Interferon gamma: a master key in the immune system.
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Animals↗
Biomedical subjects
A Billiau
Publications and source records attributed to A Billiau.
Interleukin 1 induces human marrow stromal cells in long-term culture to produce granulocyte colony-stimulating factor and macrophage colony-stimulating factor.
Pure interleukin 1 (IL 1) was found to stimulate established human bone marrow stromal layers in long-term culture to produce colony-stimulating activity (CSA). Maximal concentrations in the culture medium were reached 24 hours after a single IL 1 pulse. The effect could be neutralized by a specific rabbit anti-IL 1 antiserum. Stromal layers, once stimulated by IL 1, continued to release CSA into the culture medium in the absence of exogenous IL 1. A second IL 1 pulse induced CSA release in an identical manner, as did the primary stimulation, indicating that the CSA released was actively produced. Using specific immunologic assays, both granulocyte colony-stimulating factor (G-CSF) and macrophage CSF (M-CSF) could be identified in the culture supernatants, and production of both factors was inducible by IL 1. Shortly after initiation of the long-term marrow cultures "spontaneous" G-CSF and M-CSF release occurred. The release of G-CSF diminished following addition of the anti-IL 1 antiserum, indicating that endogenous production of IL 1 by stromal cells had contributed to this effect. These results further support the role of IL 1 as an important modulator of CSF production by cells of the hematopoietic microenvironment.
Bone Marrow↗
Human fibroblasts produce granulocyte-CSF, macrophage-CSF, and granulocyte-macrophage-CSF following stimulation by interleukin-1 and poly(rI).poly(rC).
Electrophoretically pure human interleukin-1 (IL-1) beta was found to stimulate human fibroblasts in a monolayer culture to elaborate colony-stimulating activity (CSA). Supernatant fluids from cultures induced with increasing concentrations of IL-1 were found to stimulate colony formation of myeloid (CFU-GM), erythroid (BFU-E), and multipotent (CFU-GEMM) progenitor cells in a dose-dependent fashion. The effect on mixed colony formation, however, was less than on CFU-GM and BFU-E growth. Similar to IL-1, the synthetical double-stranded RNA poly(rI).poly(rC) also stimulated release of CSA by fibroblasts. The kinetics of IL-1- and poly(rI).poly(rC)-induced CSA release were found to be different, in that poly(rI).poly(rC)-induced CSA production occurred more slowly. Anti-IL-1 antiserum was able to completely neutralize the IL-1-induced CSA release, but had no effect on poly(rI).poly(rC)-induced CSF production, suggesting that the latter effect was mediated by other mechanisms than IL-1 in supernatant. By the use of specific immunologic assays, G-CSF, M-CSF, and GM-CSF could be identified in media conditioned by fibroblasts treated with IL-1 or poly(rI).poly(rC). Poly(rI).poly(rC) appeared to be a better inducer for M-CSF than IL-1.
Colony-Forming Units Assay↗
Proliferation of myeloid progenitor cells in human long-term bone marrow cultures is stimulated by interleukin-1 beta.
To study the effect of interleukin-1 (IL-1) beta on the proliferation of hematopoietic progenitor cells (HPC) in long-term bone marrow cultures (LTBMC), stromal cell layers were established from normal human bone marrow. Autologous cryopreserved mononuclear phagocyte- and T-lymphocyte-depleted bone marrow cells were reinoculated on the stromal layers in fresh culture medium, with or without the addition of human IL-1 beta (30 U/mL). Once a week, half of the culture supernatant was replaced with fresh culture medium with or without IL-1, and all nonadherent cells were returned to the flasks. At weekly intervals during a period of 5 weeks, one culture was sacrificed to determine the total number of cells and hematopoietic progenitor cells, present in the adherent and the nonadherent cell fractions. In IL-1-stimulated cultures, the number of cells recovered during a period of 5 weeks exceeded the number of cells in unstimulated control cultures by 1.5 times. This difference was attributed to a twofold increase in the number of adherent cells. The number of HPC recovered from IL-1-stimulated cultures was not different from that recovered from controls. The levels of colony-stimulating activity (CSA) in supernatants from IL-1-stimulated cultures were significantly higher than those in supernatants from control cultures. These results indicate that IL-1 enhances the recovery of cells in LTBMC by stimulating the proliferation of HPC with the concurrent release of CSA from stromal cells, without diminishing the number of HPC.
