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The human interferon alpha-receptor (IFNAR gene product or IFN alpha R protein) was expressed in hamster CHO cells and in mouse A9 cells. The response of the IFN alpha R cDNA transfectants to human IFNs was studied by measuring induction of (2'-5') A synthetase (2'-5' AS). In the murine cells, the IFN alpha R protein conferred response to the human IFN-alpha-8 (alpha-B) subtype, but not to huIFN-alpha-2 (alpha-A) or to huIFN-beta. In murine huIFN alpha R cDNA transfectants, containing a hygromycin B resistance gene placed under the control of the 2'-5' AS gene Interferon Response Sequence (IRS), survival and growth of the cells in the presence of hygromycin B was induced by huIFN-alpha-8 but not by huIFN-alpha-2, indicating that the effect of huIFN alpha R is transcriptional. In hamster CHO cells, the huIFN alpha R protein conferred a completely different pattern of response to human IFN subtypes. Thus, the CHO-IFN alpha R transfectants responded to huIFN-beta by 2'-5' AS induction as well as by activation of the ISGF3 and IRF-1 transcription factors. In contrast, the CHO-IFN alpha R cells showed no response to huIFN-alpha-8. The differential response conferred by the huIFN alpha R protein in the two types of rodent cells, indicates that IFN subtype recognition is influenced by another component contributed by the rodent host cell. The ability of human cells, and of human-mouse hybrid cells containing human chromosome 21, to respond to all IFN subtypes is likely to depend also on interactions of the IFN alpha R protein with additional receptor components.

Mice lacking the known subunit of the type I interferon (IFN) receptor were completely unresponsive to type I IFNs, suggesting that this receptor chain is essential for type I IFN-mediated signal transduction. These mice showed no overt anomalies but were unable to cope with viral infections, despite otherwise normal immune responses. Comparison of mice lacking either type I or type II IFN receptors showed that, at least in response to some viruses, both IFN systems are essential for antiviral defense and are functionally nonredundant.

A subunit of the interferon alpha receptor (IFN alpha R) that confers biologic response to and specific "binding" for IFN alpha 8 has recently been cloned. We have explored the biological consequences of expressing the cloned IFN alpha R subunit in human cells resistant to IFN alpha and in mouse cell lines nonresponsive to human IFN alpha. The expression of the cloned IFN alpha R subunit in the human IFN alpha-resistant K-562 cell line restored sensitivity to the antiviral effect of not only IFN alpha 8 but also IFN alpha 2 and IFN alpha Con1. In mouse L-929 cells the expression of the cloned receptor subunit markedly increased antiviral sensitivity to human type I IFNs. In either human K-562 or mouse L-929 cells these effects were observed without a detectable increase in the binding for any of the subtypes of IFN alpha tested. We propose that the cloned IFN alpha R subunit functions as a transducer subunit for the IFN alpha R. This concept is supported by the finding that the cloned receptor protein, when it is expressed in Cos cells, has an M(r) of 75 kDa, which is different from the main IFN alpha-binding proteins, the alpha and beta subunits of the IFN alpha R. This report also suggests that alterations at the receptor level could be involved in IFN alpha resistance in some cell lines.

Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by induction of class I human leukocyte antigen (HLA) and protection against encephalomyocarditis virus (EMCV) infection. Whereas human chromosome 6 encodes the Hu-IFN-gamma receptor, human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. Probes from a genomic clone were used to identity cDNA clones expressing a species-specific accessory factor. These cDNA clones are able to substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma receptor-mediated induction of class I HLA antigens. However, the factor encoded by the cDNA does not confer full antiviral protection against EMCV, confirming that an additional factor encoded on human chromosome 21 is required for reconstitution of antiviral activity against EMCV. We conclude that this accessory factor belongs to a family of such accessory factors responsible for different actions of IFN-gamma.

