Interferon-gamma: mechanism of action and therapeutic potential.
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Animals↗
Biomedical subjects
A Billiau
Publications and source records attributed to A Billiau.
Human hepatoma cells produce an 85 kDa gelatinase regulated by phorbol 12-myristate 13-acetate.
Several human cell lines were studied for the production of gelatinases. Diploid fibroblasts, the melanoma cell line Bowes, the MG-63 osteosarcoma cell line and the human hepatoma cell line Malavu all constitutively produced a 67 kDa gelatinase. Gelatinolytic enzymes were quantified by a sensitive zymographic substrate conversion assay. Upon induction with phorbol 12-myristate 13-acetate (PMA), the human hepatoma cell line secreted considerable amounts of an 85 kDa gelatinase activity. The induction process was time- and dose-dependent. It represented a true increase in production per individual cell and was associated by a marked change of the cell morphology. The effect of various proteinase inhibitors and the maximal activity of the enzyme near neutral pH demonstrate that it is a neutral metalloproteinase. Characterization studies showed the 85 kDa gelatinase to be transformed to lower molecular weight, active forms by treatment with p-aminophenylmercuric acetate (APMA) or trypsin.
Carcinoma, Hepatocellular↗
Sequence of the porcine interferon-gamma (IFN-gamma) gene.
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Amino Acid Sequence↗
Interferon gamma, a mediator of lethal lipopolysaccharide-induced Shwartzman-like shock reactions in mice.
The involvement of cytokines in the pathogenesis of a generalized, Shwartzman-like lethal inflammatory response to bacterial lipopolysaccharides (LPS) was studied by testing the ability of cytokines or neutralizing anticytokine antibodies to modify the course of the syndrome. The reaction was elicitable in non-SPF NMRI mice by two consecutive injections of S. marcescens LPS: a first injection in the footpad, followed after 24 h by an intravenous dose; the size and route of the preparatory LPS dose were found to be critical. Treatment with mAbs against IFN-gamma was found to completely prevent the reaction. Treatment with IFN-gamma on the other hand, rendered the mice more sensitive to elicitation of the reaction. In contrast, systemic administration of IFN-alpha/beta exerted a desensitizing effect. The role of endogenous cytokines in the pathogenesis of this generalized Shwartzman reaction was also documented by a study of the cytokine levels in the serum of the mice. In comparisons between mice given lethal and nonlethal induction schedules, a good correlation was found between mortality rates and height of IFN or TNF levels, but no correlation was seen with IL-6 levels. Also, in mice that were protected by anti-IFN-gamma antibody, serum IFN and TNF were undetectable, whereas IL-6 levels were as high as in unprotected mice. These data provide evidence that among the cytokines that govern the inflammatory response to LPS, endogenous IFN-gamma occupies a key position. These findings therefore also open perspectives for clinical application of IFN-gamma antagonists.
Animals↗
Identification of the monocyte chemotactic protein from human osteosarcoma cells and monocytes: detection of a novel N-terminally processed form.
The chemotactic activity for monocytes in culture supernatants from double-stranded RNA-stimulated human MG-63 osteosarcoma cells and from LPS-stimulated human monocytes was purified to homogeneity and characterized by amino acid sequence analysis. The chemotactic protein derived from the fibroblastoid osteosarcoma cells had a blocked N-terminus but sequencing of tryptic fragments showed that it was identical with a recently identified monocyte chemoattractant designated MCP-1 or MCAF isolated from glioma or myelomonocytic cells, respectively. Preparations of monocyte -derived chemotactic activity appeared to contain not only the blocked protein, but also a novel N-terminally processed form of this molecule, lacking 5 amino acid residues.
Amino Acid Sequence↗
Interleukin 6 production in the central nervous system during experimental autoimmune encephalomyelitis.
Interleukin 6 (IL6) is one of the major inflammation-associated cytokines. Elevated serum or tissue levels of IL6 have been reported to occur in several human diseases, including infections of the central nervous system (CNS), but not in non-infectious CNS inflammation, e.g. multiple sclerosis. While studying experimental autoimmune encephalomyelitis (EAE) as an animal model for autoimmune inflammation of the CNS, we found increased IL6 levels in the CNS of mice suffering from a lethal form of the disease. IL6 levels in the spleens and sera were not significantly increased. These findings are indicative of local production of IL6 in the CNS during EAE, and represent the first demonstration of IL6 production in non-infectious CNS inflammatory disease.