Bone Marrow Cells↗
Cellular immunity changes caused by LDH virus: analogy with observations of neuroblastoma-bearing mice.
Evidence is presented that LDH virus infection of mice results in drastic changes in several immune activities. Serum IFN titer and splenic NK activity are increased during the acute phase of infection. NK stimulation is mediated by IFN-alpha,beta since injection of an antibody against murine IFN-alpha,beta is able to abolish the effect. IL-2 production is inhibited throughout the study period following injection of LDH virus (14 days), although a partial recovery is observed during the second week. Similarly, IL-2 receptor expression and MLC responsiveness are suppressed. This suppression lasts for 2 and 7 days respectively after injection. Addition of recombinant IL-2, but not of indomethacin, to the MLC cultures restores the proliferation rate. Not only proliferation but also cytotoxic cell generation in MLC is diminished during the first week after LDH virus injection. Again, this response is normalized at day 14. Additional observations indicate that LDH virus is present in murine neuroblastoma. This explains some of the previously described effects of this tumor on the cellular immune system of the host.
Animals↗
Purification and characterization of human fibroblast-derived hybridoma growth factor identical to T-cell-derived B-cell stimulatory factor-2 (interleukin-6).
Human fibroblast cultures, when stimulated with interleukin-1 (IL-1) produce a growth factor for B-cell hybridoma and plasmocytoma cell lines. The availability of both a fast-growing and high-producer cell line (MG-63 osteosarcoma cells) and of a highly sensitive and specific assay system for this hybridoma growth factor (HGF) allowed us to obtain analytically pure preparations. Crude HGF from MG-63 cells was processed through a five-step concentration and purification schedule. Sequential adsorption to controlled pore glass (CPG) beads, antibody affinity chromatography and gel filtration resulted in a 10,000-fold purification to a specific activity of 10(9) units/mg HGF. Electrophoretically pure HGF was obtained after additional purification by cation-exchange chromatography and reversed-phase HPLC. The purification procedure revealed two distinct biologically active HGF components. The amino-terminal sequence of one of the two components was determined and found to correspond to that already predicted from cDNA clones of a protein alternatively called 26-kDa protein, interferon-beta 2 (IFN-beta 2) or B-cell stimulating factor-2 (BSF-2). The first two designations (26-kDa protein and IFN-beta 2) refer to a postulated fibroblast secretory protein with so far no unambiguously defined function; the latter designation (BSF-2) refers to a T-cell product possessing differentiation stimulatory effect on B-cell lines. The reported results firmly establish that the protein is secreted by fibroblasts and reveal that it possesses B-cell growth stimulatory activity. The new designation interleukin-6 (IL-6) is proposed to resolve prescribing nomenclature confusion.
Cell Cycle↗
Identification of an interleukin HP1-like plasmacytoma growth factor produced by L cells in response to viral infection.
We have recently purified and partially sequenced a new T cell-derived lymphokine with growth factor activity for B cell hybridomas and plasmacytomas, which we named interleukin HP1 (HP1). Here we show that, in response to viral infection or after treatment with poly(rI).poly(rC), L cells produce a factor that is capable of supporting the in vitro growth and survival of HP1-dependent cell lines. Serologic and structural evidence is presented in favor of the identity between the fibroblast factor and HP1, demonstrating that non-T cells can make HP1-related molecules.
Animals↗
The role of interferon-beta 1 and the 26-kDa protein (interferon-beta 2) as mediators of the antiviral effect of interleukin 1 and tumor necrosis factor.
