Biomedical subjects

G R Klimpel

Publications and source records attributed to G R Klimpel.

At least 37 records · Page 2Linked to original sources

PubMed1996

Extracellular activities of human granzymes. I. Granzyme A induces IL6 and IL8 production in fibroblast and epithelial cell lines.

Situated in secretory granules of cytotoxic cells, granzymes are essential for induction of target cell apoptosis with perforin. However, since cytotoxic cells constitutively secrete a portion of their synthesized granzymes, these proteases could mediate extracellular functions independently of their role in the lytic event. Thrombin has been shown to function as an activation molecule by cleaving its receptor. We hypothesize that granzymes may act similarly. In this report, we show that purified human granzyme A can induce human lung fibroblasts to produce IL6 and IL8. Cytokine induction is abrogated by treating the serine protease with the suicide serine protease inhibitor 3,4-dichloroisocoumarin. Other fibroblast lines as well as epithelial cells produced cytokines in response to granzyme A. Our data suggest that granzyme A can function as an activation molecule with potentially important immunoregulatory functions.

Cell Line↗

PubMed1996

A role for stem cell factor (SCF): c-kit interaction(s) in the intestinal tract response to Salmonella typhimurium infection.

Cholera toxin (CT) has been shown to induce stem cell factor (SCF) production in mouse ligated intestinal loops. Further, SCF interaction(s) with its receptor (c-kit) was shown to be important for the intestinal tract secretory response after CT exposure. In this study, we have investigated whether SCF production is induced in the intestinal tract after exposure to Salmonella typhimurium and whether this production could be an important intestinal tract response to Salmonella infection. Using a mouse ligated intestinal loop model, increased levels of SCF mRNA were detected at 2-4 h post-Salmonella challenge. Intestinal fluid obtained from Salmonella-challenged loops contained high levels of SCF by ELISA. Human and murine intestinal epithelial cell lines were also shown to have increased levels of SCF mRNA after exposure to Salmonella. Inhibition of Salmonella invasion of epithelial cells was shown to be one potentially important role for SCF:c-kit interactions in host defense to Salmonella infection. Pretreatment of human or murine intestinal cell lines with SCF resulted in a cellular state that was resistant to Salmonella invasion. Finally, mice having mutations in the white spotting (W) locus, which encodes the SCF-receptor (c-kit), were significantly more susceptible to oral Salmonella challenge than their control littermates. Taken together, the above results suggest that an important intestinal tract response to Salmonella infection is an enhanced production of SCF and its subsequent interactions with c-kit.

Animals↗

PubMed1996

Extracellular activities of human granzyme A. Monocyte activation by granzyme A versus alpha-thrombin.

Granzymes, serine proteases located in the granules of cytotoxic T cel ls and NK cells, are essential for induction of target cell apoptosis. However, since cytotoxic cells constitutively secrete a portion of their synthesized granzymes, these proteases could mediate extracellular functions independent of their role in the lytic event. Thrombin, another serine protease, can induce cytokine production in a number of different cell types. In this study, we test the hypothesis that granzymes, like thrombin, can regulate cell-mediated immunity by inducing the production of different cytokines. We show that granzyme A (GA) stimulates IL-6, IL-8, and TNF-alpha production by human PBMC and purified monocytes. In contrast, monocytes exposed to thrombin had enhanced IL-8 production with no induction of IL-6 or TNF-alpha production. However, monocytes exposed to either GA or thrombin had enhanced phagocytic activity. The enzymatic activity of GA and thrombin was required for the induction of cytokine production and for the enhancement of phagocytic activity. The induction of different cytokine profiles by GA vs thrombin suggested that GA activates monocytes via a receptor that was different from the thrombin receptor. This conclusion was strengthened by the fact that GA was incapable of inducing Ca2+ mobilization in insect cells transfected with the thrombin receptor. These results suggest that enzymatically active GA mediates important immunoregulatory functions through signaling pathways that does not involve thrombin receptor activation.

Animals↗

PubMed1996

Yersinia enterocolitica envelope proteins that are crossreactive with the thyrotropin receptor (TSHR) also have B-cell mitogenic activity.