It has been previously reported that inhibition of human erythroid colony-forming units (CFU-E) in vitro by interleukin-1 (IL-1) is an indirect effect, occurring through the production of interferon gamma (IFN gamma). IFN gamma, in turn, inhibits CFU-E colony formation directly, and its inhibitory effect can be overcome by exposure to high concentrations of erythropoietin (EPO). To develop an in vitro animal model for investigating inhibition of erythropoiesis by IFN gamma, the effects of recombinant murine (rm) IFN gamma on highly purified CFU-E from the spleens of mice infected with the anemia strain of the Friend virus (FVA) were studied. rmIFN gamma inhibited CFU-E colony formation in a dose-dependent manner. This inhibition occurred with large (> or = 8 cell) colonies only; smaller colonies were not affected. The inhibitory effect was corrected to 72% of control by high EPO concentrations of 64 U/mL. Murine CFU-E were then cultured with rmIFN gamma in the presence of a soluble murine IFN gamma receptor fused to the hinge and Fc domains of the human IgG1 heavy chain (mIFN gamma R-IgG). Inhibition of CFU-E colony formation by rmIFN gamma (100 U/mL) was corrected by mIFN gamma R-IgG in a dose-dependent manner, with an approximate IC50 of 0.05 nmol/L, and complete or near complete correction at 0.5 nmol/L. Similarly, a human IFN gamma R-IgG greatly reduced the inhibitory effect of recombinant human IFN gamma on human CFU-E. These experiments provide an in vitro animal model for studying the inhibitory effects of IFN gamma on erythropoiesis and indicate that IFN gamma R-IgG may be a useful agent for reducing the toxicity of IFN gamma in vivo.

We have described conditions by which MHC class II (I-A) glycoproteins can be induced to be differentially expressed after treatment of macrophages with rIFN-gamma. Treatment of macrophages from BCG-resistant mice with 1 U of rIFN-gamma induced transient I-A expression that decayed in the presence of cycloheximide. Subsequent treatment of these macrophages with 100 U of rIFN-gamma induced the persistence of I-A that was not affected by cycloheximide. The aim of this investigation was to define, by pharmacologic intervention, the second signals that resulted in the induction of persistence of I-A. Treatment of the macrophages that transiently expressed I-A with PMA resulted in the induction of persistence. When we compared the effect of different protein kinase C (PKC) inhibitors with the induction of persistence by rIFN-gamma, we found that H-7 blocked the induction of persistence only when added before or at the same time as the addition of a high dose of rIFN-gamma. In contrast, the addition of staurosporine to macrophages as late as 2 h after treatment with high doses of rIFN-gamma inhibited the induction of I-A persistence. The addition of a high dose of rIFN-gamma to macrophages previously treated with a low dose of rIFN-gamma resulted in the synergistic activation of PKC. The effect of H-7 and of staurosporine on the activation of PKC activity coincided with the effect of these inhibitors on the induction of persistent I-A expression. Tyrosine kinase inhibitors genistein and herbimycin did not affect the induction of I-A persistence nor of PKC activation. Antibody to the IFN-gamma receptor inhibited PKC activation. Finally, the addition of the high dose of rIFN-gamma to macrophages from BALB/c.Bcgs mice, previously treated with the low dose of rIFN-gamma, failed to activate high levels of PKC activity attained after similar treatment of macrophages from BALB/c.Bcgr mice. One effect of the Bcg gene may be to regulate the activation of PKC activity.

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-Guérin (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.

Interferon-gamma (IFN-gamma) is a potent growth-inhibitory cytokine also endowed with differentiating activity on neural cells. Binding of IFN-gamma to its high-affinity receptor induces a rapid and transient activation of phospholipase A2 (PLA2). The mechanism coupling the IFN-gamma receptor (IFN-gamma-R) to PLA2 activation is not clearly defined, and no information is available on this mechanism in neuroblast cells. We have tested the hypothesis that GTP-binding proteins (G-proteins) may couple the IFN-gamma-R to PLA2 in the human neuroblastoma (NB) cell line LAN-5. Incubation of NB cells with IFN-gamma resulted in a rapid increase in [3H]arachidonic acid (AA) release, and this effect was blocked by pretreatment with anti-IFN-gamma antibodies. IFN-gamma-stimulated AA release was still observed in permeabilized cells that were blocked by pretreatment with anti-IFN-gamma-R antibodies. Exposure of permeabilized LAN-5 cells to guanosine 5'-[gamma-thio]triphosphate (GTP[S]), a non-hydrolysable GTP analogue, induced a dose-dependent release of [3H]AA. A non-specific nucleotide effect was excluded, since similar stimulatory effects on AA mobilization were not observed by GTP, ATP, CTP, ADP and GDP. IFN-gamma-stimulated AA release was completely blocked by the guanine nucleotide analogue that inhibits G-protein function, guanosine 5'-[beta-thio]diphosphate (GDP[S]). A role for G-proteins in IFN-gamma-R coupling to PLA2 was further supported by the inhibition of IFN-gamma-induced [3H]AA release by treatment of permeabilized cells with pertussis toxin and with the antiserum against the common alpha-subunits of G-proteins. To determine a possible contribution to AA mobilization by the phospholipase C and diacyglycerol lipase pathway or by protein kinase C activation, the effects of neomycin, a phospholipase C inhibitor, and PMA (phorbol 12-myristate 13-acetate), a direct activator of protein kinase C, were investigated. Neither neomycin nor PMA affected either basal or IFN-gamma-stimulated AA release. Ca2+ concentration, which has been shown to regulate the activity of some PLA2s, does not appear to play an important role in the regulation of the IFN-gamma-stimulated PLA2 activity, since incubating permeabilized cells in different concentrations of Ca2+ induced AA release without affecting the IFN-gamma response. Altogether, these findings suggest the existence of IFN-gamma-R, which couples a Ca(2+)-independent PLA2 activation via pertussis-toxin-sensitive G-proteins.