Animals↗
The neutrophil-activating proteins interleukin 8 and beta-thromboglobulin: in vitro and in vivo comparison of NH2-terminally processed forms.
Isolation of the human neutrophil activating protein (NAP) interleukin 8 (IL8) from leukocytes has revealed that it is structurally related to beta-thromboglobulin (beta TG) from platelets. Both these proteins occur as natural mixtures of multiple forms, differing from each other by unequal truncation at the NH2 terminus. In this study we have compared IL8 and beta TG forms for in vitro and in vivo neutrophil activation. In contrast to IL8, none of the beta TG forms were found to exert granulocyte chemotactic activity in vitro, as measured in the agarose assay. However, fractions rich in the most extensively processed forms of beta TG (e.g. NAP-2) as well as pure NAP-2 did induce lactoferrin release from granulocytes, whereas fractions containing only the longer forms (e.g. connective tissue-activating peptide III) were inactive. In order to observe this in vitro effect, about 10-fold less IL8 (10 nM) than NAP-2 was required. In the presence of a vasodilator substance low doses (2-20 pmol) of IL8 and the shorter forms of beta TG caused granulocyte accumulation and plasma leakage in rabbit skin whereas the longer forms of beta TG again failed to do so. Finally, granulocytosis induction following i.v. injection was found to occur with NAP-2. At the maximal dose tested (250 pmol), this in vivo effect of NAP-2 was less pronounced than that of IL8. In the case of IL8, NH2-terminal processing did not seem to affect granulocyte stimulatory activity. It should be noted, however, that the extent of processing of IL8 is less than that occurring with beta TG. It can be concluded that the platelet factor beta TG, structurally related to the monokine IL8, can also play a role in neutrophil activation during inflammatory reactions.
Amino Acid Sequence↗
Enhanced release of plasminogen activator inhibitor(s) but not of plasminogen activators by cultured rat glial cells treated with interleukin-1.
Astroglial cells are known to proliferate during development of the nervous system, as well as during post-traumatic gliosis. We have previously shown that the proliferation of cultured astrocytes can be stimulated by the urokinase-type (uPA) of plasminogen activator (PA) and that astrocytes are able to release such uPA upon stimulation with basic fibroblast growth factor, which is known to act as a mitogen for these cells. Here we report studies on the effects of human interleukin-1 (IL-1) on the release of PA activity by cultured newborn rat astroglial cells. Whereas there is controversy in the literature as to whether IL-1 stimulates multiplication of astroglial cells, we failed to observe such an effect in our system. We did observe, however, a dose-dependent decrease in PA activity in the supernatant of the IL-1 treated cultures. Further analysis revealed that this apparent decrease in PA release was in fact due to an increased release of plasminogen activator inhibitor (PAI). A similar IL-1 induced increase in PAI release was also found to occur in cultures of transformed astrocytes (human glioma LN18) and in cultured Schwann cells, but not in cultures of neurons or neuronal tumour cells. Since protease inhibitors are known to possess neuritogenic properties, our results suggest that IL-1, by its capacity to induce PAI, may promote neuritogenesis.
Adrenal Gland Neoplasms↗
Cooperation between bovine leukaemia virus transactivator protein and Ha-ras oncogene product in cellular transformation.
Human T-lymphotropic viruses (HTLV-I and -II) and bovine leukaemia virus (BLV) express transactivator proteins able to increase long terminal repeat (LTR) directed viral expression. These transacting factors are though to be involved in the induction of leukaemia by these viruses. Transfection of BLV transactivator p34tax together with Ha-ras immortalizes and transforms rat embryo fibroblasts, in vitro. The transformed cell induce tumours in nude mice. These data emphasize the causal role exerted by p34tax in in vivo tumorigenesis.
Animals↗
Chicken macrophage activation by interferon: do birds lack the molecular homologue of mammalian interferon-gamma?
