Current prospects of type II interferon γ signaling and autoimmunity.

Interferon γ (IFNγ) is a pleiotropic protein secreted by immune cells. IFNγ signals through the IFNγ receptor, a protein complex that mediates downstream signaling events. Studies into IFNγ signaling have provided insight into the general concepts of receptor signaling, receptor internalization, regulation of distinct signaling pathways, and transcriptional regulation. Although IFNγ is the central mediator of the adaptive immune response to pathogens, it has been shown to be involved in several non-infectious physiological processes. This review will provide an introduction into IFNγ signaling biology and the functional roles of IFNγ in the autoimmune response.

Interferon-γ constrains cytokine production of group 2 innate lymphoid cells.

Group 2 innate lymphoid cells (ILC2s) produce a significant amount of interleukin-5 (IL-5), which supports eosinophil responses in various tissues; they also produce IL-13, which induces mucus production and contributes to tissue repair or fibrosis. The ILC2s are activated by alarmins, such as IL-33 released from epithelia, macrophages and natural killer T (NKT) cells in response to infection and allergen exposure, leading to epithelial injury. We examined gene expression in lung ILC2s and found that ILC2s expressed Ifngr1, the receptor for interferon-γ (IFN-γ). Interferon-γ severely inhibited IL-5 and IL-13 production by lung and kidney ILC2s. To evaluate the effects in vivo, we used α-galactosylceramide (α-GalCer) to induce NKT cells to produce IL-33 and IFN-γ. Intraperitoneal injection of α-GalCer in mice induced NKT cell activation resulting in IL-5 and IL-13 production by ILC2s. Administration of anti-IFN-γ together with α-GalCer significantly enhanced the production of IL-5 and IL-13 by ILC2s in lung and kidney. Conversely, cytokine production from ILC2s was markedly suppressed after injection of exogenous IL-33 in Il33(-/-) mice pre-treated with α-GalCer. Hence, IFN-γ induced or already present in tissues can impact downstream pleiotropic functions mediated by ILC2s, such as inflammation and tissue repair.

Non-steroidal antiandrogens act as AF-1 agonists under conditions of high androgen-receptor expression.

BACKGROUND: The mechanism of resistance acquisition to antiandrogens in prostate cancer is not fully understood. Numerous clinical and basic research studies have shown expression of androgen receptors (ARs) increases in hormone-refractory prostate cancer and therefore we explored possible molecular mechanisms by which prostate cancer acquires resistance to antiandrogens under conditions of increased AR expression. METHODS: In order to study resistance to antiandrogens at the AR transactivation level we used a human AR (hAR) reporter assay system. In addition, we utilized an hAR deletion mutant to determine the functional domain responsible for the acquisition of resistance. RESULTS: Increased hAR protein expression enhanced the sensitivity of AR transactivation to low concentrations of DHT, and also reduced the inhibitory activity of the non-steroidal antiandrogens, hydroxyflutamide, and bicalutamide on DHT-induced AR transactivation. Moreover, these antiandrogens acquired agonistic activity under conditions of high hAR protein expression. Such agonistic activity of antiandrogens was not detected in an hAR deletion mutant (hAR-DeltaA/B) that lacked an A/B domain with AF-1 activity. CONCLUSIONS: We found that non-steroidal antiandrogens act as AF-1 agonists under conditions of high AR protein expression. This partial antagonistic property of antiandrogens may be a molecular mechanism by which prostate cancer develops resistance to these drugs.

Interferon-gamma differentially regulates TGF-beta1 and TGF-beta2 expression in human retinal pigment epithelial cells through JAK-STAT pathway.

Retinal pigment epithelium (RPE) and transforming growth factor-beta (TGF-beta) have been shown to be involved in various retinal diseases. We have studied the role of inflammatory cytokines on the expression and secretion of TGF-beta in human RPE cells (HRPE). Confluent cultures of HRPE derived from donor eyes were used. RT-PCR analyses showed that TNF-alpha and IL-1beta increased the mRNA levels of both TGF-beta1 and TGF-beta2. IFN-gamma enhanced constitutively expressed, as well as, TNF-alpha-and IL-1beta-induced TGF-beta1 mRNA levels but decreased TGF-beta2 mRNA. The effects of these cytokines on TGF-beta1 and TGF-beta2 secretion correlated with the mRNA levels. TGF-beta1 was always produced as the latent form while 21-31% of TGF-beta2 was in the active form. IFN-gamma reduced the production of active form of TGF-beta2 to 4-9%. TGF-beta3 secretion was not detectable under any of the conditions. The Real-Time PCR analysis of TGF-beta mRNAs confirmed the observed results. The TGF-beta1 and TGF-beta2 secretion was induced by TGF-beta2 and TGF-beta1, respectively. Under these conditions, the contrasting effects of IFN-gamma on TGF-beta1 and TGF-beta2 secretion were also observed. JAK inhibitor selectively inhibited IFN-gamma induced TGF-beta1 secretion and mRNA levels while reversing the inhibitory effects of IFN-gamma on TGF-beta2. Analyses of transcription factor activity strongly indicated the role of STAT-1 but not NFkappaB, C-Myc, C-Jun, SP-1, MEF-2. Our data demonstrate that IFN-gamma differentially regulates constitutively expressed, as well as, cytokine-induced TGF-beta1 and TGF-beta2 mRNA levels and secretion of TGF-betas by HRPE.

Synthesis and biological properties of chimeric interferon-alpha2b peptides.

