Faculty Bibliography

Mice with a targeted disruption in the IFN-gamma receptor gene (IFN-gamma R0/0) provided a useful model to ask to what extent other cytokines could replace IFN-gamma in macrophage activation. In thioglycollate-elicited peritoneal macrophages from wild-typy (WT) mice, TNF enhanced nitric oxide (NO) release in the presence of IFN-gamma, though TNF alone was not effective. In macrophages from IFN-gamma R0/0 mice, which are not responsive to IFN-gamma, TNF completely failed to stimulate NO release. The NO inducing effects of IFN-alpha/beta were indistinguishable in IFN-gamma R0/0 and WT macrophages. The important role of IFN-gamma in the regulation of the induced expression of MHC class II antigen (Ia) was confirmed by showing that after systemic infection with the BCG strain of Mycobacterium bovis, peritoneal macrophages from IFN-gamma R0/0 mice had a lower level of Ia expression than macrophages from WT mice. BCG infection was not lethal for WT mice whereas all IFN-gamma R0/0 mice died 7-9 weeks after infection. It is well known that BCG infection greatly sensitizes mice to lethal action of LPS. Injection of LPS 2 weeks after BCG inoculation was significantly less lethal for IFN-gamma R0/0 mice than for WT mice. Reduced lethality of LPS correlated with a drastically reduced TNF-alpha production in the IFN-gamma R0/0 mice after BCG infection and LPS challenge. The greatly reduced ability of BCG-infected IFN-gamma R0/0 mice to produce TNF-alpha may be an important factor in their inability to resist BCG infection.(ABSTRACT TRUNCATED AT 250 WORDS)

It has been known for a long time that the two major classes of interferons, IFN-alpha/beta and IFN-gamma, are similar in many of their actions despite the fact that they are structurally unrelated and bind to separate receptors. Recent studies revealed overlapping, common components in IFN-alpha/beta and IFN-gamma signaling. The second chain of the IFN-gamma receptor was recently identified. The generation of mice with targeted disruptions of the genes for IFN-gamma or the IFN-gamma receptor is helping to understand the essential functions of IFN-gamma in host defenses. Other studies have shown that the transcription factor IRF-1 is essential for the IFN-gamma-mediated activation of the gene for the inducible nitric oxide synthase

Twenty-four patients with solid malignancies were treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) on a Phase 1b trial. The objective of the study was to evaluate the effects of GM-CSF on peripheral blood monocyte activation. GM-CSF was administered by subcutaneous injection daily for 14 days. Immune parameters measured were monocyte cytotoxicity against the human colon carcinoma (HT29) cell line, serum tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and in vitro TNF-alpha and IL-1 beta induction. All patients were evaluable for toxicity. Fifteen patients were evaluable for immunologic response. Treatment with GM-CSF led to a statistically significant enhancement in direct monocyte cytotoxicity against HT29 cells. There was no increase in serum TNF-alpha or IL-1 beta and no consistent in vitro induction of TNF-alpha or IL-1 beta from monocytes posttreatment. Treatment was well tolerated overall. We conclude that treatment with GM-CSF can lead to enhanced monocyte cytotoxicity. Further studies are in progress to evaluate the effect of GM-CSF on other parameters of monocyte functions

