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At least 757 records · Page 42Linked to original sources
PubMed2003

9-cis retinoic acid enhances the antiviral effect of interferon on hepatitis C virus replication through increased expression of type I interferon receptor.

The concentration of type I interferon receptor (IFN-Rc) in the liver is a crucial factor in determining the efficacy of interferon (IFN) therapy in patients with chronic hepatitis C. Retinoic acids (RAs) can enhance the expression of type I IFN-Rc expression. The aim of this study was to investigate whether RAs increase the anti-hepatitis C virus (HCV) effect of IFN through an increase in IFN-Rc. The hepatocellular carcinoma cell line HuH-7 was treated with 10(-7) mol/L all-trans RA (ATRA) and 9-cis RA (9-CRA). Expression of type I IFN-Rc was investigated at both the mRNA and protein levels with the use of real-time quantitative polymerase chain reaction and flow cytometry, respectively. We investigated the anti-HCV effect, using in vitro HCV transfection, by monitoring the level of HCV RNA in the culture medium. ATRA and 9-CRA enhanced the expression of type I IFN-Rc at both the mRNA and protein levels. After IFN-alpha treatment, the activity of 2,5'-oligoadenylate synthetase was enhanced by RAs, and this enhancement was abolished when blocking antibodies had previously been bound to the surface receptors. IFN treatment decreased the concentration of HCV RNA, and this effect was enhanced by treatment with RAs. Our findings suggest that RAs enhance the anti-HCV replication effect of IFN-alpha through up-regulation of type I IFN-Rc in HuH-7 cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1995

Chicken interferon consensus sequence-binding protein (ICSBP) and interferon regulatory factor (IRF) 1 genes reveal evolutionary conservation in the IRF gene family.

Members of the IRF family mediate transcriptional responses to interferons (IFNs) and to virus infection. So far, proteins of this family have been studied only among mammalian species. Here we report the isolation of cDNA clones encoding two members of this family from chicken, interferon consensus sequence-binding protein (ICSBP) and IRF-1. The predicted chicken ICSBP and IRF-1 proteins show high levels of sequence similarity to their corresponding human and mouse counterparts. Sequence identities in the putative DNA-binding domains of chicken and human ICSBP and IRF-1 were 97% and 89%, respectively, whereas the C-terminal regions showed identities of 64% and 51%; sequence relationships with mouse ICSBP and IRF-1 are very similar. Chicken ICSBP was found to be expressed in several embryonic tissues, and both chicken IRF-1 and ICSBP were strongly induced in chicken fibroblasts by IFN treatment, supporting the involvement of these factors in IFN-regulated gene expression. The presence of proteins homologous to mammalian IRF family members, together with earlier observations on the occurrence of functionally homologous IFN-responsive elements in chicken and mammalian genes, highlights the conservation of transcriptional mechanisms in the IFN system, a finding that contrasts with the extensive sequence and functional divergence of the IFNs.

Amino Acid Sequence↗
PubMed1999

Dimerization of the interferon type I receptor IFNaR2-2 is sufficient for induction of interferon effector genes but not for full antiviral activity.

We constructed chimeric receptors wherein the extracellular domain of the erythropoietin receptor (EpoR) was fused to the transmembrane and intracellular domains of the interferon (IFN) type I receptor subunits, IFNaR1 or IFNaR2-2. Transfection into 2fTGH and Tyk2-deficient 11,1 cells showed that EpoR/IFNaR2-2 alone was able to transduce a signal upon stimulation with erythropoietin (Epo), as judged by induction of the interferon type I-inducible 6-16 promoter. In contrast, protection against infection with encephalomyocarditis virus or vesicular stomatitis virus was reduced or absent, respectively. To further investigate the role of IFNaR1 in the induction of an antiviral state, we analyzed the Epo- versus IFNalpha-induced transcription of a set of genes, involved in antiviral protection. Up to 24 h after stimulation with Epo or IFNalpha, comparable transcription of the p56, dsRNA-dependent protein kinase, 2'-5'A synthetase, and MxA genes was seen. However, at later time points, only in the case of Epo induction, a sharp decrease of mRNA levels was observed. Western blotting analysis of dsRNA-dependent protein kinase showed a similar pattern at the protein level. Taken together, our results imply a role for IFNaR1 in the induction of sustained mRNA and protein levels that are likely required for optimal antiviral activity.

