Serguei V. Kotenko

Identification and functional characterization of a second chain of the interleukin-10 receptor complex.

Interleukin‐10 (IL‐10) is a pleiotropic cytokine which signals through a specific cell surface receptor complex. Only one chain, that for ligand binding (IL‐10Rα or IL‐10R1), was identified previously. We report here that, although human IL‐10 binds to the human IL‐10R1 chain expressed in hamster cells, it does not induce signal transduction. However, the co‐expression of CRFB4, a transmembrane protein of previously unknown function belonging to the class II cytokine receptor family, together with the IL‐10R1 chain renders hamster cells sensitive to IL‐10. The IL‐10:CRFB4 complex was detected by cross‐linking to labeled IL‐10. In addition, the IL‐10R1 chain was able to be co‐immunoprecipitated with anti‐CRF antibody when peripheral blood mononuclear cells were treated with IL‐10. These results demonstrate that the CRFB4 chain is part of the IL‐10 receptor signaling complex. Thus, the CRFB4 chain, which we designate as the IL‐10R2 or IL‐10Rβ chain, serves as an accessory chain essential for the active IL‐10 receptor complex and to initiate IL‐10‐induced signal transduction events.

Tumor Cell Responses to IFNγ Affect Tumorigenicity and Response to IL-12 Therapy and Antiangiogenesis

Expression of a dominant negative mutant IFNgammaR1 in murine SCK and K1735 tumor cells rendered them relatively unresponsive to IFNgamma in vitro and more tumorigenic and less responsive to IL-12 therapy in vivo. IL-12 induced histologic evidence of ischemic damage only in IFNgamma-responsive tumors, and in vivo Matrigel vascularization assays revealed that while IFNgamma-responsive and -unresponsive tumor cells induced angiogenesis equally well, IL-12 and its downstream mediator IFNgamma only inhibited angiogenesis induced by the responsive cells. IL-12 induced angiogenesis inhibitory activity in the responsive cells, which may be attributable to production of the chemokine IP-10. Thus, IL-12 and IFNgamma inhibit tumor growth by inducing tumor cells to generate antiangiogenic activity.

Identification and sequence of an accessory factor required for activation of the human interferon γ receptor

Abstract Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon γ (Hu-IFN-γ), 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-γ receptor, human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-γ 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-γ 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-γ.

A JAK1/JAK2 chimera can sustain alpha and gamma interferon responses.

Cell lines that are mutated in interferon (IFN) responses have been critical in establishing an essential role for the JAK family of nonreceptor tyrosine kinases in interferon signalling. Mutant gamma1A cells have previously been shown to be complemented by overexpression of JAK2. Here, it is shown that these cells carry a defect in, and can also be complemented by, the beta-subunit of the IFN-gamma receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant gamma2A cells lack detectable JAK2 mRNA and protein. By using gamma2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN-gamma signalling was examined. Individual mutation of six conserved tyrosine residues, mutation of a potential phosphatase binding site, or mutation of the arginine residue in the proposed SH2-like domain had no apparent effect on signalling in response to IFN-gamma. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-gammaR2 subunit requires the amino-terminal region but not the pseudokinase domain. Consistent with this, in chimeras with JAK1, the JAK2 amino-terminal region was required for receptor association and STAT1 activation. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-alpha and -gamma in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different receptor and signalling proteins rather than in the substrate specificity of their kinase domains.

Interaction between the components of the interferon gamma receptor complex

Interferon γ (IFN-γ) signals through a multimeric receptor complex consisting of two different chains: the IFN-γ receptor binding subunit (IFN-γR, IFN-γR1), and a transmembrane accessory factor (AF-1, IFN-γR2) necessary for signal transduction. Using cell lines expressing different cloned components of the IFN-γ receptor complex, we examined the function of the receptor components in signal transduction upon IFN-γ treatment. A specific IFN-γR2:IFN-γ cross-linked complex was observed in cells expressing both IFN-γR1 and IFN-γR2 indicating that IFN-γR2 (AF-1) interacts with IFN-γ and is closely associated with IFN-γR1. We show that the intracellular domain of IFN-γR2 is necessary for signaling. Cells coexpressing IFN-γR1 and truncated IFN-γR2, lacking the COOH-terminal 51 amino acids (residues 286-337), or cells expressing IFN-γR1 alone were unresponsive to IFN-γ treatment as measured by MHC class I antigen induction. Jak1, Jak2, and Stat1α were activated, and IFN-γR1 was phosphorylated only in cells expressing both IFN-γR1 and IFN-γR2. Jak2 kinase was shown to associate with the intracellular domain of the IFN-γR2.