We have previously reported the antiproliferative activity of synthetic sequences 29-35 and 122-139 of the interferon-alpha2b (IFN-alpha2b), both probably representing a common receptor recognition domain. In the search of new peptidic agonists, we designed and synthesized the linear peptide (Gly)2-122-137-Gly138-Gly29-30-35-(Gly)2, in which Gly residues replaced the 138 and 29 Cys bound through a disulfide bridge in the native cytokine. Additionally, a cyclic analog was obtained by reaction of the N- and C-terminal ends of the linear fragment. Thus, the distance that separates residues 122 and 35 in the crystalline structure of the IFN-alpha2b was maintained through a (Gly)4 bridge. When the influence of chimeric peptides on the proliferation of WISH cells was studied, it was shown that both derivatives significantly diminished cell growth. A more evident inhibitory effect on (125)I-IFN-alpha2b binding to WISH cell-membrane receptors was observed for both peptides. Results indicated that chimeric IFN-alpha2b peptides behaved as partial agonists of the IFN-alpha2b molecule and may be of interest for drug design purposes.

Intestinal Na+-K+-ATPase activity and molecular events downstream of interferon-gamma receptor stimulation.

This study evaluated the effects of human interferon-gamma (IFN-gamma) on Na(+)-K(+)-ATPase activity and the intracellular signaling pathways involved in human intestinal epithelial Caco-2 cells. Na(+)-K(+)-ATPase activity was determined as the difference between total and ouabain-sensitive ATPase. p38 MAP kinase activity was analyzed by Western blotting using the p38 MAP kinase assay kit. Total and phosphorylated STAT1 protein levels were detected using the PhosphoPlus Stat1. IFN-gamma decreased Na(+)-K(+)-ATPase activity in a time- and concentration-dependent manner. The IFN-gamma-induced decrease in Na(+)-K(+)-ATPase activity was accompanied by no changes in the abundance of alpha(1) subunit Na(+)-K(+)-ATPase. Downregulation of protein kinase C (PKC) with phorbol-12,13-dibutyrate (PDBu) prevented the inhibitory effect of IFN-gamma on Na(+)-K(+)-ATPase activity. Inhibition of Raf-1, mitogen-activated protein kinase kinase (MAPKK/MEK), p38 MAPK and STAT1 with, respectively, GW 5074, PD 98059, SB 203580 and epigallocatechin gallate prevented inhibition of Na(+)-K(+)-ATPase activity by IFN-gamma. Treatment with IFN-gamma markedly increased the expression of total and phospho-STAT1, this being accompanied by activation of p38 MAPK. Activation of phospho-STAT1 by IFN-gamma was almost abolished by epigallocatechin gallate and markedly reduced by SB 203580, but insensitive to downregulation of PKC. The increase in short circuit current (I(sc)) by 1.0 and 2.5 micrograms ml(-1) amphotericin B was markedly attenuated in IFN-gamma-treated cells. However, the inhibitory effect of PDBu on the amphotericin B-induced increase in I(sc) was of similar magnitude in vehicle- and IFN-gamma-treated cells. It is concluded that IFN-gamma markedly attenuates Na(+)-K(+)-ATPase activity. The transduction mechanisms set into motion by IFN-gamma involve the activation of PKC downstream STAT1 phosphorylation and Raf-1, MEK, ERK2 and p38 MAPK pathways, in a complex sequence of events.

Class II cytokine receptor ligands inhibit human vascular smooth muscle proliferation.

BACKGROUND: Vessel injury provokes the release of proinflammatory cytokines and growth factors that influence vascular smooth muscle cell (VSMC) proliferation and migration. Produced by T lymphocytes, interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) both have immunoregulatory functions and act on similar receptors, designated class II cytokine receptors. We hypothesized that the class II cytokine receptor participates in vascular remodeling by inhibiting VSMC proliferation. The purposes of this study were to determine the influence of class II cytokine receptor stimulation on (1) unstimulated, (2) cytokine-stimulated, and (3) growth factor-stimulated VSMC proliferation. METHODS: Human aortic VSMCs were isolated and cultured. VSMCs were treated with IL-10 or IFN with or without tumor necrosis factor-alpha (TNF-alpha) or basic fibroblast growth factor (FGF). Proliferation was quantified by colormetric assay. RESULTS: Compared to control, both TNF and FGF stimulated concentration-dependent VSMC proliferation (P < .005). IL-10 and IFN alone had no effect on unstimulated cell growth. With TNF or FGF stimulation, both IL-10, at a dose as low as 10 fg/ml, and IFN, at a dose as low as 1.0 U/ml, inhibited cell growth (P < .001). CONCLUSIONS: The class II cytokine receptor ligands, IL-10 and IFN, inhibit cytokine-(TNF) and growth factor-(FGF) induced VSMC proliferation. The class II cytokine receptor may provide a novel therapeutic target in regulating vessel wall remodeling after vascular injury.

Unrestricted agonist activity on murine and human cells of a lipopeptide derived from IFN-gamma.

We describe the isolation of a synthetic agonist of IFN-gamma (L-mIFN-gamma-CT) active on mouse and human cells. Its biological activity is the result of the ability of the C-terminal extremity of murine IFN-gamma to interact with the intracellular part of IFNgamma-R and the observation that the modification of peptides by a palmitic acid enables their cytoplasmic delivery. L-mIFN-gamma-CT stimulated murine cells exhibited an increase in MHC class II molecules and FcgammaRII/III expression and conferred protection against viral lysis. Unresponsiveness to L-mIFN-gamma-CT of cells recovered from IFNgamma-R alpha-chain knockout mice indicated the involvement of IFNgamma-R in the biological activities observed. Induction of VCAM-1, ICAM-1, and HLA-DR expression on human cells stimulated with L-mIFN-gamma-CT demonstrated an abrogation of species specificity. These results describe the development of a new synthetic agonist of IFN-gamma, which substitutes for the native cytokine in any IFN-gamma responsive cells, by acting intracellularly on IFN-gammaR.