Alkaline Phosphatase↗
PubMed2004

Transcriptional regulation of the 230-kDa bullous pemphigoid antigen gene expression by interferon regulatory factor 1 and interferon regulatory factor 2 in normal human epidermal keratinocytes.

Interferon regulatory factors (IRFs) are a family of transcriptional factors induced by interferon-gamma (IFN-gamma). Recent studies have indicated that the deregulation of IRF system in keratinocytes is responsible, at least in part, for aberrant proliferation and the differentiation of the psoriatic epidermis. Previously, we reported that the expression of 230-kDa bullous pemphigoid antigen (BPAG1) gene, which is strictly restricted to basal keratinocytes, is transcriptionally suppressed by IFN-gamma, but the contribution of IRFs in such suppression is still unclear. In this study, we investigated the role of IRFs in the regulation of BPAG1 gene expression. Computer analysis identified IRF1 and IRF2 consensus sequences between -135 and -123 on BPAG1 promoter region. Transient transfection studies with BPAG1 promoter-luciferase reporter gene plasmids and IRF1 and IRF2 expression plasmids revealed that IRF1 and IRF2 directly down-regulated BPAG1 gene transcription in cultured normal human epidermal keratinocytes. Several sets of gel retardation assays with the BPAG1-IRF binding sequence as a probe indicated that IRF1 and IRF2 could bind to the BPAG1-IRF sequence, but some other protein(s), which was induced by IFN-gamma stimulation and possessed binding activity to IRF consensus sequence, showed preferential binding to the BPAG1-IRF sequence. Our results suggest that IFN-gamma-IRF system is involved in BPAG1 gene regulation in type-1 helper T-cell inflammatory skin conditions, such as psoriasis vulgaris.

Autoantigens↗
PubMed1994

Structure and regulation of the human interferon regulatory factor 1 (IRF-1) and IRF-2 genes: implications for a gene network in the interferon system.

Interferon regulatory factor 1 (IRF-1) and IRF-2 are structurally similar DNA-binding factors which were originally identified as regulators of the type I interferon (IFN) system; the former functions as a transcriptional activator, and the latter represses IRF-1 function by competing for the same cis elements. More recent studies have revealed new roles of the two factors in the regulation of cell growth; IRF-1 and IRF-2 manifest antioncogenic and oncogenic activities, respectively. In this study, we determined the structures and chromosomal locations of the human IRF-1 and IRF-2 genes and further characterized the promoters of the respective genes. Comparison of exon-intron organization of the two genes revealed a common evolutionary structure, notably within the exons encoding the N-terminal portions of the two factors. We confirmed the chromosomal mapping of the human IRF-1 gene to 5q31.1 and newly assigned the IRF-2 gene to 4q35.1, using fluorescence in situ hybridization. The 5' regulatory regions of both genes contain highly GC-rich sequences and consensus binding sequences for several known transcription factors, including NF-kappa B. Interestingly, one IRF binding site was found within the IRF-2 promoter, and expression of the IRF-2 gene was affected by both transient and stable IRF-1 expression. In addition, one potential IFN-gamma-activated sequence was found within the IRF-1 promoter. Thus, these results may shed light on the complex gene network involved in regulation of the IFN system.

Animals↗
PubMed1996

A novel interferon regulatory factor family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes.