Autoantibodies to the thyrotropin receptor (TSHR) have been shown to mediate the hyperthyroidism associated with Graves' disease (GD). A number of hypotheses have been proposed which link an infectious agent to the mechanism(s) involved in the induction of GD. Several studies have suggested that the development of GD may be linked to infection with the enteric pathogen Yersinia enterocolitica. We have recently identified two low molecular weight (5.5 and 8 kDa) envelope proteins of Y. enterocolitica that are cross-reactive with the extracellular domain of human TSHR (ETSHR). In this study, we have purified these ETSHR-crossreactive Yersinia proteins (TSHR-CRP) and have further characterized their immunoreactivity. Both the 5.5 and 8 kDa TSHR-CRPs were shown to be mitogenic for mouse spleen cells. This mitogenic activity was specific for B cells and was not due to lipopolysaccharide (LPS) contamination. TSHR-CRPs were mitogenic for LPS-non-responsive spleen cells obtained from C3H/Hej mice, and polymyxin B did not inhibit the mitogenic activity of the TSHR-CRPs. TSHR-CRPs also induced high levels of IL-6 production in B cells and induced production and secretion of significant levels of IgG and IgM. Finally, culture supernatants from TSHR-CRP-stimulated spleen cells were shown by Western blot analysis to contain antibodies that recognized the ETSHR These results identify for the first time two envelope proteins of Yersinia that have mitogenic activity and therefore could represent important proteins involved in the pathogenesis of Yersinia infections. Because these mitogenic proteins also contain epitopes crossreactive with the TSHR, they are potentially important for advancing our understanding of the role molecular mimicry plays in the induction of autoimmunity to the TSHR.

Animals↗

PubMed1996

Cholera toxin induces synthesis of phospholipase A2-activating protein.

The mechanism of cholera toxin (CT)-stimulated arachidonate metabolism was evaluated. CT caused rapid in vitro synthesis of prostaglandin E2 (PGE2) in murine smooth muscle-like cells (BC3H1), reaching maximal levels within 3 to 4 min. In comparison, cyclic AMP (cAMP) levels were unchanged, and addition of dibutyryl cAMP did not affect PGE2 synthesis. CT-induced PGE2 synthesis was prevented by actinomycin D or cycloheximide, indicating a need for de novo protein synthesis. Northern blot analysis of total RNA from BC3H1 cells revealed that exposure to CT resulted in an increase in abundance of mRNA encoding phospholipase A2 (PLA2)-activating protein (PLAP). PLAP is a regulatory protein that increases the enzymatic activity of cellular PLA(2), which in turn causes increased hydrolysis of arachidonate from membrane phospholipids. Furthermore, CT evoked the accumulation of PLAP mRNA in J774 (murine monocyte/macrophage) and Caco-2 (human intestinal epithelial) cells in vitro, but the responses were more delayed than that of BC3H1 cells. A protein band of approximately 35 kDa, which corresponded to the size of PLAP, was observed in sodium dodecyl sulfate extracts of Caco-2 cells by Western blot (immunoblot) analysis using affinity-purified antibodies to PLAP synthetic peptides. Synthesis of PLAP protein was increased after 2 h of exposure to CT. Exposure of mouse intestinal loops to either CT or live Salmonella typhimurium for 3 h increased mucosal PLAP mRNA levels. The role of PLAP in CT-induced PGE2 synthesis provides an attractive explanation for the reported suppression of CT-induced intestinal secretion by inhibitors of protein synthesis.

Amino Acid Sequence↗

PubMed1996

Phospholipase A2 activating protein and idiopathic inflammatory bowel disease.