In mammalian species, interferon-gamma (IFN-gamma) is a lymphokine with a wide range of biological effects, of which the antiviral and macrophage-activating capacities are those best characterized. In birds, no equivalent with a similar range of actions has as yet been isolated. Chicken splenocytes were stimulated by mitogens in conditions that were similar to those used for the induction of mammalian IFN-gamma. Culture fluids were assayed for antiviral and macrophage-activating capacities. As much as 1000 units/ml of an interferon-like antiviral activity was found in the culture fluid of Staphylococcus aureus lysate-induced spleen cells. Seroneutralization assays with a polyclonal antiserum against purified interferon and physicochemical studies revealed that the antiviral activity is identical to or closely related to type I interferon (interferon-alpha/beta). The presence of macrophage activating factors (MAF) in the splenocyte medium was demonstrated by measuring increased production of H2O2 by chicken peritoneal macrophage cultures and a chicken macrophage cell line (HD11). The heat stability of this MAF activity was similar to that of the antiviral factor, and was completely neutralized by the anti-IFN-alpha/beta antiserum. These results show that when the classical procedure used for the production of mammalian IFN-gamma is applied to chicken splenocytes, it does not yield an equivalent for IFN-gamma/MAF. This suggests that the classification of interferons into types (alpha, beta and gamma), while generally applicable in mammals, may not be applicable in birds.
Animals↗
Bacterial lipopolysaccharide potentiates gamma interferon-induced cytotoxicity for normal mouse and rat fibroblasts.
Gamma interferon (IFN-gamma) can be cytolytic for normal mouse fibroblasts isolated from embryonic or adult tissue (R. Dijkmas, B. Decock, H. Heremans, J. Van Damme, and A. Billiau, Lymphokine Res. 8:25-34, 1989). This cytotoxicity has been shown to be transcription and translation dependent, thereby suggesting involvement of a suicidelike mechanism. The dose of IFN-gamma required for cytotoxicity is higher than that needed for antiviral and macrophage activation but can be reduced 10- to 100-fold by cotreatment of the cells with tumor necrosis factor or interleukin-1 (IL-1) or both, two cytokines that by themselves are not toxic for these cells. Here, we show that bacterial lipopolysaccharide (LPS), which alone has no effect on the viability of mouse fibroblasts, stimulates cell suicide induced by IFN-gamma. The effect was observed in cultures that were virtually free of nonfibroblastoid cells. LPS showed its toxicity-enhancing effect only if applied on the cells simultaneously with or immediately after treatment with IFN-gamma. Pretreatment of the cells with LPS was ineffective. Inclusion of antibodies directed against tumor necrosis factor alpha or IL-1 alpha in the culture medium did not block the cytotoxic effect of combined IFN-gamma plus LPS treatment. The time courses of cell toxicity appearance in fibroblasts treated with combined IFN-gamma plus LPS or IFN-gamma plus IL-1 were similar. In addition to LPS, heat-killed gram-negative (Escherichia coli) but also gram-positive (Staphylococcus aureus, Listeria monocytogenes) bacteria were found to enhance IFN-gamma-induced cell death. These findings suggest that IFN-gamma formed in vivo during infectious processes directly aggravates tissue destruction.
Animals↗
Interferon-gamma inhibits stimulated adrenocorticotropin, prolactin, and growth hormone secretion in normal rat anterior pituitary cell cultures.
Preincubation of rat anterior pituitary (AP) cells with homologous interferon-gamma (IFN-gamma) caused a dose-dependent inhibition of ACTH secretion stimulated by CRF. The effect was seen in both monolayer and aggregate AP cell cultures and was not due to cytotoxicity. In monolayer cultures IFN-gamma also inhibited PRL and GH release stimulated by various hypothalamic releasing factors. IFN-gamma did not affect the time kinetics of the ACTH response to CRF. The dose needed for half-maximal inhibition amounted to approximately 1 (antiviral) U/ml. The effect of IFN-gamma was abrogated by an IFN-gamma-neutralizing monoclonal antibody. Furthermore, ACTH secretion by the AP cells was not affected by the anti-IFN-gamma antibody added alone, indicating that in the culture system no endogenous IFN-gamma is operational in regulating the ACTH response studied. Of the other cytokines tested [interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interferon-alpha/beta (IFN-alpha/beta)] only TNF-alpha and IL-6 were found to inhibit CRF-stimulated ACTH release, although this inhibition was less pronounced than that caused by IFN-gamma. Lipopolysaccharide, even at high doses, did not significantly inhibit the ACTH response to CRF. These results identify IFN-gamma as one of the inflammatory cytokines that, like IL-1, TNF-alpha, and IL-6, have the potential to regulate pituitary function.