We have isolated a novel cDNA clone encoding interferon (IFN) consensus sequence-binding protein in adult T-cell leukemia cell line or activated T cells (ICSAT); this protein is the human homolog of the recently cloned Pip/LSIRF. ICSAT is structurally most closely related to the previously cloned ICSBP, a member of the IFN regulatory factor (IRF) family of proteins that binds to interferon consensus sequences (ICSs) found in many promoters of the IFN-regulated genes. Among T-cell lines investigated, ICSAT was abundantly expressed in human T-cell leukemia virus type 1 (HTLV-1)-infected T cells. When the HTLV-1 tax gene was expressed or phorbol myristake acetate-A23187 stimulation was used, ICSAT expression was induced in Jurkat cells which otherwise do not express ICSAT. When the binding of ICSAT to four different ICSs was tested, the relative differences in binding affinities for those ICSs were determined. To study the functional role of ICSAT, we performed cotransfection experiments with the human embryonal carcinoma cell line N-Tera2. ICSAT was demonstrated to possess repressive function over the gene activation induced by IFN stimulation or by IRF-1 cotransfection. Such repressive function is similar to that seen in IRF-2 or ICSBP. However, we have found that ICSAT has a different repressive effect from that of IRF-2 or ICSBP in some IFN-responsive reporter constructs. These results suggest that a novel mechanism of gene regulation by "differential repression" is used by multiple members of repressor proteins with different repressive effects on the IFN-responsive genes.

Amino Acid Sequence↗
PubMed1998

Identification and regulation of testicular interferon-gamma (IFNgamma) receptor subunits: IFNgamma enhances interferon regulatory factor-1 and interleukin-1beta converting enzyme expression.

Interferon-gamma (IFNgamma) transmits its signal through a specific cell surface receptor (IFNgammaR), which consists of a primary ligand binding alpha-chain (IFNgammaR alpha) and a signaling beta-chain (IFNgammaR beta). Recent studies identified the cytokines IFNgamma, interleukin-6 (IL-6), IL-1alpha, and tumor necrosis factor-alpha in testicular cells. Therefore, we: 1) examined the expression of IFNgammaR alpha and IFNgammaR beta subunits in freshly isolated and purified rat testicular cells; 2) examined the differential regulation of receptor components by cytokines using primary cultures of Sertoli cells; 3) identified the cell signaling pathway components of testicular IFNgammaR; and 4) characterized the functional role of testicular IFNgamma using primary Sertoli cells. We demonstrated the messenger RNAs for both chains of IFNgammaR in rat testicular cells using Northern hybridization analysis. Western blot analysis and immunocytochemistry showed that both specific IFNgammaR protein subunits were present in cultured primary Leydig and Sertoli cells prepared from the testes of immature rats. The expression of both IFNgammaR component messenger RNAs in cultured Sertoli cells was increased by its specific ligand (IFNgamma), as well as IL-1alpha and tumor necrosis factor-alpha, in both a time- and dose-dependent manner. IFNgamma-activation of the Janus (JAK) tyrosine kinases, JAK1 and JAK2 proteins, indicate that IFNgammaR, expressed in the Sertoli cell, is functional. Moreover, IFNgamma modulates the expression of interferon regulatory factor (IRF)-1 and IL-1beta converting enzyme genes in Sertoli cells. Thus, our data are suggestive of a role(s) for IFN-gamma in the regulation of distinct gene expression and cell-specific sensitivity to apoptosis in the testis.

Animals↗
PubMedDate not supplied

[The functioning of the interferon system with different methods and dosages of recombinant alpha 2-interferon (reaferon) administration].

In clinical trials in 24 healthy volunteers of different doses of reaferon inoculated intramuscularly and intravenously for 3 days a pyrogenic reaction was observed the intensity of which depended upon the dose and route of inoculation. Reaferon exerted a dose-dependent effect on the recipient's interferon system: with large doses the interferon response of leukocytes and the capacity of lymphocytes for gamma-IF synthesis were markedly inhibited, whereas low doses (2 million IU/day) caused no significant changes. The intravenous inoculation of reaferon produced a relatively high but short-term, and the intramuscular inoculation a relatively low but of longer duration antiviral activity in the blood serum.

Adolescent↗
PubMed1993

Differential regulation of human indoleamine 2,3-dioxygenase gene expression by interferons-gamma and -alpha. Analysis of the regulatory region of the gene and identification of an interferon-gamma-inducible DNA-binding factor.