Background: Although pokeweed mitogen (PWEM) can induce peripheral blood mononuclear cells (PBMCs) to synthesize IgG, IgA, and IgM, such cultures fail to induce IgE synthesis. The present study examined the possibility that the stimulation of interferon-gamma (IFN-gamma) production plays a role in the failure of PWM to induce IgE synthesis., Methods: We examined PBMCs from eight normal control subjects for IFN-gamma and IL-4 production. Since IFN-gamma synthesis is known to inhibit IgE synthesis, we also examined the effect of a neutralizing anti-IFN-gamma antibody and of two anti-IFN-gamma receptor antibodies, monoclonal antibody (mAb) GIR208, which blocks cellular binding of IFN-gamma, and mAB GIR94.5, which binds to the IFN-gamma receptor but does not block the binding of IFN-gamma to its receptor on PWM-stimulated PBMCs., Results: After stimulation with PWM, culture supernatants contained significantly more IFN-gamma (p = 0.001) and IL-4 (P = 0.001) compared with supernatants from nonstimulated cultures. PWM-stimulated PBMCs also expressed higher levels of IFN-gamma and IL-4 gene transcripts than unstimulated cells. When cultured in the presence of anti-IFN-gamma, supernatants from PWM-stimulated cultures also induced CD23 on Ramos B cells in an IL-4-dependent manner. In the presence of mAB GIR208 and a neutralizing anti-IFN-gamma antibody, but not mAb GIR94.5, PWM stimulated PBMCs from eight normal control subjects and six patients with atopic dermatitis to produce IgE. Monoclonal antibody GIR208, however, did not enhance IgG synthesis. Furthermore, the exogenous addition of IFN-gamma inhibited the IgE-stimulatory effect of mAb GIR208. Monoclonal antibody GIR208 was unable to induce purified B cells to synthesize IgE in the presence of IL-4., Conclusions: Thus unlike anti-CD40, mAb GIR208 does not act as a second signal for the induction of IgE synthesis. These results demonstrate that the induction of IFN-gamma production contributes to the failure of PWM to stimulate synthesis of IgE in PBMCs from atopic and nonatopic donors.

Interferon-gamma (IFN-gamma) exerts pleiotropic effects, including antiviral activity, stimulation of macrophages and natural killer cells, and increased expression of major histocompatibility complex antigens. Mice without the IFN-gamma receptor had no overt anomalies, and their immune system appeared to develop normally. However, mutant mice had a defective natural resistance, they had increased susceptibility to infection by Listeria monocytogenes and vaccinia virus despite normal cytotoxic and T helper cell responses. Immunoglobulin isotype analysis revealed that IFN-gamma is necessary for a normal antigen-specific immunoglobulin G2a response. These mutant mice offer the possibility for the further elucidation of IFN-gamma-mediated functions by transgenic cell- or tissue-specific reconstitution of a functional receptor.

The single cell-thick intestinal epithelium forms a crucial barrier between the host and environment, and is modeled in vitro by a monolayer of polarized, highly differentiated T84 epithelial cells impermeable to most macromolecules because of functional intercellular tight junctions. Absence of a permeability defect across the monolayer, either transcellular or paracellular, is indicated by development of a transepithelial electrical resistance of > or = 1000 ohm-cm2, reported to be markedly diminished by exposure to a T lymphocyte cytokine, IFN-gamma. We sought to define this phenomenon in four ways by determining its duration and reversibility; the uniqueness of type II (gamma) IFN as opposed to type I (alpha) IFN; the surface of the polarized columnar epithelium likely involved in responding to IFN-gamma; and whether a specific surface membrane receptor on the epithelial cell participates in the response. Using a special apparatus that allows differential cytokine exposure of monolayer surfaces, our data demonstrate 1) only the monolayer's basolateral surface is IFN-gamma responsive, whereas the apical (microvillous) surface is no; 2) the alteration in electrical resistance of epithelium is prolonged (5 days), even after a single (24 h) exposure to IFN-gamma, but nevertheless is reversible; 3) the effect is likely receptor-ligand mediated, because it can be partially blocked by IFN-gamma receptor-specific monoclonal Ig; 4) an alteration in tight junction function (a paracellular pathway) rather than cell necrosis or a transcellular pathway is responsible for IFN-gamma-induced monolayer dysfunction because permeability to a 44,000-Da macromolecule (horseradish peroxidase) did not increase, and intracytoplasmic T84 cell enzymes were not released into the media; and 5) the biologic phenomenon could not be induced by a species (alpha) of class I IFN, making IFN-gamma reasonably unique in this regard. Given the proximity; activation status, and capacity of T lymphocytes for cytokine production in mucosa, we suggest that IFN-gamma-induced changes in epithelial permeability may be a major cause of altered intestinal barrier function in vivo.