Adrenocorticotropic Hormone↗
Molecular cloning of the baboon interferon-gamma cDNA.
Interferon-gamma (IFN-gamma) is a cytokine produced by T lymphocytes and Natural Killer cells which has a key function in resistance against infections. Baboon (Papio anubis) IFN-gamma was produced by stimulation of baboon splenocytes with a lysate of Staphylococcus aureus. This interferon was active on human cells and could be seroneutralized with a polyclonal antiserum against human IFN-gamma, but not with antisera against human interferon-alpha and interferon-beta. Poly(A)(+)-RNA was isolated from baboon splenocytes and fractionated according to its sedimentation coefficient by sucrose density centrifugation. BaIFN-gamma mRNA was present in the 15 S fraction as was shown by hybridization with a human IFN-gamma cDNA probe. A cDNA library was constructed and a clone containing the complete BaIFN-gamma cDNA was isolated. The cDNA codes for a polypeptide of 165 amino acids of which the 23 N-terminal may serve as signal peptide. BaIFN-gamma differs at 11 residues from human IFN-gamma. Southern analysis of chromosomal DNA confirmed some of the nucleotide sequence differences between baboon and human IFN-gamma. The baboon IFN-gamma cDNA was placed under control of a trc promoter and brought to expression in Escherichia coli cells. Recombinant baboon IFN-gamma could be seroneutralized with certain monoclonal anti-human IFN-gamma antibodies. The presented work leads to the availability of recombinant baboon IFN-gamma for animal experiments but also yields new insight in the structure-function relationship of IFN-gamma.
Amino Acid Sequence↗
Clinical potential of monoclonal antibodies against gamma interferon.
Interferon-gamma (IFN-gamma) is a cytokine discovered by its ability to inhibit virus replication in cells. Although it exerts many different actions on different cells, the most prominent action is its macrophage activating effect. Macrophages, in turn, fulfill various regulatory and effector functions in inflammation. It can therefore be expected that interference with the action of IFN-gamma will affect inflammatory reactions. The studies reported here show that administration of neutralizing monoclonal antibodies directed against murine IFN-gamma modifies inflammatory reactions in various experimental models, in particular local LPS-induced inflammation in a footpad swelling model, LPS-induced lethal shock in a generalized Shwartzman type model, and experimental allergic encephalitis. These and other observations justify the hope that administration of monoclonal antibodies against IFN-gamma will be useful to treat or prevent certain inflammatory disorders.
Animals↗
Cholera and pertussis exotoxins protect mice against the lethal Schwartzman reaction and antagonize the effects of lipopolysaccharide on second messenger systems.
Pertussis toxin, and also cholera toxin are capable of inhibiting the effects of LPS in the elicitation of the generalized Schwartzman reaction. This is a potentially lethal generalized thrombo-haemorrhagic hypersensitivity and inflammatory-type response that occurs after two consecutive injections of LPS. The two exotoxins furnish significant protection against the lethal outcome of this reaction. It is known that the acute haematological and haemodynamic changes are accompanied by alterations in the levels of various endogenous mediators: glucocorticoid hormones, prostaglandins, arachidonic acid metabolites, cytokines and proteases. In vitro effects of LPS on murine leukocyte cell lines can be antagonized by pertussis toxin, implicating a Gi-like regulatory protein in the mediation of these effects. Experiments designed to study the involvement of particular second messenger systems (cAMP and phosphatidylinositol) used by LPS in vivo, revealed that the protective effects conferred by these exotoxins are associated with the antagonization of alterations caused by LPS. No correlation was found between the levels of IL-6 and the mortality rate in this experimental mouse model. The results indicate that G proteins play a role in the generation of the Schwartzman reaction and open a new approach for pharmacological intervention in endotoxemia and in clinical settings with Gram-negative sepsis.
