Molecular cloning and expression of the human interferon-gamma receptor

A cDNA encoding the human interferon-gamma receptor was isolated from a lambda gt11 expression library using a polyclonal antireceptor antiserum. The gene for this receptor was identified in a cosmid library and transfected into mouse cells. The human interferon-gamma receptor expressed in mouse cells displayed the same binding properties as in human cells. However, transfected cells were not sensitive to human IFN-gamma, suggesting the need for species-specific cofactors in receptor function. As inferred from the cDNA sequence, the human interferon-gamma receptor shows no similarities to known proteins and represents a novel transmembrane receptor. It is most likely the product of a single mRNA and a gene located on chromosome 6q.     

Modulation of human natural killer cell cytotoxic activity, lymphokine production, and interleukin 2 receptor expression by thymic hormones

Thymic hormone preparations have been shown to modulate natural killer (NK) activity in vivo in mice. We have investigated the effects of thymosin fraction 5 (TF5) on the in vitro NK cell activity of highly purified human large granular lymphocytes (LGL). The results indicate that TF5 but not kidney fraction 5 (a preparation used as control) is able to enhance the spontaneous NK activity of normal LGL. In addition, TF5 exhibited additive effects with recombinant interferon-alpha in enhancing NK activity in vitro. TF5 also enhanced interleukin 2 production and interleukin 2 receptor expression as well as interferon-gamma production in mitogen-stimulated LGL. Thymosin-alpha 1, a synthetic polypeptide originally isolated in its native form from TF5, also exhibited enhancing effects on LGL activities, suggesting that it is the active species in TF5. These results indicate that thymic hormones might regulate NK activity through the induction of lymphokine production and receptor expression by LGL.     

Lipopolysaccharide and interleukin 1 inhibit interferon-gamma-induced Fc receptor expression on human monocytes

The objectives of these studies were to study the effects of bacterial lipopolysaccharide (LPS) on interferon-gamma (IFN-gamma)-induced Fc receptor expression on human monocytes and to examine whether these effects were mediated through stimulation of interleukin 1 (IL-1) production. Fc receptor expression was determined by binding of monomeric monoclonal murine immunoglobulin (Ig)G2a and cytofluorographic analysis. IL-1 activity in monocyte supernatants and lysates was assayed by augmentation of mitogen-induced murine thymocyte proliferation. IFN-gamma induced the expression of Fc receptors on human monocytes that were specific for murine IgG2a. This induction was inhibited by the addition of LPS in amounts as low as 2 to 8 pg/ml. LPS inhibition of IFN-gamma-induced Fc receptor expression was paralleled by the appearance of IL-1 in monocyte lysates and supernatants. The addition of purified human or recombinant IL-1 beta at the initiation of culture similarly inhibited the expression of IFN-gamma-induced Fc receptors on the monocytes. LPS also inhibited Fc receptor expression on the human myelomonocytic cell line THP-1 after induction with IFN-gamma or phorbol myristate acetate alone or with both agents together. This inhibition also was paralleled by the production of IL-1 but the addition of exogenous IL-1 to the THP-1 cells had no effect on IFN-gamma-induced Fc receptor expression. Tumor necrosis factor (TNF) inhibited IFN-gamma-induced Fc receptor expression on human monocytes but was much less potent than comparable amounts of IL-1. TNF also did not inhibit Fc receptor expression on THP-1 cells. In fact, IL-1 or TNF led to an enhancement in IFN-gamma-induced Fc receptor expression on THP-1 cells. These results indicate that LPS can inhibit IFN-gamma-induced Fc receptor expression on human monocytes and that IL-1 and TNF may mediate these effects of LPS. Thus, an autocrine or paracrine role is suggested for these cytokines. The possibility exists that intracellular IL-1 resulting from LPS stimulation may be at least in part responsible for inhibition of Fc receptor expression.     

Bacterial lipopolysaccharide down-regulates expression of GTP cyclohydrolase I feedback regulatory protein.