The induction of indoleamine 2,3-dioxygenase (IDO) activity has been implicated in the antiproliferative action of interferon (IFN)-gamma on tumor cells and the inhibition of intracellular pathogens. Earlier studies have demonstrated that the expression of the IDO gene is induced strongly by IFN-gamma, but very poorly by IFN-alpha despite the presence of a sequence highly homologous to the IFN-alpha-responsive sequence element (interferon-stimulated response element (ISRE)) in its IFN-gamma-responsive control region. In addition, a sequence with a partial homology to the IFN-gamma-responsive sequence (GAS) identified by Lew et al. (Lew, D.J., Decker, T., Strehlow, I., and Darnell, J.E., Jr. (1991) Mol. Cell. Biol. 11, 182-191) in a human gene for a guanylate-binding protein and to the X box sequence found in all major histocompatibility complex class II genes was found. Deletion experiments have indicated that the ISRE homolog (but not the GAS-related or the X box-related sequence) was essential for an inducibility by IFN-gamma. To investigate the lack of inducibility by IFN-alpha despite the presence of an ISRE homolog, the binding of this ISRE homolog to the IFN-alpha-stimulated gene factor 3 (ISGF3) was examined. Gel mobility shift experiments and competition experiments indicated that this ISRE homolog did not form a stable complex with ISGF3. This may account for a poor inducibility by IFN-alpha. This inability to bind ISGF3 appears to be (at least in part) due to minor differences between the nucleotide sequence of the ISRE homolog present in the IDO gene promoter and the ISRE consensus sequence found in IFN-alpha-inducible genes. An IFN-gamma-inducible DNA-binding factor was identified with characteristics different from ISGF3: (i) the IFN-gamma-inducible factor was detected in the nuclear extracts, but not in the cytoplasmic extracts; and (ii) the appearance of this DNA-binding factor required new protein synthesis, which could explain the dependence on new protein synthesis for the induction of IDO by IFN-gamma.

Amino Acid Sequence↗
PubMed1998

Expression of interferon-gamma receptors and interferon-gamma-induced up-regulation of intercellular adhesion molecule-1 in basal cell carcinoma; decreased expression of IFN-gamma R and shedding of ICAM-1 as a means to escape immune surveillance.

The peritumoural inflammatory infiltrate in basal cell carcinoma (BCC) of the skin consists mainly of T lymphocytes which hardly invade the tumour nests. The absence of intercellular adhesion molecule-1 (ICAM-1) on BCC cells may explain the lack of tumour-infiltrating cells and the lack of an active cell-mediated immune response in this tumour. In this study, the induction of ICAM-1 was investigated in BCC biopsies using recombinant human interferon-gamma (rHuIFN-gamma). The expression of interferon-gamma receptors (IFN-gamma R) in the biopsies was also investigated. The results showed that BCC cells expressed ICAM-1 after incubation with rHuIFN-gamma, but to a lesser degree than normal epidermal cells. The levels of shed ICAM-1 were significantly increased in the culture supernatants of tumour biopsies compared with those from normal skin biopsies, after culturing in the presence of rHuIFN-gamma. The expression of IFN-gamma R was significantly decreased on the tumour cells compared with the overlying epidermis. The decreased expression of IFN-gamma R on the tumour cells and the shedding of ICAM-1 into the peritumoural stroma may be a plausible mechanism by which the tumour cells are protected against an active cell-mediated immune response.

Adult↗
PubMed1999

Interferon alfa receptor expression and growth inhibition by interferon alfa in human liver cancer cell lines.