BACKGROUND: Crohn's disease and ulcerative colitis are idiopathic inflammatory bowel diseases (IBD) involving synthesis of eicosanoids from arachidonic acid (AA), which is released from membrane phospholipids by phospholipase A2 (PLA2). A potentially important regulator of the production of these mediators is a protein activator of PLA2, referred to as PLA2 activating protein (PLAP). AIMS: The purpose of this investigation was to discover if PLAP values might be increased in the inflamed intestinal tissue of patients with IBD and in intestinal tissue of mice with colitis. PATIENTS: Biopsy specimens were taken from patients with ulcerative colitis and Crohn's disease undergoing diagnostic colonoscopy, and normal colonic mucosa was obtained from patients without IBD after surgical resection. METHODS: Immunocytochemistry with affinity purified antibodies to PLAP synthetic peptides was used to locate PLAP antigen in sections of intestinal biopsy specimens from IBD patients compared with that of normal intestinal tissue. Northern blot analysis with a murine [32P] labelled plap cDNA probe was performed on RNA extracted from the colons of mice fed dextran sulphate sodium (DSS) and cultured HT-29 cells exposed to lipopolysaccharide (LPS). RESULTS: PLAP antigen was localised predominantly within monocytes and granulocytes in intestinal tissue sections from IBD patients, and additional deposition of extracellular PLAP antigen was associated with blood vessels and oedema fluid in the inflamed tissues. In contrast, tissue sections from normal human intestine were devoid of PLAP reactive antigen, except for some weak cytoplasmic reaction of luminal intestinal epithelial cells. Similarly, colonic tissue from DSS treated mice contained an increased amount of PLAP antigen compared with controls. The stroma of the lamina propria of the colonic mucosa from the DSS treated mice reacted intensely with antibodies to PLAP synthetic peptides, while no reaction was observed with control mouse colons. These data were supported by northern analysis which showed that PLAP mRNA was increased in the colons of DSS treated mice and cultured HT-29 cells exposed to LPS. CONCLUSIONS: As PLAP values were increased in the intestinal mucosa of IBD patients and mice with colitis, as well as in LPS treated cultured HT-29 cells, a role was postulated for PLAP in increasing PLA2 activity, which leads to the increased synthesis of eicosanoids in intestinal tissues of patients with these inflammatory diseases.

Animals↗

PubMed1995

A role for stem cell factor and c-kit in the murine intestinal tract secretory response to cholera toxin.

The role of stem cell factor (SCF) and its receptor (c-kit) in the intestinal secretory response to cholera toxin (CT) was investigated using a ligated intestinal loop model in mice having mutations in the dominant white spotting (W) locus and the steel (Sl) locus. W/Wv mice, which express an aberrant form of the c-kit protein, failed to give an intestinal secretory response after luminal CT challenge. In contrast, W/Wv mice and their control littermates had equivalent intestinal secretory responses to Escherichia coli heat-stable enterotoxin (STa). Sl/Sld mice, which express only a soluble truncated form of SCF, also gave a significantly reduced intestinal secretory response to CT when compared to the secretory response of their littermate controls. The unresponsiveness of W/Wv mice to CT was restricted to the intestinal tract since these mice had foot pad swelling responses to CT challenge that were equivalent to their littermate controls. Restoration of mast cells in W/Wv mice by bone marrow transplantation of control littermate bone marrow did not reverse the CT-unresponsiveness of the intestinal tract. Histological evaluation of the gastrointestinal tract from W/Wv mice showed a normal distribution of enterochromaffin cells (ECC). CT challenge of either ligated intestinal loops from C57B1/6 mice or a mouse intestinal epithelial cell line (MODE-K) resulted in elevated levels of mRNA for SCF. MODE-K cells exposed to CT also had enhanced expression of c-kit. Finally, fluid obtained from CT-challenged ligated intestinal loops from C57B1/6 mice contained significant levels of SCF. Taken together, the above results suggest that CT-induced intestinal secretory responses are dependent upon SCF-c-kit interactions. These interactions appear to be induced as a consequence of CT stimulation of the intestinal tract and may also play a role in the development or functionality of the enteric nervous system.