A cDNA encoding a 63-kDa human IFN-alpha R (hIFN-alpha R) has recently been cloned. Mouse cells transfected with this cDNA failed to show any signal transmission for IFN-beta, suggesting the involvement of additional chains in the receptor complex. We have expressed in Escherichia coli and COS 7 cells a soluble recombinant protein (sIFN-alpha R) comprising the extracellular domain of the hIFN-alpha R fused at the carboxyl terminus to a sequence of five histidine residues. The sIFN-alpha R was affinity purified and used to generate mAb. One mAb, 64G12, which recognized the cellular receptor as well as the recombinant proteins was found to neutralize the biologic activity of IFN-alpha, -beta, and -omega and natural type I IFN, but not IFN-gamma. To our knowledge, this is the first report of a neutralizing mAb against the human IFN-alpha R. Interestingly, we found that the affinity of this antibody for the cellular receptor depended on the cell line used. These results provide strong evidence that the cloned hIFN-alpha R chain is required for binding and signal transmission of all subspecies of type I IFN and suggest a structural heterogeneity of the receptor at the cell surface.

Recombinant human tumor necrosis factor and recombinant human gamma interferon (IFN-gamma) exert synergistic growth inhibitory effects in WiDR human colorectal carcinoma cells. In this cell line, tumor necrosis factor increases IFN-gamma binding. Interleukin 1 (IL-1) is a cytokine that mimics many of the biological actions of TNF. Therefore, in the present study, we investigated the effects of recombinant human IL-1 on cell growth and IFN-gamma receptor expression in WiDR cells. IL-1 slightly inhibited the growth of WiDR cells, and exerted additive growth inhibitory effects in the presence of IFN-gamma. IL-1 caused a time- and dose-dependent increase in 125I-labeled IFN-gamma binding that was maximal at 6 h, persisted for at least 24 h, and was blocked by both actinomycin D and cycloheximide. The increase in binding was associated with an increase in cell surface IFN-gamma receptor protein expression as determined by Scatchard analysis of equilibrium binding data and by immunofluorescent staining with an anti-human IFN-gamma receptor monoclonal antibody. IL-1 also produced a time- and dose-dependent increase in IFN-gamma receptor mRNA levels that was maximal at 3 h and persisted for at least 24 h. Actinomycin D, but not cycloheximide, completely blocked the IL-1-mediated increase in IFN-gamma receptor mRNA levels. However, IL-1 did not alter IFN-gamma receptor mRNA half-life. These data indicate that IL-1 and IFN-gamma exert additive growth inhibitory effects on colon cancer cell growth, and suggest that IL-1 increases IFN-gamma receptor expression in these cells by enhancing IFN-gamma mRNA levels.

The last ten years have seen an explosive growth in our understanding of IFN gamma. The cloning of the cDNAs for IFN gamma and its receptor have made available large amounts of highly purified recombinant IFN gamma and soluble IFN gamma receptor. In addition, highly specific neutralizing monoclonal antibodies have been generated to both of these proteins. Using these reagents, IFN gamma and the IFN gamma receptor have been characterized on a molecular basis. Structure-function studies carried out on these proteins have identified key molecular regions that are required for biologic activity. Moreover, a great deal is now known concerning the physiologic role that IFN gamma plays in vivo. In this review we focus on the new developments in the areas of IFN gamma biochemistry and biology and pay particular attention to the IFN gamma receptor and three aspects of IFN gamma biology that are of special interest to immunologists: host defense, inflammation, and autoimmunity.