Type I interferon (IFN) receptor consists of two chains (Hu-IFN-alphaR1 and Hu-IFN-alphaR2), and Hu-IFN-alphaR2 takes a soluble (Hu-IFN-alphaR2a), short (Hu-IFN-alphaR2b), or long (Hu-IFN-alphaR2c) form. We examined the expression of type I IFN receptor, the growth-suppression effect of IFN-alpha, and their relationship in 13 liver cancer cell lines. With reverse-transcription polymerase chain reaction (RT-PCR) analysis, the expressions of Hu-IFN-alphaR1, Hu-IFN-alphaR2a, and Hu-IFN-alphaR2c were confirmed in all cell lines, and that of Hu-IFN-alphaR2b in 12 cell lines. All cell lines expressed mRNAs of a transcriptional activator, interferon regulatory factor (IRF)-1, and its antagonistic repressor (IRF-2). Flow cytometry revealed weak expression of Hu-IFN-alphaR2 on the cell surface in 12 cell lines. The soluble-form protein of Hu-IFN-alphaR2 was detected at varying levels in culture supernatants of all cell lines with enzyme-linked immunosorbent assay (ELISA). Cell proliferation was suppressed in proportion to the dose of human natural IFN-alpha at 96 hours of culture, but it was not clearly related to the expression of Hu-IFN-alphaR2 protein on the cell surface. Investigations on the morphology, DNA, and cell cycle presented four growth suppression patterns as a result of IFN-alpha: 1) induction of apoptosis and blockage of cell cycle at the S phase (9 cell lines); 2) blockage at the S phase (2 cell lines); 3) induction of apoptosis and blockage at the G2/M phase (1 cell line); and 4) blockage at the G1 phase (1 cell line). There was no evidence showing that changes in the expressions of Bcl-2, Bcl-xL, Bak, and Bax lead directly to IFN-alpha-mediated apoptosis. Our findings demonstrated that IFN-alpha would express growth-suppression effects at varying degrees by inducing inhibition of cell-cycle progression with or without apoptosis, regardless of the expression level of Hu-IFN-alphaR2 protein on the cell surface.

Apoptosis↗
PubMed2002

Interferon regulatory factor-1 mediates interferon-gamma-induced apoptosis in ovarian carcinoma cells.

Interferon-gamma (IFN-gamma), as one of interferon family that regulates antiviral, antiproliferative, and immunomodulatory responses, has been implicated for the growth regulation of ovarian cancer cells. However, the molecular mechanisms are not yet fully defined. To analyze detailed mechanisms, the ovarian cancer cell lines (2774, PA-1, OVCAR-3, and SKOV-3) were treated with IFN-gamma. The growth of 2774 was most effectively suppressed than that of other cells in both time-course and dose-dependent experiments. The order of sensitivity in other cells was PA-1 >> OVCAR-3 > SKOV-3 (not responded at all). The DNA fragmentation and DAPI staining assays suggested that the IFN-gamma-mediated cytotoxicity could be triggered by apoptosis. The treatment induced IFN regulatory factor-1 (IRF-1) in two IFN-gamma-sensitive cells (2774, PA-1), whereas IRF-1 was not induced in two IFN-gamma-resistant cells (OVCAR-3, SKOV-3). The levels of p53 and p21WAF1 were not strikingly changed in all four cells. Interestingly, the expression of interleukin-converting enzyme (ICE, or caspase-1) was increased by the treatment in a kinetically consistent manner to the induction of IRF-1. However, CD95 (Fas/APO-1) was not changed. Apoptosis was greatly induced, when IRF-1 was transiently expressed in PA-1 without the treatment of IFN-gamma. However, it was repressed when IRF-1 together with IRF-2, an antagonist of IRF-1, were coexpressed. In addition, the effect of IFN-gamma was reduced in the 2774 and PA-1 cells stably expressing either IRF-1 antisense or IRF-2 sense, as shown by the cytotoxicity and FACS analysis. Furthermore, the IFN-gamma-induced apoptosis was greatly reduced, when inhibitors of ICE were treated into PA-1 cells. Taken together, these results suggest that IRF-1 directly mediates the IFN-gamma-induced apoptosis via the activation of caspase-1 gene expression in IFN-gamma-sensitive ovarian cancer cells.

Apoptosis↗
PubMed1993

Purification of interferon gamma-interferon gamma receptor complexes by preparative electrophoresis on native gels.

Cytokine-receptor complexes are required for certain studies including crystallization, NMR spectra, and investigation of the biological response mechanism to the cytokine. The purity of the ligand-receptor complex is critical for most of these applications. We investigated the possibility of purifying protein-protein complexes by electrophoresis on native gels. Starting with partially purified mouse and highly purified human proteins, we prepared milligram amounts of interferon gamma-interferon gamma receptor complexes by preparative electrophoresis on nondenaturing polyacrylamide gels. In both cases, pure ligand-receptor complexes with the correct stoichiometry of binding were recovered. Electrophoresis on preparative native gels may prove to be of general interest for the preparation of protein-protein complexes to be used in diverse studies.