Animals↗

PubMed1995

Thrombin induces IL-6 production in fibroblasts and epithelial cells. Evidence for the involvement of the seven-transmembrane domain (STD) receptor for alpha-thrombin.

alpha-Thrombin is a multifunctional serine protease that has an important role in the coagulation cascade, wound healing, and inflammatory response. In this study, we show that thrombin induces IL-6 production in human epithelial cells and fibroblasts. ELISA and Northern blot analyses showed that physiologic concentrations of thrombin (0.1-1 micrograms/ml) induced IL-6 production in human lung fibroblasts, skin fibroblasts, and epithelial cells. Hirudin, a thrombin inhibitor, completely blocked IL-6 induction by thrombin. Treatment of fibroblasts with inactivated diisopropylphosphofluoridate (DIP)-alpha-thrombin, gamma-thrombin, or trypsin had no effect on IL-6 production. In contrast, treatment with the thrombin-tethered ligand receptor peptide TRP-7 (SFLLRNP) induced IL-6 production, but at lower levels than that induced by native alpha-thrombin. Finally, IL-6 pretreatment of lung or skin fibroblasts resulted in the enhanced production of IL-6 following exposure to thrombin. These results suggest that fibroblasts and epithelial cells may represent a significant source of IL-6 in the inflammatory response to tissue injury, and that cytokine production is an important biologic consequence of thrombin's interaction with its seven-transmembrane domain (STD) receptor.

Amino Acid Sequence↗

PubMed1995

Cholera toxin and Salmonella typhimurium induce different cytokine profiles in the gastrointestinal tract.

Salmonella infection of the gastrointestinal tract (GT) results in fluid secretion and inflammation. In contrast, cholera toxin (CT) induces fluid secretion but no inflammation. Using a murine ligated intestinal loop model, we investigated cytokine production (interleukin-1 [IL-1], IL-2, IL-4, IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha) in the GT following exposure to these agents. Salmonella typhimurium induced a Th1-like cytokine profile in loops obtained from either nonimmune mice or Salmonella-immunized mice. CT induced only IL-6 and IL-10 production in ligated loops from nonimmune mice but induced a Th2-like cytokine profile in ligated loops obtained from CT-immunized mice. These results show that CT and S. typhimurium induce very different cytokine profiles in the GT.

Animals↗

PubMed1995

Human T-cell recognition of Listeria monocytogenes: recognition of listeriolysin O by TcR alpha beta + and TcR gamma delta + T cells.

The cell-mediated immune response to Listeria monocytogenes has been well characterized in the mouse. Listeriolysin O (LLO) is a major antigen in murine T-cell recognition of L. monocytogenes. In this study, we show that LLO is also recognized by human TcR alpha beta T cells and TcR gamma delta T cells. Human peripheral blood mononuclear cells (PBMC) cultured in vitro with live listeriae and then expanded with interleukin 2 were shown to respond to purified LLO. The generation of LLO-responsive T cells was dependent on the use of live bacteria during the initial in vitro challenge. LLO-induced proliferation of T cells expanded by exposure of PBMC to live listeriae was major histocompatibility complex restricted. PBMC cultured with formalin-fixed listeriae and subsequently expanded by interleukin 2 gave high proliferative responses to fixed bacteria but failed to respond to LLO. PBMC stimulated in vitro with fixed listeriae contained predominantly TcR alpha beta + T cells. In contrast, PBMC obtained from 85% of the donors studied generated high numbers of TcR gamma delta + T cells following in vitro culture with live listeriae. Using a panel of synthetic amphipathic LLO peptides, we found that LLO-specific T cells from different individuals recognized both common and unique peptides. LLO 470-508 was recognized by three of five individuals, while LLO 203-226 and LLO 107-126 were recognized by two of six individuals. A TcR gamma delta + T-cell line was established from PBMC stimulated with live listeriae and was shown to recognize LLO 470-508. Proliferative responses could be induced in this cell line by peptide-pulsed autologous PBMC but not by peptide-pulsed allogeneic PBMC. Our results establish the importance of LLO in human T-cell recognition of listeriae and show that both TcR alpha beta + T cells and TcR gamma delta + T cells recognize this antigen. Finally, since LLO 470-508 has a high degree of homology with other gram-positive bacterial toxins, the recognition of this peptide by TcR gamma delta + T cells suggests that an important role of these T cells in host defense is the recognition of bacterium-derived toxins.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of Yersinia enterocolitica envelope proteins which induce antibodies that react with human thyrotropin receptor.