Other kinases can substitute for Jak2 in signal transduction by interferon-gamma.

Each cytokine which utilizes the Jak-Stat signal transduction pathway activates a distinct combination of members of the Jak and Stat families. Thus, either the Jaks, the Stats, or both could contribute to the specificity of ligand action. With the use of chimeric receptors involving the interferon γ receptor (IFN-γR) complex as a model system, we demonstrate that Jak2 activation is not an absolute requirement for IFN-γ signaling. Other members of the Jak family can functionally substitute for Jak2. IFN-γ can signal through the activation of Jak family members other than Jak2 as measured by Statlα homodimerization and major histocompatibility complex class I antigen expression. This indicates that Jaks are interchangeable and indiscriminative in the Jak-Stat signal transduction pathway. The necessity for the activation of one particular kinase during signaling can be overcome by recruiting another kinase to the receptor complex. The results may suggest that the Jaks do not contribute to the specificity of signal transduction in the Jak-Stat pathway to the same degree as Stats.

Chimeric erythropoietin-interferon gamma receptors reveal differences in functional architecture of intracellular domains for signal transduction.

Binding of interferon gamma (IFN-γ) causes oligomerization of the two interferon γ receptor (IFN-γR) subunits, receptor chain 1 (IFN-γR1, the ligand-binding chain) and the second chain of the receptor (IFN-γR2), and causes activation of two Jak kinases (Jak1 and Jak2). In contrast, the erythropoietin receptor (EpoR) requires only one receptor chain and one Jak kinase (Jak2). Chimeras between the EpoR and the IFN-γR1 and IFN-γR2 chains demonstrate that the architecture of the EpoR and the IFN-γR complexes differ significantly. Although IFN-γR1 alone cannot initiate signal transduction, the chimera EpoR/γR1 (extracellular/intracellular) generates slight responses characteristic of IFN-γ in response to Epo and the EpoR/γR1·;EpoR/γR2 heterodimer is a fully functional receptor complex. The results demonstrate that the configuration of the extracellular domains influences the architecture of the intracellular domains.

Mouse Macrophages Carrying Both Subunits of the Human Interferon-γ (IFN-γ) Receptor Respond to Human IFN-γ but Do Not Acquire Full Protection against Viral Cytopathic Effect

Studies of hamster-human and mouse-human somatic fibroblast hybrids and transfected mouse fibroblasts have demonstrated that signaling through the human interferon-γ receptor (hu-IFN-γR) requires the formation of a complex consisting of ligand (IFN-γ), a ligand binding receptor chain (IFN-γR1), and a signal transducing receptor chain (IFN-γR2). To date, the ability of this receptor complex to transduce the full repertoire of biological signals has been difficult to assess due to the limited number of activities that IFN-γ can exert on fibroblasts. The current report assesses the ability of hu-IFN-γR chains to transduce signals in the absence of background human gene products by expressing hu-IFN-γR2 in a transformed macrophage cell line (F10/96) derived from a hu-IFN-γR1 transgenic mouse. Our results indicate that F10/96 clones expressing both human receptor proteins bind hu-IFN-γ with an affinity comparable to that of human cells. Binding of either human or mouse IFN-γ to its respective receptor elicits classic IFN-γ responses such as up-regulation of major histocompatibility complex antigens, enhanced expression of IRF-1, and increased production of NO2− radicals, interleukin-6, tumor necrosis factor-α, and granulocyte macrophage-colony stimulating factor. However, hu-IFN-γ could not fully protect the clones from cytopathic effects of encephalomyocarditis virus and vesicular stomatitis virus while mo-IFN-γ could. These results demonstrate that while co-expression of hu-IFN-γR1 and hu-IFN-γR2 is necessary and sufficient for most IFN-γ-induced responses, it is not sufficient to confer a generalized antiviral state. These findings further suggest that additional species-specific accessory factor(s) are necessary for full signaling potential through the IFN-γ receptor complex. The nature and potential role of such factors in IFN-γR signaling is discussed.