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IFN-gamma-activated inhibitor of translation complex
Neutrophil extracellular trap-forming death
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Gamma-Interferon, abbr. gIFN. Approved gene symbol IFNG.
Antigen induced interferon;
Immune interferon (IIF);
Type 2 interferon;
Mitogen induced interferon;
Many of the activities described in the older publications as MAF (macrophage activating factor) or TRF (T-cell replacing factor) are due to IFN-gamma.
IFN-gamma is produced mainly by T-cells and natural killer cells activated by antigens, mitogens, or alloantigens. It is produced by lymphocytes expressing the surface antigens CD4 and CD8. The synthesis of IFN-gamma is induced, among other things, by IL2, bFGF, and EGF. The synthesis of IFN-gamma is inhibited by 1-alpha,25-Dihydroxy vitamin D3, dexamethasone and CsA (Cyclosporin A).
B-cells also produce IFN-gamma, and constitutive synthesis has been observed in many established human B-cell lines.
IFN-gamma is a dimeric protein with subunits of 146 amino acids. The protein is glycosylated at two sites. The pI is 8.3-8.5.
IFN-gamma is synthesized as a precursor protein of 166 amino acids including a secretory signal sequence of 23 amino acids. Two molecular forms of the biologically active protein of 20 and 25 kDa have been described. Both of them are glycosylated at position 25. The 25 kDa form is also glycosylated at position 97. The observed differences of natural IFN-gamma with respect to molecular mass and charge are due to variable glycosylation patterns. 40-60 kDa forms observed under non-denaturing conditions are dimers and tetramers of IFN-gamma.
Recombinant IFN-gamma isolated from Escherichia coli is also biologically active and glycosylation therefore is not required for biological activity (see also: Recombinant cytokines). IFN-gamma contains two cysteine residues that are not involved in disulfide bonding.
At least six different variants of naturally occurring IFN-gamma have been described. They differ from each other by variable lengths of the carboxyterminal ends. The biological activities of these variants do not differ from recombinant IFN-gamma obtained from Escherichia coli. It has been proposed that at least some of these variants are the result of proteolytic cleavage by exopeptidases and hence constitute purification artifacts. In contrast to IFN-alpha and IFN-beta IFN-gamma is labile at pH 2.
IFN-gamma can exist in a form associated with the extracellular matrix and may, therefore, exert juxtacrine growth control.
IFN-gamma does not display significant homology with the other two interferons, IFN-alpha and IFN-beta. Murine and human IFN-gamma show approximately 40 % sequence homology at the protein level.
The human gene has a length of approximately 6 kb. It contains four exons and maps to chromosome 12q24.1.
A number of binding proteins with molecular masses between 70 and 160 kDa have been described for IFN-gamma. They are expressed on all types of human cells with the exception of mature erythrocytes. The receptor expressed in monocytes and other hematopoietic cells (see also: hematopoiesis) has a molecular mass of 140 kDa. A 54 kDa protein is observed in other cell types. The expression of the IFN-gamma receptor on human peripheral blood monocytes is significantly increased by GM-CSF.
IFN-gamma receptors are N- and O-glycosylated and bind IFN-gamma with an affinity of 10**-10 - 10**-11 M. The extracellular domain of the human 54 kDa receptor has a length of 229 amino acids and a transmembrane domain of 20 amino acids. The intracellular domain has a length of 222 amino acids and is involved probably in signal transduction.
The gene encoding this receptor maps to human chromosome 6. This receptor is not related to the receptors for IFN-alpha and IFN-beta. At the protein level the murine and the human receptor show a sequence homology of 52 %.
IFN-gamma/ receptor complexes are rapidly internalized by endocytosis. In mouse/human cellular hybrids carrying human chromosome 6 and therefore expressing the receptor IFN-gamma binds to this receptor without eliciting any biological responses. The cells respond to IFN-gamma if human chromosome 21 is also introduced into these cells. These observations suggest that another still unknown protein encoded by a gene on human chromosome 21 is required to generate a functional receptor molecule. This protein interacts with the extracellular domain of the receptor and, like the 54 kDa molecule, is also species-specific.