Amino Acids↗
PubMed1998

The antiviral action of interferon is potentiated by removal of the conserved IRTAM domain of the IFNAR1 chain of the interferon alpha/beta receptor: effects on JAK-STAT activation and receptor down-regulation.

The first cloned chain (IFNAR1) of the human interferon-alpha (IFN alpha) receptor acts as a species-specific transducer for type 1 IFN action when transfected into heterologous mouse cells. Stably transfected mouse L929 cell lines expressing truncation mutants of the intracellular domain of the human IFNAR1 chain were tested for biological responses to human IFN alpha. Deletion of the intracellular domain resulted in a complete loss of sensitivity to the biological activity of human IFN but markedly increased IFNAR1 cell surface expression, demonstrating that the intracellular domain is required for biological function and contains a domain that negatively regulates its cell surface expression. Removal of the conserved membrane distal 16-amino-acid IRTAM (Interferon Receptor Tyrosine Activation Motif) sequence: (1) increased sensitivity to IFN alpha's antiviral activity, (2) increased the rapid IFN alpha-dependent formation of STAT-containing DNA-binding complexes, (3) prolonged tyrosine phosphorylation kinetics of the JAK-STAT pathway, and (4) blocked the IFN-dependent down-regulation of the IFNAR1 chain. These results indicate that the IRTAM negatively regulates signalling events required for the induction of IFN's biological actions via regulating receptor down-regulation.

Amino Acid Sequence↗
PubMed1983

Interferon induction of (2'-5') oligoisoadenylate synthetase in diploid and trisomy 21 human fibroblasts: relation to dosage of the interferon receptor gene (IRFC).

Trisomy 21 human fibroblasts are more sensitive to human interferon-alpha (IFN-alpha) than are diploid controls, consistent with the location of the gene (IFRC) which codes for the IFN-alpha receptor on chromosome 21. When compared in the antiviral assay, the difference in sensitivity is five- to tenfold, much greater than the 50% difference in IFRC gene dosage. An understanding of the mechanism by which this amplification of gene dosage occurs is relevant to the specific pathology of Down's syndrome and as a model system for studying the pathogenic effects of chromosomal aneuploidy. The enzyme (2'-5') oligoisoadenylate synthetase (2-5A synthetase), which is believed to be central to the interferon-induced antiviral response, is induced 50% more in trisomy 21 fibroblasts than in diploid controls. Thus the amplification in response occurs subsequent to the binding of IFN-alpha to its receptor and the triggering of the first set of intracellular events, the latter exemplified by the induction of 2-5A synthetase. Similar results were obtained with IFN-gamma, consistent with other evidence which indicates that a gene coding for a separate IFN-gamma receptor is also located on chromosome 21.

Diploidy↗
PubMed1990

Sublocalization on chromosome 21 of human interferon-alpha receptor gene and the gene for an interferon-gamma response protein.

The cellular responses to alpha and beta interferons (IFN-alpha and -beta) are mediated through the IFN-alpha/beta (type I) receptor, while the response to IFN-gamma is mediated through the IFN-gamma (type II) receptor. The receptors for IFN-alpha/beta and IFN-gamma are encoded by genes on human chromosomes 21 and 6q, respectively. The presence of chromosome 21q confers both ligand binding and responsiveness to human IFN-alpha/beta, whereas chromosome 6q confers binding of Hu-IFN-gamma, but not cellular responsiveness on somatic cell hybrids. Chromosome 6q (i.e., the Hu-IFN-gamma receptor gene) and chromosome 21q are both necessary for the cellular response of somatic cell hybrids (from fibroblasts) to Hu-IFN-gamma. It is conceivable that the factor mediating activity through the IFN-gamma receptor is, in fact, the IFN-alpha receptor, or that the two genes are distinct but part of an "interferon response" region. Here we more precisely localize on human chromosome 21 the genes for the IFN-alpha receptor and for the factor(s) mediating the action of IFN-gamma through the chromosome 6-encoded receptor. Hamster-human somatic cell hybrids containing various fragments of human chromosome 21 were used. The presence of the human IFN-alpha/beta receptor was determined by binding 32P-labeled human IFN-alpha to cells, covalently cross-linking the [32P]IFN-alpha-receptor complex, and analyzing it by SDS-polyacrylamide gel electrophoresis. The presence of the IFN-gamma receptor-related factor mediating cellular responsiveness was determined by HLA induction in hybrid cells containing the IFN-gamma receptor (chromosome 6q), a transfected copy of the human HLA-B7 gene, and various portions of chromosome 21. In all hybrids examined, the two genes cosegregate. Specifically, both genes are localized to the region of chromosome 21 containing the markers D21S58, D21S65, and GART and appear to be proximal to D21S58. The implications for IFN action are discussed.