Graves' disease is an autoimmune disease mediated by autoantibodies to the thyrotropin receptor (TSHR). Several studies have suggested that the development of Graves' disease may be linked to infection with the enteric pathogen Yersinia enterocolitica. Using the purified recombinant extracellular domain of human TSHR (ETSHR), we have recently shown that immunization of mice with Y. enterocolitica results in the production of antibodies capable of reacting with the ETSHR. In this study, we identify two low molecular weight (5.5 kDa and 8 kDa) envelope proteins of Yersinia containing epitopes that are crossreactive with the TSHR. Identification of these crossreactive envelope proteins was achieved by Western blotting using affinity-purified anti-Y. enterocolitica antibodies that specifically react with the TSHR and, conversely, for envelope proteins of Yersinia. Confirmation that these Yersinia proteins contained crossreactive epitopes with the ETSHR was obtained by immunizing mice with partially purified envelope proteins, which resulted in the production of Abs that recognized the ETSHR. Further, some of the cross-reactive envelope proteins were purified with SDS-PAGE and HPLC. The crossreactive envelope proteins were shown to be chromosomally encoded, exposed on the surface of bacteria, and produced by virulent as well as avirulent strains of Yersinia (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica VW+, and Y. enterocolitica VW-). These results identify for the first time the Yersinia envelope proteins that are crossreactive with the ETSHR. Availability of these proteins will allow future studies to determine whether patients with Graves' disease have a unique immune response against these proteins when compared with healthy individuals.

Animals↗

PubMed1994

Thymic hypoplasia and T-cell deficiency in ectodermal dysplasia: case report and review of the literature.

Ectodermal dysplasia is a heterogeneous disorder that includes a constellation of congenital malformations occasionally associated with mild to moderate immune dysfunction. In this report, we describe a female infant with ectodermal dysplasia who was found to have thymic hypoplasia but no other phenotypic features of the DiGeorge anomalad. She experienced Candida parapsilosis sepsis at 1 week of age and a skin infection with Mycobacterium chelonii at 6 months. The numbers of blood B cells were normal and serum immunoglobulins normal to slightly reduced, but serum antibody responses of all immunoglobulin isotypes to protein immunogens were absent. Blood T cells were profoundly reduced and proliferative responses of T cells to mitogens were blunted. In contrast, there was an increased number of natural killer (NK) cells and increased NK activity in the blood. Over the first year of life, some of the immunodeficiencies resolved. Although the numbers of blood T cells (17% of total lymphocytes) remained low, proliferative responses to mitogens normalized and specific antibody responses improved. It seems likely that the thymic hypoplasia in this case was due to a paucity of ectodermal elements in the developing thymus, and that the immune defects were largely secondary to that event. In that respect, this human model of ectodermal dysplasia and thymic hypoplasia resembled the ectodermal/thymic defects found in the nude mouse.

Cell Line↗

PubMed1994

Characterization of functional neurotensin receptors on human lymphocytes.

BACKGROUND: Neurotensin, a tridecapeptide, regulates gut motility, secretion, and mucosal growth; an immunomodulatory role for neurotensin has been postulated but not clearly defined. The purpose of this study was to determine whether neurotensin receptors (NTR) are present on peripheral blood lymphocytes (PBLs) and to characterize binding, functional, and molecular properties. METHODS: Iodine 125 labeled-neurotensin binding was determined for both human PBLs and the T-cell lines, Molt-4 and Jurkat, by Scatchard analysis. To analyze functional capacity of the NTR, PBLs were cultured in the presence of phytohemagglutinin (1 microgram/ml) with or without neurotensin and harvested at 48 hours after an 8-hour pulse of tritiated thymidine. For molecular analyses, RNA extracted from PBLs and T-cell lines was analyzed by either Northern hybridization with a labeled NTR cDNA or ribonuclease protection with an antisense human NTR probe. RESULTS: Scatchard analyses of neurotensin binding to human PBLs and MOLT-4 showed two classes of binding sites with different affinities. Incubation of phytohemagglutinin-stimulated PBLs with neurotensin significantly enhanced proliferation. Northern hybridization showed mRNA of the authentic NTR or a receptor subtype of close homology in PBLs and the T-cell lines; ribonuclease protection analysis identified the authentic human NTR in the MOLT-4 cell line. CONCLUSIONS: Using a combination of molecular techniques and Scatchard analysis, we have demonstrated the presence of a cell surface NTR with a high affinity for neurotensin on human PBLS and the MOLT-4 cell line. The functional role of NTR has been established by enhanced proliferation with addition of neurotensin. These data provide further evidence for a link between neurotensin and the immune system and suggest that neurotensin may play an important regulatory role in gut mucosal immune responses in vivo.