A soluble form of the IFN-gamma receptor has been described also. Soluble receptors have been found also for IL1 (see: IL1ra, IL1 receptor antagonist), IL2, IL4, IL6, IL7, IGF and TNF-alpha. They probably function as physiological regulators of cytokine activities (see also: Cytokine inhibitors) by inhibiting receptor binding or act as transport proteins.
IFN-gamma has antiviral and antiparasitic activities and also inhibits the proliferation of a number of normal and transformed cells. IFN-gamma synergises with TNF-alpha and TNF-beta in inhibiting the proliferation of various cell types. The growth inhibitory activities of IFN-gamma are more pronounced than those of the other interferons (see: IFN). However, the main biological activity of IFN-gamma appears to be immunomodulatory in contrast to the other interferons that are mainly antiviral.
In T-helper cells IL2 induces the synthesis of IFN-gamma and other cytokines. IFN-gamma acts synergistically with IL1 and IL2 and appears to be required for the expression of IL2 receptors on the cell surface of T-lymphocytes. Blocking of the IL2 receptor by specific antibodies also inhibits the synthesis of IFN-gamma. IFN-gamma thus influences cell mediated mechanisms of cytotoxicity. IFN-gamma is a modulator of T-cell growth and functional differentiation. It is a growth-promoting factor for T-lymphocytes and potentiates the response of these cells to mitogens or growth factors.
The human promyelocytic leukemia cell line HL-60 can be induced to differentiate by a number of stimuli. IFN-gamma, but not other interferons (see: IFN), specifically induces differentiation of these cells into monocytes.
IFN-gamma inhibits the growth of B-cells induced by IL4. IFN-gamma and Anti-Ig costimulate the proliferation of human B-cells but not of murine B-cells. IFN-gamma also inhibits the production of IgG1 and IgE elicited by IL4 in B-cells stimulated by bacterial lipopolysaccharides. IFN-gamma regulates the expression of MHC class 2 genes and is the only interferon that stimulates the expression of these proteins.
IFN-gamma also stimulates the expression of Ia antigens on the cell surface, the expression of CD4 in T-helper cells, and the expression of high-affinity receptors for IgG (see also: CD16, CD32, CD64) in myeloid cell lines, neutrophils, and monocytes. In monocytes and macrophages IFN-gamma induces the secretion of TNF-alpha and the transcription of genes encoding G-CSF and M-CSF. In macrophages IFN-gamma stimulates the release of reactive oxygen species. IFN-gamma is involved also in processes of bone growth and inhibits bone resorption probably by partial inhibition of the formation of osteoclasts.
IFN-gamma inhibits the proliferation of smooth muscle cells of the arterial intima in vitro and in vivo and therefore probably functions as an endogenous inhibitor for vascular overreactions such as stenosis following injuries of arteries. IFN-gamma inhibits the proliferation of endothelial cells and the synthesis of collagens by myofibroblasts. It thus functions as an inhibitor of capillary growth mediated by myofibroblasts and fibroblast growth factors (see: FGF) and PDGF.
IFN-gamma specifically induces the transcription of a number of genes. These genes contain regulatory DNA sequences within their promoter regions (ISRE; Interferon-stimulated response element; see: IRS, interferon response sequence) that function as binding sites for a number of transcription factors (see: GAF; gamma-Interferon activation factor). Some of these genes are expressed also in response to other interferons (see: IFN).
TRANSGENIC ANIMALS, KNOCK-OUT, AND ANTISENSE STUDIES
The expression of IFN-gamma in the pancreas of transgenic mice has been shown to precipitate autoimmune diabetes. IFN-gamma produced locally in the pancreas of transgenic animals causes infiltration of lymphocytes and islet cell destruction. However, new islet cells are formed continuously from duct cells. The IFN-gamma induced islet neogenesis is similar to embryonic islet morphogenesis and offers a model system for studying factors modulating islet development.