The cytokine interleukin-8 (IL-8) is an important mediator of neutrophil, lymphocyte, and basophil chemotaxis and activation. Earlier we demonstrated that beta interferon (IFN-beta) can inhibit tumor necrosis factor (TNF)-induced IL-8 gene expression at the transcriptional level, apparently by a novel mechanism. To define the cis-acting elements and trans-acting factors involved in this inhibition, DNA constructs containing portions of the 5'-flanking region of the IL-8 gene were linked to the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into human diploid FS-4 fibroblasts. The region spanning positions -98 to +44 was sufficient to confer both inducibility by TNF and inhibition by simultaneous treatment with IFN-beta. Inhibition of TNF- or IL-1-induced CAT activity by IFN-beta or IFN-alpha was also observed when a DNA fragment containing only the NF-IL-6 and NF-kappa B sites (positions -94 to -70) was placed upstream of the homologous or a heterologous minimal promoter. A construct containing three copies of the NF-kappa B element in front of the CAT gene also was inducible by TNF, and this stimulatory effect too was inhibited by IFN-beta, indicating that the NF-kappa B element is sufficient to confer inhibition by IFN-beta. This inhibitory effect was specific for the NF-kappa B site of the IL-8 gene since it was less marked with constructs containing three copies of the NF-kappa B site from the HLA-B7 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated the role of the transcription factor NF-kappa B in the induction of H-2 antigens by tumor necrosis factor (TNF) in murine J558L myeloma cells. An earlier report suggested that J558L cells may have a defect in NF-kappa B activation in response to some stimuli. Treatment of J558L cells with either TNF or lipopolysaccharide (LPS) resulted in nuclear translocation of NF-kappa B, as demonstrated by electrophoretic mobility shift assay. Both TNF and LPS activated the same NF-kappa B nuclear complexes, composed of the p50 and p65 subunits. LPS mediated a stronger and more sustained activation of NF-kappa B than TNF. In contrast, TNF induced higher levels of H-2 antigen surface expression than did LPS, suggesting that activation of NF-kappa B is not sufficient for optimal enhancement of H-2 expression. An inhibitor of NF-kappa B activation, pyrrolidinedithiocarbamate (PDTC), dramatically reduced the induction of H-2 antigen by TNF, supporting the view that NF-kappa B is required for TNF-induced H-2 antigen expression. Constitutive levels of H-2 antigen expression on the cell surface and of nuclear NF-kappa B also decreased after PDTC treatment. However, PDTC had a smaller inhibitory effect on LPS-induced NF-kappa B activation and H-2 antigen expression, suggesting that TNF and LPS activate NF-kappa B by somewhat different pathways

Mice with a targeted disruption of the interferon gamma receptor gene (IFN-gamma R0/0 mice) and control wild-type mice were inoculated with the Bacillus Calmette-Guerin (BCG) strain of Mycobacterium bovis. BCG infection was not lethal for wild-type mice whereas all IFN-gamma R0/0 mice died approximately 7-9 wk after inoculation. Histological examination at 2 and 6 wk after BCG inoculation showed that livers of IFN-gamma R0/0 mice had higher numbers of acid-fast bacteria than wild-type mice, especially at 6 wk. In parallel, the livers of IFN-gamma R0/0 mice showed a reduction in the formation of characteristic granulomas at 2 wk after inoculation. Injection of lipopolysaccharide (LPS) 2 wk after BCG inoculation was significantly less lethal for IFN-gamma R0/0 mice than for wild-type mice. Reduced lethality of LPS correlated with a drastically reduced production of tumor necrosis factor alpha (TNF-alpha) in the IFN-gamma R0/0 mice. Interleukin 1 alpha (IL-1 alpha) and IL-6 levels in the serum were also significantly reduced in the IFN-gamma R0/0 mice after BCG infection and LPS challenge. The greatly reduced capacity of BCG-infected IFN-gamma R0/0 mice to produce TNF-alpha may be an important factor in their inability to resist BCG infection. These results show that the presence of a functional IFN-gamma receptor is essential for the recovery of mice from BCG infection, and that IFN-gamma is a key element in the complex process whereby BCG infection leads to the sensitization to endotoxin

Tumor necrosis factor (TNF) is a pleiotropic cytokine whose many demonstrated actions include effects on cell growth and differentiation. TNF treatment of cells is known to lead to a rapid increase in serine/threonine phosphorylation of many cellular proteins, but the kinases responsible remain largely unidentified. We show that TNF treatment induces a rapid and transient increase in mitogen-activated protein kinase (MAPK) activity in the human diploid FS-4 cell line, for which TNF is known to be mitogenic. TNF-induced activation of MAPK was demonstrated by its enhanced ability to phosphorylate myelin basic protein in vitro and by a characteristic shift in the electrophoretic mobility of MAPK proteins. MAPK activation was accompanied by a significant increase of MAPK phosphorylation on tyrosine residues, which was demonstrated by 32P labeling of cells and isolation of the labeled proteins after immunoprecipitation with antibodies to phosphotyrosine, and by direct immunoblotting of SDS-polyacrylamide gel electrophoresis-fractionated unlabeled cell lysates with antibodies to phosphotyrosine. The pp42 and pp44 MAPK were the only proteins whose tyrosine phosphorylation was demonstrably increased in FS-4 cells after TNF treatment. MAPK activation is likely to represent an important component in the cascade of signals that link TNF receptors to various TNF-elicited cellular responses