Blotting, Northern↗

PubMed1993

Tumor necrosis factor (TNF alpha) regulates intestinal mucus production during salmonellosis.

Mucus production by goblet cells in the gastrointestinal tract following Salmonella typhimurium infection using a ligated ileal loop model in mice was investigated. Assessment of the morphology of the loop tissue after Salmonella challenge revealed generalized tissue inflammation, characterized by edema and an inflammatory cell infiltrate. Villi were shortened and blunted, and crypts contained an increased number of cells with mitotic figures. Production of TNF alpha in the loops followed Salmonella challenge and occurred at the same time as the pathological sequelae. A nearly 50% decrease in the number of goblet cells in infected tissue compared to tissue from noninfected controls was observed at these same times. The sulfation of mucins produced by the goblet cells in infected tissues was increased in the villi but was unchanged in the crypts compared to uninfected tissues. Treatment of mice with antibody to TNF alpha before Salmonella challenge abrogated tissue pathology and returned goblet cell numbers and mucin profiles to those observed in noninfected controls. Our results indicate that TNF alpha may mediate changes in goblet cell expression and mucin sulfation in response to Salmonella challenge.

Animals↗

PubMed1993

Immunization of mice with Yersinia enterocolitica leads to the induction of antithyrotropin receptor antibodies.

It is well established that autoimmune Graves' disease, which is characterized by hyperthyroidism, is mediated by autoantibodies to the thyrotropin receptor (TSHr). Although what initially triggers this autoantibody response is not known, a number of studies have suggested that Yersinia enterocolitica, an enterobacteria, could initiate the immune response against the TSHr. In this study, we produced antibodies against purified extracellular domain of human TSHr (ETSHr) and showed that anti-ETSHr antibodies reacted with envelope preparations from Y. enterocolitica. This reactivity was specifically blocked by preincubating sera with purified ETSHr. Moreover, antibodies reactive with ETSHr were induced by immunizing mice with Y. enterocolitica but not with Shigella flexneri SA100, Salmonella typhimurium TML, and Listeria monocytogenes. Anti-Y. enterocolitica antisera specifically reacted with the ETSHr protein and the reactivity could be blocked both by ETSHr and Y. enterocolitica envelope proteins. Our studies provide the first direct evidence that immunization with Y. enterocolitica can lead to the production of antibodies capable of reacting with TSHr and might provide the initial stimulus necessary for breakdown of self-tolerance to TSHr, eventually leading to the development of autoimmunity to TSHr.

Animals↗

PubMed1993

Ity influences the production of IFN-gamma by murine splenocytes stimulated in vitro with Salmonella typhimurium.

The Ity-Lsh-Bcg genetic locus in the mouse has been documented to confer innate resistance to at least three intracellular pathogens: Salmonella typhimurium, Leishmania donovani, and Mycobacterium. Expression of the resistance gene(s) results in a slower net growth of these pathogens in the reticuloendothelial system early postinfection. Although it is clear that the resident macrophages in resistant mice are functionally superior with regard to antimicrobial activity, the exact mechanism(s) underlying the control exerted by this gene is not understood. Using S. typhimurium infection as a model, we have examined the influence of this resistance gene(s) on the production of IFN-gamma, a cytokine known to play an important role in host-defense against several intracellular pathogens. We compared IFN-gamma production by splenocytes from resistant (Ity(r)) and sensitive (Ity(s)) inbred mouse strains after stimulation in vitro with S. typhimurium. Spleen cells from Ity(r) mouse strains produced significantly higher levels of IFN-gamma when compared to spleen cells obtained from Ity(s) mouse strains. Enhanced IFN-gamma production was not a generalized response to bacteria. Listeria monocytogenes induced comparable levels of IFN-gamma production from both Ity(r) (CBA/J) and Ity(s) (C57BL/6) mice. Splenocytes from Ity congenic mouse strains displayed similar differences in the level of IFN-gamma produced after S. typhimurium stimulation, with spleen cells from the Ity(r) strain producing significantly higher levels of IFN-gamma when compared to spleen cells from the Ity(s) strain. A requirement for adherent cells and/or adherent cell-derived factors has been documented for IFN-gamma production by S. typhimurium-stimulated splenocytes. Interestingly, supernatant from adherent cells obtained from Ity(r) mouse strains was found to induce the production of significantly higher levels of IFN-gamma when compared to adherent cell supernatant from Ity(s) strains. Nylon wool nonadherent cells from Ity(s) mouse strains produced high levels of IFN-gamma when exposed to supernatants obtained from adherent cells of Ity(r) mouse strains. In contrast, nylon wool nonadherent cells from Ity(r) mouse strains produced reduced levels of IFN-gamma when exposed to supernatant obtained from adherent cells of Ity(s) mouse strains. Thus, modulation of IFN-gamma production appears to be a function of the Ity(r) gene(s). This study documents for the first time that the Ity locus may play a role in controlling resistance to Salmonella infection by regulating IFN-gamma production by NK cells.