Mice with a targeted disruption of the IFN-gamma gene have been developed by homologous recombination in ES cells. These knock-out animals develop normally and are healthy in the absence of pathogens. They are characterized, however, by an impaired production of macrophage antimicrobial products and reduced expression of macrophage major histocompatibility complex class 2 antigens. IFN-gamma deficient mice are killed by normally sublethal doses of the intracellular pathogen Mycobacterium bovis. Splenocytes show uncontrolled proliferation in response to mitogen and alloantigen. After a mixed lymphocyte reaction, T-cell cytolytic activity is found to be enhanced against allogeneic target cells. Resting splenic natural killer cell activity is reduced in these mice.
Mice lacking the IFN-gamma receptor have been created also by homologous recombination. These animals have no overt anomalies and show normal cytotoxic and T-helper cell responses. However, mutant mice show an increased susceptibility to infection by Listeria monocytogenes and vaccinia virus. Immunoglobulin isotype analysis reveals that IFN-gamma is required for a normal antigen-specific immunoglobulin G2a response.
DETECTION AND ASSAY METHODS
IFN-gamma can be detected by sensitive immunoassays. A specific ELISA test allows detection of individual cells producing IFN-gamma. Minute amounts of IFN-gamma can be detected indirectly by measuring IFN induced proteins such as Mx protein. The induction of the synthesis of IP-10 has been used also to measure IFN-gamma concentrations. A new bioassay employs induction of indoleamine 2,3-dioxygenase activity in 2D9 cells. A sensitive radioreceptor assay is also available. Lewis describes a bioassay based on induction of a reporter gene linked to a promoter element that responds to interferons (see: IFN). Production of the reporter gene product is dose-dependent in the range of 1 to approximately 100 U/mL of IFN and sensitivity is comparable to standard cytopathic effect assays.
NO production by RAW264.7 cells in response to murine IFN-gamma can be used also as a bioassay. Schein describes a quantitative rapid assay for IFN-gamma that is based on its ability to stimulate the activity of ds-RNases while inhibiting e. g., RNase A.
For an immunoassay allowing rapid determination of the rate of IFN-gamma production under conditions preventing consumption/degradation see also: Cytokine immunotrapping.
For further information see also subentry "Assays" in the reference section. For further information on assays for cytokines see also: bioassays, cytokine assays.
CLINICAL USE AND SIGNIFICANCE
Like the other interferons (see: IFN) IFN-gamma can be used as an antiviral and antiparasitic agent. IFN-gamma has been shown to be effective in the treatment of chronic polyarthritis. This treatment, which probably involves a modulation of macrophage activities, significantly reduces joint aches and improves various clinical parameters and allows reduction of corticosteroid doses.
In spite of the antiproliferative activities of IFN-gamma hopes that this interferon may be of use in the treatment of various malignancies have been disappointed. In many instances a combination treatment with other interferons (see: IFN) and cytokines, and also with chemotherapy has been moderately effective only.
IFN-gamma may be of value in the treatment of opportunistic infections in AIDS patients. It has been shown also to reduce inflammation, clinical symptoms, and eosinophilia in severe atopic dermatitis.
Experiments are in progress to insert IFN-gamma genes into human tumor cells by genetic engineering as a means for generating autologous or HLA-matched allogeneic tumor cell vaccines for the treatment of patients with renal cell carcinoma and other cancers. The transduction of murine tumor cells with a functional IFN-gamma gene has been shown to lead to the rejection of the genetically modified cells by syngeneic hosts (for cancer vaccines see also: Cytokine gene transfer).
Treatment with IFN-gamma of a subgroup of patients with sepsis (see also: Systemic inflammatory response syndrome) characterized by an impaired function of monocytes and a particular poor prognosis has been shown to augment functions of monocytes and to result in clearance of sepsis in the majority of these patients.
See REFERENCES for entry IFN-gamma.
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