This study confirms our earlier finding that human interleukin (IL)-1 beta exerts an antiviral effect on diploid fibroblasts and on MG-63 osteosarcoma cells. It also extends the observation in that a similar effect was noted on aged but not freshly trypsinized HEp-2 cells, and that not only IL-1 beta but also IL-1 alpha and tumor necrosis factor (TNF)-alpha exerted similar antiviral effects on cells. The antiviral effects of these cytokines were neutralized by addition to the assay system of an antibody that was specific for interferon (IFN)-beta 1, indicating that IFN-beta 1 or a structurally or functionally related substance is involved in the antiviral activity observed. Both IL-1 and TNF were able to induce production of the 26-kDa protein, also known as IFN-beta 2, hybridoma/plasmacytoma growth factor (HPGF) or B-cell stimulatory factor-2 (BSF-2) and previously proposed as an alternative to IFN-beta 1 for mediating the antiviral effect of TNF. However, no good correlation was found between the antiviral effects of TNF and its potential to induce production of the 26-kDa protein. Furthermore, the anti-IFN-beta 1 serum which neutralized the antiviral activity of IL-1 and TNF did not cross-react with the 26-kDa protein. Conversely, the antiviral effect of IL-1 and TNF was only weakly neutralized by an antibody that did react with the 26-kDa protein and showed low cross-reactivity with IFN-beta 1. These observations, together with the low specific activity of the 26-kDa protein as an antiviral agent (less than 10(5) U/mg protein) provide strong arguments against this protein and in favor of IFN-beta 1 (or still another IFN-beta 1-related molecule) as the ultimate mediator of the antiviral effect of IL-1 and TNF.
Antiviral Agents↗
Regulation by interferons of the local inflammatory response to bacterial lipopolysaccharide.
Footpad swelling developing in mice after local injection of LPS (S. marcescens) was found to consist of two phases with peaks occurring on days 2 to 3 and 6 to 8, respectively. Histopathologically, the reaction was characterized by edema and mononuclear cell infiltration; the second peak was associated with intravascular thrombosis as is typically described for the Shwartzman reaction to LPS. Recombinant DNA-derived IFN-gamma, administered by i.p. injection, had a suppressive effect on the development of the reaction. The same effect was seen with recombinant DNA-derived IFN-alpha 1 and with the natural mixture of IFN-alpha and -beta. In mice pretreated with neutralizing monoclonal antibodies to IFN-gamma, the footpad response to LPS was modified in that a delayed monophasic rather than a biphasic response occurred. These data indicate that LPS induces local production of IFN-gamma, which acts as a trigger or positive regulator of the reaction. The effect of a single pretreatment with neutralizing anti-IFN-gamma antibody was found to last for as long as 6 wk. Experiments in which antibody administration was delayed till after LPS challenge indicated that endogenous IFN-gamma was also involved in the late phases of the inflammation. The results show that regulation of inflammation by interferons is complex in that local IFN-gamma acts as a positive factor, whereas systemic IFN-alpha 1 and -gamma, probably through indirect mechanisms, downregulate inflammation.
Animals↗
Structure and expression of mos sequences in spontaneous and Moloney murine sarcoma virus-induced yolk sac carcinomas in rats.
Facilitation of yolk-sac carcinoma (YSCa) development in fetectomized rats by the Moloney murine sarcoma virus/murine leukemia virus (Mo-MSV/MLV) complex was found to be closely associated with the presence of Mo-MSV sequences in the genomes of the YSCa cells. The virus-induced YSCas consisted of cells of mono- or oligoclonal origin which always contained in their genomes at least I randomly integrated Mo-MSV provirus. In YSCas which developed in the absence of virus, no rearrangement or amplification of c-mos could be detected. In addition, blot hybridization analysis of cellular RNA failed to detect mos-related RNA in cell lines derived from Mo-MSV-induced as well as from non-virally induced YSCas. The methylation level of c-mos DNA was low in all YSCa cell lines. In contrast, v-mos DNA in cell lines derived from Mo-MSV-induced YSCas was heavily methylated.
Animals↗
Identification of the human 26-kD protein, interferon beta 2 (IFN-beta 2), as a B cell hybridoma/plasmacytoma growth factor induced by interleukin 1 and tumor necrosis factor.
A factor that promotes the growth of certain B cell hybridomas and of plasmacytomas is shown to be produced by normal human fibroblasts and by a line of human osteosarcoma cells (MG-63) after treatment with IL-1 or TNF. The hybridoma-plasmacytoma growth factor (HPGF) is identified with a 26 kD protein whose mRNA was previously shown to be induced in the same cells by the same inducers. First, poly(A)-rich RNA extracted from IL-1-treated cells could be enriched in HPGF-mRNA content by hybridization to 26 kD cDNA. Second, MG-63-derived HPGF purified to electrophoretic homogeneity was subjected to amino acid sequence analysis, whereby the NH2-terminal sequence was found to match the nucleotide sequence of a 26 kD cDNA clone.
