocyte proliferation by phosphorothioate DNA oligonucleotides.

DNA is a complex macromolecule the immunological properties of which depend on short sequence motifs called CpG motifs or immunostimulatory sequences (ISS). These sequences are mitogenic for B cells and can stimulate macrophage cytokine production. While these sequences do not directly activate T cells, they can augment effects of stimulation via the TCR. Furthermore, ISS can affect T cells because of macrophage production of IL-12 and IFN-alpha/beta. In these studies, we further evaluated the immune effects of DNA on T cells, testing the possibility that certain T cell populations can respond directly to this stimulus. We therefore tested the in vitro responses of thymocytes to a series of phosphodiester (Po) and phosphorothioate (Ps) oligonucleotides (ODNs) varying in sequence. In in vitro cultures, phosphorothioate ODNs (sODNs) containing CpG motifs induced significant proliferation of murine thymocytes, although phosphodiester compounds lacked activity. The magnitude of stimulation varied with sequences flanking the CpG motifs, as both dA and dT sequences enhanced the stimulatory capacity of the CpG motif. Furthermore, CpG sODNs were strong costimulators of anti-CD3-mediated thymocyte activation, increasing proliferation compared to anti-CD3 in the absence of DNA. This activation was only partially inhibited by cyclosporine A and was not dependent on a calcium influx. Together, these results indicate that phosphorothioate oligonucleotides containing CpG motifs can directly induce thymocyte proliferation as well as augment TCR activation. These observations thus extend the range of actions of CpG DNA and suggest additional mechanisms for its function as an immunomodulatory agent or adjuvant.

Adjuvants, Immunologic↗
PubMed1999

The adjacent flanking region plays a critical role in facilitating the presentation of the Listeria monocytogenes product lemA to H2 M3wt-restricted, peptide-specific murine CD8 cells.

Mice infected with Listeria monocytogenes (LM) generate CD8 effectors specific for f-MIGWII, the amino terminus of the bacterial product lemA presented by the class Ib MHC molecule H2 M3wt. lemA has several distinctive properties: 1) it is readily presented as an exogenous Ag in the absence of bacterial infection; 2) it is processed by a TAP-independent pathway, which is sensitive to chloroquine, pepstatin, and brefeldin; and 3) the immunogenic portion of the molecule is extremely resistant to proteolytic degradation even by proteinase K. To assess the structural basis for these findings, we expressed a truncated variant (t-lemA) containing the amino-terminal hexapeptide and the subsequent 27 amino acids linked to a histidine tail in Escherichia coli, and purified the product by affinity chromatography. Purified t-lemA could be presented to f-MIGWII-specific effectors by macrophages and fibroblasts at 1-10 nM. Unlike f-MIGWII, which binds directly to H2 M3wt, t-lemA required processing by a chloroquine-, pepstatin-, and brefeldin-sensitive pathway. Brefeldin sensitivity often implies endogenous processing in the cytoplasm, but several lines of evidence suggest translocation to the cytoplasm and proteosomal degradation are not critical for t-lemA presentation. Unlike f-MIGWII, t-lemA was profoundly resistant to proteinase K, and, using 35S-labeled t-lemA, we could identify the region from position 1 to approximately 30 as the protease-resistant element. Thus, the hydrophobic peptide sequence following f-MIGWII can account for the unusual properties of lemA noted above. Analogous modification could be used to alter the properties of other peptide Ags presented by class I MHC products.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Intraperitoneal immunity and pneumoperitoneum.

BACKGROUND: Carbon dioxide (CO(2)) pneumoperitoneum has been implicated as a possible factor in depressed intraperitoneal immunity. Using in vitro functional assays, CO(2) has been shown to decrease the function of peritoneal macrophages harvested from insufflated mice. However, an effective in vivo assessment is lacking. Listeria monocytogenes (LM), an intracellular pathogen, has served as a well-established in vivo model to study cell-mediated immune responses in mice. This study examines the immune competence of mice based on their ability to clear intraperitoneally administered LM following CO(2) vs helium (He) insufflation. METHODS: Eighty-five mice (C57Bl/6, males, 4-6 weeks old) were divided between the following four treatment groups: CO(2) insufflation, He insufflation, abdominal laparotomy (Lap), and control (anesthesia only). Immediately postoperatively, each group was inoculated percutaneously and intraperitoneally with a sublethal dose (.015 x 10(6) org) of virulent LM (EGD strain). Half of the animals were killed on postoperative day 3 and half on day 5. Spleens and livers (sites of bacterial predilection) were harvested, homogenized, and plated on TSB agar. The amount of bacteria (1 x 10(6) LM/spleen and liver) from each group was then compared. Statistical significance was set at p </= 0.05. RESULTS: Control animals had nominal bacteria on day 3 (0.016 x 10(6) LM/spleen and liver), and the bacterial burden remained low at day 5 (0.038 x 10(6) LM/spleen and liver) postchallenge. On day 3, the bacterial burden was significantly higher in the CO(2) group (5.46 x 10(6) LM/spleen and liver) as compared to He (0.093 x 10(6) LM/spleen and liver) and controls. The Lap group (3.44 x 10(6) LM/spleen and liver) had significantly more bacteria than the controls. There were no significant differences between any of the groups on day 5. CONCLUSIONS: In this animal model, CO(2) pneumoperitoneum impaired cell-mediated intraperitoneal immunity significantly more than He pneumoperitoneum and controls on day 3. Also on day 3, laparotomy caused impairment of intraperitoneal immunity when compared to controls. Finally, intraperitoneal immunosuppression resolved by day 5.