Availability of mice with a targeted disruption of the interferon gamma (IFN-gamma) receptor gene (IFN-gamma R0/0 mice) made it possible to examine parameters of macrophage activation in the absence of a functional IFN-gamma receptor. We asked to what extent other cytokines could replace IFN-gamma in the induction of nitric oxide or major histocompatibility complex class II antigen (Ia) expression in peritoneal macrophages. In thioglycollate-elicited macrophages from wild-type mice, tumor necrosis factor (TNF) alone was virtually ineffective in inducing release of NO2- (the endproduct of nitric oxide generation), but TNF enhanced NO2- release in the presence of IFN-gamma. In macrophages from IFN-gamma R0/0 mice, which were unresponsive to IFN-gamma, TNF completely failed to stimulate NO2- release. The stimulatory actions of IFN-alpha/beta on NO2- release were indistinguishable in wild-type and IFN-gamma R0/0 macrophages: IFN-alpha/beta was ineffective on its own, showed marginal stimulation of NO2- release in combination with TNF, and was moderately effective in the presence of lipopolysaccharide. The level of constitutive Ia antigen expression was not significantly different in peritoneal macrophages from wild-type and IFN-gamma R0/0 mice. An increased Ia expression was induced by IL-4 and granulocyte-macrophage colony-stimulating factor in both wild-type and IFN-gamma R0/0 macrophages, but the magnitude of this induction was less than with optimal concentrations of IFN-gamma in macrophages from wild-type mice. IFN-alpha/beta showed only a minor stimulatory effect on Ia expression in both wild-type and IFN-gamma R0/0 macrophages. Simultaneous treatment of wild-type macrophages with IFN-alpha/beta and IFN-gamma reduced the IFN-gamma-induced Ia expression in wild-type macrophages, but IFN-alpha/beta did not show an inhibitory effect on IL-4- or granulocyte-macrophage-colony-stimulating factor-induced Ia expression in either wild-type or IFN-gamma R0/0 macrophages. The important role of IFN-gamma in the regulation of the induced expression of major histocompatibility complex class II antigen was confirmed by showing that after systemic infection with the BCG strain of Mycobacterium bovis resident peritoneal macrophages from IFN-gamma R0/0 mice had a lower level of Ia expression than macrophages from wild-type mice. The inability of other cytokines to substitute fully for IFN-gamma in macrophage activation helps to explain the earlier observed decreased resistance of IFN-gamma R0/0 mice to some infections

TSG-6 (TNF-stimulated gene 6) was originally discovered by differential screening of a cDNA library prepared from TNF-stimulated human diploid FS-4 fibroblasts. We show that the 35-kDa protein encoded by TSG-6 was undetectable in the medium of untreated FS-4 cultures, whereas its production reached approximately 1400 and 700 ng/10(6) cells after 24-h treatment with IL-1 or TNF, respectively. Stimulation of TSG-6 protein and mRNA levels was also demonstrated in normal human mononuclear cells by treatment with TNF and, especially, by LPS. In view of the inducibility of TSG-6 by inflammatory cytokines and its earlier demonstrated affinity for hyaluronan, we examined the presence of TSG-6 protein in the synovial fluids from patients with various forms of arthritis. TSG-6 protein was undetectable in the joint fluids of persons with no known history of arthritis, but high levels of TSG-6 oere demonstrated in the synovial fluids of a majority of arthritis patients. TSG-6 protein was also detected in the sera of some of the arthritis patients, albeit at concentrations that were less than in the joint fluids. To investigate the source of TSG-6 in the synovial fluids, we examined the production of TSG-6 protein in cultures of synovial cells. Synoviocytes from rheumatoid arthritis patients produced TSG-6 protein constitutively, and this production was increased by treatment with TNF or IL-1, but not with TGF-beta. Steady-state levels of TSG-6 mRNA were also increased in synoviocytes after treatment with TNF or IL-1. The presence of high levels of TSG-6 protein in the synovial fluids of arthritis patients and its inducibility by inflammatory cytokines in fibroblasts, mononuclear cells, synoviocytes, and chondrocytes suggest a role for TSG-6 in arthritis and inflammation