Amino Acid Sequence↗
Heterogeneity of Chinese hamster ovary cell-produced recombinant murine interferon-gamma.
Chinese hamster ovary cells transformed with a hybrid expression plasmid containing both the murine interferon-gamma (MuIFN-gamma) and the murine dihydrofolate reductase-coding sequences were subjected to selection in stepwise increasing concentrations of methotrexate. By this procedure the production rate of MuIFN-gamma was increased from an initial level of approximately 20,000 to approximately 500,000 antiviral units per milliliter of culture supernatant. [35S]Methionine-labeled proteins secreted by these cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without prior immunoprecipitation with polyclonal or monoclonal antibodies against splenocyte-derived MuIFN-gamma. Besides two major components of Mr 19,000 and 38,000, a multiplicity of minor components were immunoprecipitated. Cells treated with tunicamycin, an inhibitor of N-glycosylation, secrete two major components of Mr 14,000 and 27,000 and only two minor components of Mr 12,000 and 13,000. When the proteins were labeled with [35S]cysteine, a residue that is only present at the carboxyl terminus of the mature MuIFN-gamma, no minor components could be detected in the growth medium of tunicamycin-treated cells. The presented results indicate that the heterogeneity of the recombinant Chinese hamster ovary-produced MuIFN-gamma is due to at least three cumulative modifications of the Mr 14,000 MuIFN-gamma peptide: carboxyl-terminal proteolytic processing (the Mr 13,000 and 12,000 components), variations in N-glycosylation (components ranging in size from Mr 12,000 to 26,500), and dimerization (components ranging from Mr 27,000 to 50,000).
Animals↗
Interleukin 1 and poly(rI).poly(rC) induce production of a hybridoma growth factor by human fibroblasts.
Cultures of normal diploid fibroblasts and of a human osteosarcoma cell line (MG-63) are shown to be able to produce a factor which promotes the growth of B cell hybridomas (hybridoma growth factor, HGF). The induction is stimulated by treatment of the cells with interleukin 1 (IL 1) (alpha or beta) or polyriboinosinic-polyribocytidylic acid [poly(rI).poly(rC)]. Combined treatment with cycloheximide and actinomycin D also stimulates production and enhances production induced by IL 1 or poly(rI).poly(rC). Extremely small doses of IL 1 (0.1 units/ml) are active as inducer of HGF. Also, under optimal conditions the yield of HGF can attain as much as 10(4) units/ml. Tumor necrosis factor (TNF-alpha), which otherwise shares various properties with IL 1, is a weak inducer of HGF. Although there is a superficial resemblance between induction of HGF and that of interferon-beta, the two activities are serologically distinct and conditions for their induction are quite different. In fact, conditions for induction of HGF are indistinguishable from those described for the induction of the mRNA of the so-called 26-kDa protein (also known as interferon-beta 2). Finally, the HGF derived from IL 1- or poly(rI).poly(rC)-treated fibroblasts is serologically not distinguishable from that produced by mitogen-stimulated peripheral blood leukocytes.
Animals↗
Anti-interferon-gamma antibody protects mice against the generalized Shwartzman reaction.
A generalized Shwartzman reaction was found to occur in non-SPF-NMRI mice given a local injection of S. marcescens lipopolysaccharide (LPS) in the footpad, followed, with an interval of 24 h, by an i.v. injection. The reaction occurred several hours after the second injection. It was characterized by disseminated subcutaneous bleedings at the mouth, anus, conjuctiva, nose tip and tail end. Most mice died within 24-48 h. The size of the LPS dose given in the footpad (5 micrograms) was found to be critical in that the reaction failed to occur with higher as well as lower doses. The reaction was found to be completely absent in mice that had received one or more systemic injections of monoclonal antibodies with neutralizing potential for murine interferon-gamma (IFN-gamma). Mice treated with control preparations, including a monoclonal antibody with binding but no neutralizing activity for murine IFN-gamma, remained sensitive to induction of the reaction. These observations assign a crucial role to endogenous IFN-gamma in the elicitation of the generalized Shwartzman reaction and open perspectives for the prevention or therapy of clinical conditions related to the Shwartzman reaction by the use of antibodies to IFN-gamma.