GTP cyclohydrolase I feedback regulatory protein (GFRP) is a 9.7-kDa protein regulating GTP cyclohydrolase I activity in dependence of tetrahydrobiopterin and phenylalanine concentrations, thus enabling stimulation of tetrahydrobiopterin biosynthesis by phenylalanine to ensure its efficient metabolism by phenylalanine hydroxylase. Here, we were interested in regulation of GFRP expression by proinflammatory cytokines and stimuli, which are known to induce GTP cyclohydrolase I expression. Recombinant human GFRP stimulated recombinant human GTP cyclohydrolase I in the presence of phenylalanine and mediated feedback inhibition by tetrahydrobiopterin. Levels of GFRP mRNA in human myelomonocytoma (THP-1) cells remained unaltered by treatment of cells with interferon-gamma or interleukin-1beta, but were significantly down-regulated by bacterial lipopolysaccharide (LPS, 1 microg/ml), without or with cotreatment by interferon-gamma, which strongly up-regulated GTP cyclohydrolase I expression and activity. GFRP expression was also suppressed in human umbilical vein endothelial cells treated with 1 microg/ml LPS, as well as in rat tissues 7 h post intraperitoneal injection of 10 mg/kg LPS. THP-1 cells stimulated with interferon-gamma alone showed increased pteridine synthesis by addition of phenylalanine to the culture medium. Cells stimulated with interferon-gamma plus LPS, in contrast, showed phenylalanine-independent pteridine synthesis. These results demonstrate that LPS down-regulates expression of GFRP, thus rendering pteridine synthesis independent of metabolic control by phenylalanine.     

Interferon-gamma mediates gene expression of IL-18 binding protein in nonleukocytic cells

Interleukin-18 (IL-18) binding protein is a soluble decoy receptor for IL-18 which efficiently antagonizes biological functions of IL-18 in vitro and in vivo. Since regulation of IL-18 activity likely contributes to the pathogenesis of inflammatory diseases as well as malignancies, we investigated gene expression of IL-18 binding protein (IL-18BP) in different human cell systems, namely in the keratinocyte cell line HaCaT, in the colon carcinoma cell line DLD-1, and in primary renal mesangial cells. In unstimulated cells only minute amounts of mRNA coding for IL-18 binding protein were detectable. However, in all three cell types gene expression was markedly upregulated by interferon-gamma (IFN-gamma). IL-18 is recognized as a pivotal mediator of IFN-gamma production. Therefore, the present data imply that activity of IL-18 is modulated by a negative feedback mechanism which is mediated by IFN-gamma-induced IL-18 binding protein.     

Chimeric interferon-gamma receptors demonstrate that an accessory factor required for activity interacts with the extracellular domain.

We determined the species specificity and function of structural domains of the interferon-gamma receptor (IFN-gamma R) by construction of human/murine chimeric IFN-gamma R cDNA clones and their expression in various cells. We demonstrate that we can reconstitute a biologically active IFN-gamma R in eukaryotic cells with chimeric receptors as long as the extracellular domain and an accessory factor are from the same species. These results indicate that the extracellular domain of the receptor interacts directly or indirectly with the species-specific accessory factor.     

Expression of HDL receptor, CLA-1 in human smooth-muscle cells and effect of interferon-gamma on its regulation.

High-density lipoprotein (HDL) exerts antiatherogenic effects by various mechanisms. The protective effect of HDL is thought to involve the reverse transport of cholesterol from cells in the arterial wall to the liver for disposal. We previously identified human scavenger receptor BI (hSR-BI/CLA-1) as a receptor for human HDL, but did not examine the expression of hSR-BI/CLA-1 in smooth-muscle cells. In this present study, a human aortic intima smooth-muscle cell line immortalized with SV 40 DNA was established, and the expression of hSR-BI/CLA-1 in this cell line analyzed by Western blot and RT-PCR. HSR-BI/CLA-1 mRNA and protein were detected in both this cell line and primary human aortic smooth-muscle cells. A cytokine, interferon-gamma (IFN-gamma) inhibited the hSR-BI/CLA-1 protein expression, but not mRNA expression. This observation confirmed that selective cholesterol ester uptake from HDL was inhibited by IFN-gamma. These results indicated that hSR-BI/CLA-1 may be expressed in human smooth-muscle cells, and the expression may be modulated by IFN-gamma. HSR-BI/CLA-1 on smooth-muscle cells could play an important role in atherogenesis.     

Proteasome from cytokine-treated human cells shows stimulated BrAAP activity and depressed PGPH activity.

The branched chain amino acid-preferring (BrAAP) activity of multicatalytic proteinase complex isolated from human umbilical vein endothelial cells and treated with interferon-gamma was increased more than 2-fold, which was associated with a marked increase in LMP7 expression and decreased peptidylglutamyl peptide-hydrolyzing activity. Increases in BrAAP activity in supernatants from cells treated with interferon-gamma, tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, or lipopolysaccharide paralleled the increases in LMP7 expression. These findings are consistent with the conclusion that the increased BrAAP activity of LMP-containing multicatalytic proteinase complex results from incorporation of LMP7 or other LMP subunits.     