Animals↗
PubMed1999

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.

Angiotensin II↗
PubMed1998

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.

Animals↗
PubMed1998

H2M3wt-restricted, Listeria monocytogenes-immune CD8 T cells respond to multiple formylated peptides and to a variety of gram-positive and gram-negative bacteria.

A subset of H2M3wt-restricted, Listeria monocytogenes (LM)-immune CD8 effectors recognize antigen-presenting cells (APC) preincubated with heat-killed LM. The responsible product, which we have previously designated heat-killed Listeria-associated antigen (HAA), is extremely hydrophobic and resistant to proteolytic degradation. Despite the protease resistance of HAA, we now report that HAA-immune clones are uniformly responsive to fMIGWII, a formylated oligopeptide derived from the recently described LM product, lemA. While fMIGWII was by far the most potent peptide tested, over half our clones also responded to the LM-derived peptide fMIVII and cross-reactive responses to two other unrelated formylated peptides at concentrations of <1 microM were frequently observed. One of these peptides (fBlaZ) did not share any amino acid in common with fMIGWII except N-formyl methionine at position 1. Unformylated variants of the same peptides were inactive. HAA-immune CD8 cells also responded in an H2M3wt-restricted manner to APC pretreated with heat-killed or live preparations of other gram-positive and gram-negative bacteria such as Streptococcus pyogenes (SP) and Proteus vulgaris (PV). Unlike fMIGWII which is water soluble and protease sensitive, the native antigens extracted from SP and PV, like HAA, were very hydrophobic and proteinase K resistant, presumably reflecting in each case the association of cross-reactive polypeptides with bacterial lipid or phospholipid. Thus, HAA/lemA-immune, H2M3wt-restricted effectors can respond to a variety of formylated peptides and bacterial antigens in vitro. Similar cross-reactions in vivo might have physiologically significant implications.

Animals↗
PubMed1997

Bovine herpesvirus 1 downregulates the expression of bovine MHC class I molecules.

The mechanism of immunosuppression induced by bovine herpesvirus 1 (BHV-1) was investigated by studying the effects of the virus on the expression of major histocompatibility complex (MHC) class I molecules. After infection with the virus, the expression of class I molecules was detected by flow cytometry and pulse-chase analysis. A selective downregulation of expression of class I molecules was seen in the infected cells, while the class II expression remained unaffected. The reduction in surface expression was evident as early as 8 hours postinfection, reaching significant levels by 12 hours. The downregulation was seen with a multiplicity of infection as low as 0.1. A modified live vaccine strain of BHV-1 also induced the downregulation of class I expression. Analysis of the viral proteins(s) involved in this downregulation with metabolic inhibitors (cycloheximide or phosphonoacetic acid), suggested that the immediate early and/or early proteins of the virus mediate this effect. Pulse-chase analysis revealed that the synthesis of the class I heavy chain, and the assembly/transport of class I molecules were affected by the virus infection. These results suggest that BHV-1 interferes with the molecular mechanisms involved in the synthesis, and assembly/transport of MHC-class I molecules. This interference with the class I antigen processing pathway might help the virus to evade the cytotoxic T-lymphocyte response of the host.

Animals↗
PubMed1997

Characterization of the murine H2-M3wt-restricted CD8 response against a hydrophobic, protease-resistant, phospholipid-associated antigen from Listeria monocytogenes.

Mice infected with Listeria monocytogenes (LM) generate protective CD8 cells of varying specificity. One subset, unlike conventional LM-immune CD8 cells, can respond to antigen-presenting cells (APC) treated with heat-killed LM (HKLM). These cells proved to have surprisingly uniform specificity, recognizing a product we designated HKLM-associated antigen (HAA) presented by the non-classical class Ib product H2-M3wt. HAA proved to be extremely hydrophobic and the bioactive portion of the molecule was highly protease-resistant, leading us initially to speculate that it might be a non-peptide. Recent studies, however, identify HAA as a complex containing lemA, a listerial protein bearing the immunogenic amino terminal peptide sequence fMIGWII, tightly associated with bacterial cardiolipin. A variety of cell types can process and present exogenous HAA/lemA, and the phospholipid component appears essential for this processing. Endosomal acidification and proteolysis are required for processing, but the site where antigen binds to H2-M3wt within APC remains uncertain. HAA/lemA-immune effectors are unusually cross-reactive. We could readily detect H2-M3wt-restricted responses to APC incubated with unrelated N-formylated peptides, and bacteria. HAA-like products represent an intriguing new set of bacterial antigens recognizable by immune CD8 cells.