Animals↗
PubMed1996

Altered synthesis of interferon-gamma and expression of interferon-gamma receptor by peripheral blood mononuclear cells from patients with IgA nephropathy and non-IgA proliferative glomerulonephritis.

Previously we reported disease-specific interaction between interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in patients with IgA nephropathy (IgAN), suggesting the existence of unusual T cell behavior in this disease. In the present study, we investigated characteristic synthesis of interferon-gamma (IFN-gamma) and expression of IFN-gamma receptor (IFN-gamma R) in the peripheral blood mononuclear cells (PBMC) from patients with IgAN and other chronic proliferative glomerulonephritis (PGN). Heparinized peripheral blood samples were obtained from 38 patients with chronic mesangial proliferative glomerulonephritis (CGN; including 24 with IgA nephropathy) and 20 healthy controls. PBMC were isolated by gradient centrifugation and fragments were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum (FCS) for 72 hr. IFN-gamma concentrations in supernatants were evaluated by the enzyme-linked immunosorbent assay (ELISA). Other parts of PBMC pellets were reacted with anti-human IFN-gamma R monoclonal antibody and FITC-labeled anti-mouse second antibody for analysis of IFN-gamma R expression on these cells by FACScan. The remaining PBMC were fractionated into CD4+ T cells, CD8+ T cells, B cells, NK, cells and macrophages using the MACS cell sorting system. The isolated cells were evaluated for IFN-gamma or IFN-gamma R mRNA expression by the semiquantitative RT-PCR method. In vitro IFN-gamma synthesis was enhanced in patients with CGN, and NK cells were revealed to be responsible for such enhancement. On the other hand, the expression of IFN-gamma R on macrophages was suppressed in CGN patients. These results suggest that impairment of regulation of the IFN-gamma system might be involved in the development of CGN.

Antigens, CD↗
PubMed1995

Variable region cDNA sequences and characterization of murine anti-human interferon gamma receptor monoclonal antibodies that inhibit receptor binding by interferon gamma.

Murine monoclonal antibodies (mAbs) are described that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R) and inhibit the binding to it of interferon gamma. The inhibitory activities (IC50s) of these mAbs, quantified by radioimmunoassay using native receptor on human Raji cells, lie in the range 0.5-24 nM, whereas their relative affinities for the immobilised recombinant extracellular receptor, determined using surface plasmon resonance technology, are in the range 0.6-40.9 nM. Nine mAbs derived from one immunization, were shown by variable region cDNA sequencing to be clonally related, with mAb A6 from this group showing the highest affinity for the receptor. Another two mAbs, gamma R38 and gamma R99, derived from a separate immunization, are clonally unrelated to each other and to those in the A6 family. From the V-region sequences, the L-chains of mAbs A6, gamma R38 and gamma R99 were shown to belong to the V kappa 34C, V kappa 34C and V kappa 1 families, whereas the H-chains belong to the 3069, J606 and J558 families, respectively. The mAbs A6 and gamma R38 recognize overlapping epitopes on the N-terminal Ig-like domain of the IFN gamma R, whereas the gamma R99 epitope is located largely in the membrane proximal Ig-like domain. Sequence comparisons with Ig structures solved by X-ray diffraction allowed deductions concerning likely CDR canonical conformations. These studies provide essential information for crystallographic and mutagenesis experiments aimed at understanding the molecular basis of the interactions of these mAbs with the extracellular IFN gamma R.

Amino Acid Sequence↗