Animals↗

PubMed1993

Salmonella typhimurium induces IFN-gamma production in murine splenocytes. Role of natural killer cells and macrophages.

IFN-gamma is a cytokine known to play an important role in host defense against Salmonella typhimurium. The lymphoid cells required for in vitro production of IFN-gamma after S. typhimurium stimulation of mouse spleen cells was investigated. Spleen cells depleted of cells bearing NK1.1, asialo GM1, Thy 1.2, or CD5 resulted in a significant reduction in IFN-gamma production after stimulation with S. typhimurium. In contrast, Con A-induced IFN-gamma production was only slightly reduced after depletion of NK1.1- or asialo GM1-bearing cells. Spleen cells from SCID mice produced elevated levels of IFN-gamma after stimulation with S. typhimurium. IFN-gamma production by SCID spleen cells was dependent upon asialo GM1+ T cells, suggesting that NK cells were the cells producing IFN-gamma in response to S. typhimurium. Splenic adherent cells were required for optimal IFN-gamma production. However, direct contact between the adherent and nylon wool nonadherent (NWNA) cell populations was not essential. IFN-gamma production was observed when the adherent and NWNA cell populations were physically separated or when supernatant from S. typhimurium-stimulated adherent cells was added to NWNA cells. Optimal IFN-gamma production was dependent on the presence of TNF-alpha, inasmuch as addition of antibody to TNF-alpha to spleen cell or NWNA cell cultures significantly reduced IFN-gamma production. However, addition of rTNF-alpha did not induce IFN-gamma production by NWNA cells. These findings document the existence of a T-independent mechanism for early IFN-gamma production in response to S. typhimurium, and show that TNF-alpha is necessary but not sufficient for the production of IFN-gamma.

Animals↗

PubMed1993

Thrombin enhances T cell proliferative responses and cytokine production.

Human alpha-thrombin, in addition to its procoagulant activity, is a mitogen for fibroblasts and endothelial cells and a chemotactic agent for monocytes. To further understand the complex physiological functions of thrombin, we investigated whether thrombin has any immunoregulatory function with regard to T cell activation. Using highly purified human alpha-thrombin and peripheral blood mononuclear cells (PBMC), we investigated whether thrombin has any effect on cytokine production and/or proliferation induced by different T cell stimuli. At physiological concentrations (1-10 micrograms/ml, 30-300 nM), thrombin significantly enhances T cell proliferation in response to mitogens, superantigens, alloantigens, and anti-CD3 stimulation. Enhanced proliferation was associated with increased IL2 and IL6 production and with an increase in the number of IL2r+ (CD25)-bearing T cells. Thrombin alone was not mitogenic nor did it induce IL2 production or increase the number of IL2r+ T cells. However, PBMC exposed to thrombin alone produced high levels of IL6. Thrombin also enhanced IL2-induced proliferation of murine and human IL2-dependent cell lines. These results suggest that thrombin may play an important role in regulating cell-mediated immunity.

Cytokines↗