Animals↗
Purification of granulocyte chemotactic peptide/interleukin-8 reveals N-terminal sequence heterogeneity similar to that of beta-thromboglobulin.
Stimulated human peripheral blood leukocytes produce a chemotactic factor for granulocytes (granulocyte chemotactic peptide/interleukin-8; GCP/IL-8), which is structurally related to platelet-derived beta-thromboglobulin. Analytically pure CGP/IL-8 and beta-thromboglobulin could be obtained after three purification steps, comprising adsorption to silicic acid, heparin-Sepharose chromatography and ion-exchange chromatography. Although GCP/IL-8 and beta-thromboglobulin had a similar affinity for heparin, they could be separated on a cation-exchange column. Both molecules were heterogeneous in that 6-7-kDa protein doublets were detected upon SDS/PAGE. N-terminal amino acid sequence analysis revealed the presence of six immunologically related but differently truncated polypeptides of beta-thromboglobulin, of which only two corresponded to previously described forms. Similarly, apart from a major polypeptide, five minor species of GCP/IL-8 were detected that also differed by N-terminal truncation. The most processed forms of beta-thromboglobulin and GCP/IL-8 were found to have their N-terminus in that region of the primary structure where a significant similarity between the two molecules starts. GCP/IL-8 was found to be chemotactic for granulocytes with a specific activity of 10(5) units/mg, whereas none of the beta-thromboglobulin species possessed detectable chemotactic activity.
Amino Acid Sequence↗
Simultaneous production of interleukin 6, interferon-beta and colony-stimulating activity by fibroblasts after viral and bacterial infection.
Different viruses were compared with the double-stranded RNA poly(rI).poly(rC) and interleukin (IL) 1 for their IL 6-inducing potential in several human and animal cell types. The laboratory viruses Sendai, Mengo and Newcastle disease virus were found to dose dependently stimulate IL 6 production in diploid fibroblasts. A similar effect was obtained with the human pathogens, measles and rubella virus. Concomitantly with IL 6, two other cytokine activities, i.e., interferon-beta and colony-stimulating activity for granulocytes and monocytes, were induced. In addition, these three activities were also produced by fibroblasts in response to Escherichia coli, whereas lipopolysaccharide was only marginally active. The specificity of the induction phenomenon was confirmed by the lack of IL 6 induction with inactivated infectious agents and by the complete neutralization of produced IL 6 by specific antibodies. This study indicates that the coordinate production of hemopoietic growth factors and interferon, originating from cells that do not classically belong to the immune system, can influence the local and systemic reactions observed during host defence against various infectious agents.
Animals↗
The chemotactic activity for granulocytes produced by virally infected fibroblasts is identical to monocyte-derived interleukin 8.
So far, the role of fibroblasts in inflammatory processes has been underestimated. We have previously shown that stimulation of fibroblasts with viruses or bacteria results in a simultaneous production of several cytokines, including interferon-beta, interleukin (IL) 6 and colony-stimulating factors. We here report that virally infected fibroblasts produce also a chemotactic factor for granulocytes. The activity is inducible not only by measles virus but also by IL 1 beta and the double-stranded RNA poly(rI).poly(rC). This factor, when purified to homogeneity, occurs as a 6-7-kDa protein doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pure protein is serologically related to a fully characterized granulocyte chemotactic peptide (GCP) from monocytes, designated IL8. Furthermore, the chemotactic factor from fibroblasts has an NH2-terminal sequence identical to that of GCP/IL8, small differences in NH2-terminal processing being observed. Finally, in addition to diploid fibroblasts, the osteosarcoma MG-63 cell line is also a producer of GCP/IL8. It can thus be concluded that GCP/IL8 can be produced by several cell types in response to infection and that fibroblasts can contribute to chemotaxis in inflammation.
Amino Acid Sequence↗