Animals↗
Human elongation factor 1 alpha: a polymorphic and conserved multigene family with multiple chromosomal localizations.
One of the genes activated in human melanoma cells by the tumor-promoting phorbol ester is that of the elongation factor 1 alpha. A cDNA clone containing the complete 3'-end untranslated region and the nucleotide sequences coding for 227 carboxyterminal amino acids was isolated. Computer-assisted comparison with known sequences of elongation factors from other species revealed homologies up to 73% and 63% on amino acid and nucleotide sequences, respectively. Northern blot analysis of mRNA from unstimulated and phorbol ester-treated cells showed a 3- to 5-fold increase in cytoplasmic elongation factor 1 alpha mRNA after phorbol ester induction. When compared with the phorbol ester-inducible single-copy gene transcripts coding for the tissue-type plasminogen activator, the cellular mRNA content of elongation factor 1 alpha is 30 times higher. By Southern blot analysis experiments on human genomic DNA, a multi-gene family was found showing polymorphisms in restriction endonuclease fragment lengths (RFLP). Several polymorphisms were studied more extensively in the population on more than 100 DNA samples from normal individuals and in three-generation families. In situ hybridization of the cDNA probe to normal human metaphase chromosomes showed multiple chromosomal localizations of the elongation factor gene(s), with peak hybridization on the chromosomes 1, 2, 4, 5, 6, 7, and 15. The estimate of the gene copy number in humans is more than ten copies per (haploid) genome.
Artemia↗
Redefining interferon: the interferon-like antiviral effects of certain cytokines (interleukin-1, interferon-beta 2, interferon-gamma) may be indirect or side effects.
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Animals↗
Interferons and inflammation.
The local inflammatory response to exogenous or endogenous agents is regulated by various humoral factors. Amongst these, the cytokines represent a prominent group to which belong not only cytotoxins, chemotactic factors, and cellular growth factors, but also the interferons. Evidence is reviewed indicating that, at the local level, interferon-gamma (IFN-gamma) constitutes a positive factor triggering or promoting the inflammatory response. Studies employing monoclonal antibodies against IFN-gamma indicate that inflammatory agents, such as bacterial endotoxin, induce local production of IFN-gamma, which acts as an up-regulator of the primary phase of the inflammatory response. Antibodies against IFN-alpha/beta, on the contrary, have so far not been found to affect inflammation. Systemic interferons, on the other hand, seem to fulfil a down-regulating role, as evidenced by the observation that exogenously administered IFN-alpha, -beta, and -gamma inhibit local inflammation.
Animals↗
The inhibition of endotoxin-induced local inflammation by LDH virus or LDH virus-infected tumors is mediated by interferon.
The footpad swelling reaction induced by local injection of S. marcescens lipopolysaccharide was found to be inhibited in mice given a transplantable tumor (TA3) or cell-free ascitic fluid from tumor-bearing mice. The tumor was shown to contain LDH virus, which is known to cause inapparent persistent infections in mice. Monoclonal antibodies directed against protein VP3 of the LDH virus could partially abrogate the anti-inflammatory effect of the TA3-ascitic fluid, and, conversely, the anti-inflammatory effect could be obtained by LDH virus isolated from the tumor and reproduced by serial passage of cell-free fluids. Inhibition of the footpad reaction was seen in the acute but not in the chronic phase of LDH virus infection, suggesting that the anti-inflammatory effect might be due to endogenous interferon (IFN) which, similarly, was only detectable in the acute phase. Newcastle disease virus, another potent interferon inducer, had a similar inhibitory effect on the footpad reactivity. Moreover, the inhibitory effect of LDH virus infection could partially be abrogated by administration of a polyclonal antibody directed against murine IFN-alpha,beta. Finally, passively administered natural murine IFN-alpha,beta or recombinant murine IFN-alpha 1 (but not recombinant murine IFN-beta) was found to cause inhibition of the footpad reaction. Since Gram-negative bacteria and their lipopolysaccharides have the ability to induce a systemic interferon response, our findings suggest that this interferon may play a modulatory role in local inflammation caused by these bacteria. Our findings also open a new perspective for interferon therapy of certain inflammatory reactions to bacterial infections.
Animals↗