17beta-estradiol inhibits NADPH oxidase activity through the regulation of p47phox mRNA and protein expression in THP-1 cells

In this report, we demonstrate that NADPH oxidase is activated by tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma) in human monocytic cells (THP-1 cells) differentiated with phorbol ester (PMA) and that physiological concentration of 17beta-estradiol inhibits NADPH oxidase activity in THP-1 cells stimulated with TNF-alpha plus IFN-gamma. This effect is mediated by estrogen receptor based on estrogen receptor antagonist (ICI 182, 780) that diminishes inhibition by 17beta-estradiol. This inhibition is specific in 17beta-estradiol because 17alpha-estradiol, testosterone and progesterone do not inhibit NADPH oxidase activity. Activation of NADPH oxidase induced by TNF-alpha plus IFN-gamma is caused by up-regulation of p47(phox) (cytosolic component of NADPH oxidase) expression. 17beta-Estradiol prevents the up-regulation of p47(phox) mRNA and protein expression. This prevention of p47(phox) expression depends on the inhibition of NF-kappaB activation. Our results implicate that 17beta-estradiol has an anti-atherosclerotic effects through the improvement of nitric oxide (NO) bioavailability caused by the regulation of superoxide (O(2)(-)) production.     

Monoclonal antibody-mediated inhibition of interferon-gamma-induced macrophage antiviral resistance and surface antigen expression

A monoclonal antibody (MAb) with specificity for murine interferon-gamma (IFN-gamma) was used as a probe for studying the effect of recombinant IFN-gamma (rIFN-gamma) on antiviral activity, Fc receptor expression, and Ia antigen induction in macrophages. Cultures of C3H/HeJ peritoneal exudate macrophages were used to allow direct comparison of all three functions in the same target cell system. Our data provide two major findings: the efficacy of the MAb is very different depending on whether murine fibroblasts or macrophages are used as the target cell in the antiviral assay, i.e., greater than 20 to 100 times more MAb was required to block antiviral activity in macrophage cultures; and 10 to 50 times more MAb was required to inhibit Fc receptor vs Ia antigen expression in response to rIFN-gamma. These latter findings confirm and extend previous observations, which indicate that the induction pathways of two important differentiation markers by IFN-gamma may be dissociable.     

Development of horse polyclonal antiserum inhibiting all in vitro biological functions of human IFN-gamma

Following a standard immunization protocol with recombinant human interferon-gamma (IFN-gamma), a horse polyclonal antiserum was obtained and evaluated for its ability to interfere with multiple IFN-gamma activities in vitro. Data obtained show that polyclonal horse antiserum neutralizes the antiproliferative activity of IFN-gamma, inhibits the binding of IFN-gamma to cellular receptors, and can up-regulate HLA-DR antigen expression and interfere with its antiviral activity. The broad neutralizing capacity of horse polyclonal antiserum has been assessed on cell lines which differ as to origin and sensitivity to IFN-gamma. Moreover, we observed that this antiserum could inhibit the binding of radiolabeled IFN-gamma to its cellular receptor, its subsequent internalization into the target cell, and its antiviral activity. As it is able to inhibit all the biological activities of IFN-gamma, this antiserum might provide new therapeutic approaches to diseases with evidence of activated cell-mediated immunity.     

Indoleamine 2,3-dioxygenase activity and L-tryptophan transport in human breast cancer cells

The activity and expression of indoleamine 2,3-dioxygenase together with L-tryptophan transport has been examined in cultured human breast cancer cells. MDA-MB-231 but not MCF-7 cells expressed mRNA for indoleamine 2,3-dioxygenase. Kynurenine production by MDA-MB-231 cells, which was taken as a measure of enzyme activity, was markedly stimulated by interferon-gamma (1000 units/ml). Accordingly, L-tryptophan utilization by MDA-MB-231 cells was enhanced by interferon-gamma. 1-Methyl-DL-tryptophan (1 mM) inhibited interferon-gamma induced kynurenine production by MBA-MB-231 cells. Kynurenine production by MCF-7 cells remained at basal levels when cultured in the presence of interferon-gamma. L-Tryptophan transport into MDA-MB-231 cells was via a Na(+)-independent, BCH-sensitive pathway. It appears that system L (LAT1/CD98) may be the only pathway for l-tryptophan transport into these cells. 1-Methyl-D,L-tryptophan trans-stimulated l-tryptophan efflux from MDA-MB-231 cells and thus appears to be a transported substrate of system L. The results suggest that system L plays an important role in providing indoleamine-2,3-dioxygenase with its main substrate, L-tryptophan, and suggest a mechanism by which estrogen receptor-negative breast cancer cells may evade the attention of the immune system.     

A promoter for Bacillus subtilis expression vector

A 117-bp EcoRI-PstI fragment with strong promoter activity (P1 promoter) was cloned from Bacillus subtilis chromosomal DNA and sequenced. The P1 promoter was shown to contain a putative -35 region (TTTACT) and -10 region (TAGATT), and promotes expression of cloned human interleukin-2 (IL-2) and human interferon-gamma (IFN-gamma) genes in B. subtilis.