Antigen Presentation↗
PubMed1996

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.

Animals↗
PubMed1996

H2-M3wt-restricted, Listeria monocytogenes-specific CD8 T cells recognize a novel, hydrophobic, protease-resistant, periodate-sensitive antigen.

Mice infected with Listeria monocytogenes (LM) generate H2-M3wt-restricted CD8 effectors which recognize a heat-killed LM-associated antigen (HAA) presented by macrophages. To characterize HAA, we extracted a bioactive component from LM using SDS or NaOH. Extracted HAA aggregated in hydrophilic solvents but dissociated in the presence of SDS into a smaller subunit which migrated in Sephadex G-200 between chymotrypsinogen (25 kDa) and cytochrome c (12.5 kDa). HAA bioactivity and size was unaffected by proteinase K under conditions which degraded virtually all detectable protein. HAA was also unaffected by other proteases, RNase and DNase, but HAA bioactivity was destroyed by periodate, an agent that degrades carbohydrates. These studies demonstrate that H2-M3wt can present a hydrophobic, non-peptide, microbial antigen, probably glycolipid in origin, to CD8 T cells.

Animals↗
PubMed1995

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.

Animals↗
PubMed1994

Bovine x murine T-cell hybridomas specific for bovine herpesvirus 1 (BHV-1) glycoproteins.

Difficulties in the isolation and long-term maintenance of bovine herpesvirus-1 (BHV-1) specific T-cell clones have hindered the analysis of bovine cell-mediated immune response to this virus. In an effort to identify the T-cell epitopes of the virus, bovine murine T-cell hybridomas specific for BHV-1 were generated as an alternative to T-cell clones. Peripheral blood lymphocytes from a calf immunized with BHV-1 were restimulated in vitro with the virus to generate bulk T-cell cultures. The antigen-specific T-cell-enriched bulk culture lymphocytes were fused with the T-cell receptor-deficient mutant of the murine thymoma cell line BW 5147. T-cell hybridomas were screened for their ability to produce interferon-gamma in response to BHV-1 stimulation. Hybridomas with various specificities were obtained. One of them was specific for the BHV-1 glycoprotein gI, two were specific for gIV, while three other hybridomas were specific for gIII. One hybridoma responded to stimulation with BHV-1, but not to any of the glycoproteins gI, gIII, or gIV, suggesting that proteins other than these major glycoproteins may be involved in the bovine T-cell response to BHV-1. Of these hybridomas, one was MHC Class I restricted, while all the others were Class II restricted.

Animals↗
PubMed1993

Down regulation of murine MHC class I expression by bovine herpesvirus 1.

The objective of this study was to investigate the effect(s) of bovine herpesvirus 1 (BHV-1) infection on the expression of MHC class I molecules in murine fibroblasts. L-cells were infected with BHV-1 at a multiplicity of infection (m.o.i.) of 10 plaque forming units (PFU) per cell, and the expression of MHC class I molecules was analyzed by flow cytometry and immunoprecipitation. Temporal studies revealed a reduction in class I expression beginning at 8 h post infection (p.i.) which reached a maximum between 10 to 16 h p.i. The loss of class I expression was restored in the presence of phosphonoacetic acid (30 micrograms/ml), a late herpesviral protein synthesis inhibitor. However, addition of cycloheximide, a total protein synthesis inhibitor (100 micrograms/ml), did not result in any difference in class I expression between virus-infected and mock-infected cells. These results suggest that the reduced class I expression is a direct consequence of BHV-1 infection, and that the late viral gene product(s) may be involved in this process. Similar phenomena may occur in natural BHV-1 infection in cattle, and this may be one of the mechanisms of immune suppression by BHV-1.

Animals↗
PubMed1990

Anti-idiotypic antibodies induce neutralizing antibodies to bovine herpesvirus 1.

A neutralizing murine monoclonal antibody (mAb) of the IgG2a isotype (MM-113), specific for bovine herpesvirus 1 (BHV-1) glycoprotein gIV, was used to develop anti-idiotypic antibodies (anti-Id) in a calf. The bovine anti-Id were isolated from the serum of the immunized calf by affinity chromatography on an MM-113-Sepharose column, followed by repeated adsorption on a murine IgG2a column. The anti-Id thus obtained specifically reacted with MM-113, but not with isotype-matched controls. They also inhibited the binding of MM-113 to BHV-1 in a concentration-dependent manner. Mice immunized with the anti-Id produced neutralizing antibodies to BHV-1. The anti-Id bound to cells permissive to BHV-1 in a cell-binding radioimmunoassay (RIA).

Animals↗