The signal transduction pathway of alpha interferon utilizes tyrosine phosphorylation to transmit a signal generated at the cell surface to the transcriptional machinery in the nucleus. Activation of the interferon pathway initiates with the binding of alpha interferon to its cell surface receptor. The ligand-receptor complex signals the activation of a latent cytoplasmic transcription factor. The active form of the interferon-stimulated gene factor (ISGF3) is phosphorylated on tyrosine residues. ISGF3 subsequently translocates to the nucleus and binds to a DNA sequence, the interferon-stimulated response element, found within the promoter of inducible genes. ISGF3 is a multicomponent factor consisting of four proteins of 113 kDa, 91 kDa, 84 kDa, and 48 kDa. Three proteins consistent with sizes of 113 kDa, 91 kDa, and 84 kDa copurify with ISGF3 and are phosphorylated on tyrosine residues after stimulation by alpha interferon. Tyrosine phosphorylation is essential for activation of ISGF3. Genistein, a tyrosine kinase inhibitor, blocks the appearance of ISGF3 and blocks the transcriptional stimulation of interferon-induced genes. This study shows that tyrosine phosphorylation provides a link between the interferon-receptor complex at the plasma membrane and specific activation of gene expression in the nucleus.

Mutations of the human interferon gamma (IFN-gamma) receptor intracellular domain have permitted us to define a restricted region of that domain as necessary for both induction of class I major histocompatibility complex antigen by IFN-gamma and protection against encephalomyocarditis virus. This region consists of five amino acids (YDKPH), all of which are conserved in the human and murine receptors. Tyr-457 and His-461 are essential for activity. Approximately 80% of the amino acids of the intracellular domain of the receptor is not required for major histocompatibility complex class I antigen induction or for antiviral protection against encephalomyocarditis virus. The observation that there was no protection by IFN-gamma against vesiculostomatitis virus indicates that other factors, in addition to chromosome 21 accessory factor(s), are required to generate the full complement of transduction signals from the human IFN-gamma receptor.

Differences in activity among the family of human IFNs alpha are much reduced if these ligands are assayed on bovine cells. In particular, the activity of IFN alpha D is much higher on bovine than on human cells. To examine these differences, the bovine counterpart of the human IFNAR has been cloned and expressed in a human cell line. The transfected cell line now recognizes the human IFN alpha D as a high-specific-activity IFN subtype, indicating that the differences in sensitivity between the bovine and human cells to the human IFN alpha lie in the structure of the IFNAR chain rather than in the other components of the functional receptor.

Interferon is a key substance of the cytokine network, and is functioning not only as the virus inhibitory factor, but also as the factor for differentiation, development, and homeostasis of animals, including man. The molecular structure, induction mechanisms, interaction with receptor, and antiviral action mechanisms of IFN were reviewed. 1. Molecular structure of IFN IFNs are now divided into 4 types--alpha, beta, gamma, omega--according to antigenicities of IFN proteins, and to base sequence of their genes. The omega type was established officially on 1990 by the Nomenclature Committee of International Society for Interferon Research (ISIR). Also, the genes for HuIFNs were named as follows; HuIFN-A for HuIFN-alpha, HuIFN-B for HuIFN-beta, HuIFN-G for HuIFN-gamma, and HuIFN-W for HuIFN-omega. Nevertheless, the names of Type I IFN and Type II IFN are still valuable, since members of Type I IFN, alpha, beta, and omega, are similar in their molecular structures, and they share the common receptor (Type I IFN receptor), on the other hand, HuIFN-gamma (the only one member of Type II IFN) has different characteristics from the other IFNs, and binds to its specific receptor (Type II IFN receptor). However, it has been reported that both N-terminal and C-terminal of IFN protein of all types, participate in the binding of IFN to its receptor. 2. Induction mechanism of IFN. The factors, IRF-1 and IRF-2, were found to play an important role in IFN induction.(ABSTRACT TRUNCATED AT 250 WORDS)

The gene coding for the alpha,beta-interferon (alpha,beta-IFN) receptor is localized to chromosome 21. Cells from patients with Down's syndrome (trisomy 21) contain an extra chromosome 21, which results in a 1.5 times increase of dosage for the genes localized to this chromosome. Trisomy 21 cells express more cell-surface alpha-IFN receptors, consistent with the increased gene dosage. Down-regulation of the alpha-IFN receptors in trisomy 21 and normal cells was studied by incubating the cells with alpha-IFN. The alpha-IFN-induced effects showed 1.6 times more internalized cell-surface alpha-IFN receptors in trisomy 21 cells compared with normal cells, but no statistically significant change in the dissociation constants. A close relationship was found between the alpha-IFN receptor number and the biological response expressed as 2',5'-oligoadenylate synthetase activity.