IFN-gamma Receptor Research Articles

Crystallization and X-ray crystallographic analysis of human STAT1

Unphosphorylated human STAT1 (1-683) has been crystallized in the presence of a phosphopeptide derived from the alpha-chain of human interferon gamma (IFNgamma) receptor. A complete data set has been collected from a KAu(CN)2-derivatized and dehydrated crystal. The crystal belonged to space group P6(1)22, with unit-cell parameters a = b = 102.6, c = 646.5 A, alpha = beta = 90, gamma = 120 degrees.

Prominent Involvement of Activated Th1-Subset of T-Cells and Increased Expression of Receptor for IFN-Gamma on Keratinocytes in Atopic Dermatitis Acute Skin Lesions

Background: Atopic dermatitis (AD) is an allergic skin disease mediated by antigen-specific IgE and an important role has been ascribed to CD4+ cells (Th cells). The objective of the study was to evaluate humoral and cellular immunological factors in the blood and the skin lesions of AD patients, and to analyze the presence of inflammatory cell-surface markers in blood and skin biopsies. Methods: The parameters for monitoring of 40 AD patients included results of prick test to inhalant allergens and epicutaneous (patch) test to contact allergens; values of total IgE, serum immunoglobulins (IgG, IgA, IgM) and different cell markers in the sera (CD3, CD4, CD8, CD20, CD21, CD23, HLA-DR). We also analyzed the presence of inflammatory cell-surface markers (CD3, CD4, CD8, CD20, CD20, CD1a, CD23, CD29, CD45Ro, IFNγ+ markers) in the biopsies of skin lesions from 10 AD patients and 5 healthy controls (HCs) by immunohistochemical analysis (method of avidin-biotin immunoperoxidase). Results: Beside increased total serum IgE and positive skin tests, a significantly higher percentage of CD23+ cells with lower percentage of CD21+ cells was revealed in peripheral blood of AD patients in comparison to HCs. A positive epidermal expression of the majority of markers of T cells (CD3+, CD4+, CD8+, CD29+, CD45Ro+, IFNγ+) and those of Langerhans’ cells (LCs) (CD1a, CD23+), without those of B cells (CD20+) were noted in AD patients, but no in the skin of HCs. Furthermore, significant difference was also found between the two groups for increased expression of CD3, CD4, CD8, CD29, CD45Ro, IFNγ+ markers (markers for IFNγ receptor) and higher intraepidermal CD23+ LCs and intradermal CD1a+ LCs in AD skin lesions. Conclusions:The obtained results suggest involvement of various humoral factors with increased production of IgE and cooperation between Th subsets and LCs, with higher production of related cytokines, and disturbed cellular immunity, including epidermal LCs with IgE receptors of high and low affinity in AD. The annotation of activated Th1 cells with increased producing of IFNγ in acute AD skin lesions is notable, and might lead to IFNγ binding to keratinocytes and consequently inflammatory skin changes in the disease.

Mice lacking the IFN-γ receptor or fyn develop severe experimental autoimmune uveoretinitis characterized by different immune responses

Endogenous interferon (IFN)-gamma negatively regulates experimental autoimmune uveoretinitis (EAU), a Th1-mediated disease. Although it is well known that IFN-gamma exerts its effects by binding to the IFN-gamma receptor (IFN-gammaR), the role that IFN-gammaR plays in the development of EAU has not been investigated. Fyn has been reported to inhibit Th2 differentiation. We aimed to investigate how endogenous IFN-gammaR and fyn, which influence Th1/Th2 differentiation, participate in the development of EAU. Sex-matched 6- to 10-week-old C57BL/6 wild-type (WT), IFN-gammaR knockout (GRKO) and fyn knockout (fyn KO) mice were compared. Mice were immunized subcutaneously with human interphotoreceptor retinoid-binding protein peptide 1-20 emulsified in Freund's complete adjuvant together with an intraperitoneal injection of Bordetella pertussis toxin. Three weeks later, mice were sacrificed, and their eyes and spleens were harvested for histopathologic analyses and examination of cellular immune responses, respectively. Cellular immune responses were evaluated by measuring the proliferative responses and cytokine production [interleukin (IL)-4, IL-5, IL-6, IL-13, IFN-gamma and tumor necrosis factor (TNF)-alpha] of splenocytes. The incidence of EAU was 40.0% in WT mice, 59.3% in GRKO mice and 78.6% in fyn KO mice. The average EAU score was 0.294 in WT mice, 0.917 in GRKO mice and 1.063 in fyn KO mice. Upon EAU induction, significant infiltration of eosinophils into the eyes was observed in GRKO and fyn KO mice compared to WT mice. Splenocytes from GRKO mice proliferated against the antigen and a mitogen more vigorously than those from WT and fyn KO mice. Stimulation of splenocytes with the antigen induced a higher production of IL-4, IL-6, IL-13 and IFN-gamma in GRKO mice compared to WT and fyn KO mice. In contrast, IL-5 and TNF-alpha were most abundantly produced by splenocytes from fyn KO mice compared to WT and GRKO mice. The incidence and mean severity of EAU were significantly higher in GRKO and fyn KO mice than in WT mice, suggesting that endogenous IFN-gammaR and fyn negatively regulate the development of EAU. The different cytokine production patterns by the GRKO and fyn KO mice indicate that the negative regulatory mechanism mediated by IFN-gammaR and fyn may differ.

Synthesis of α-Chemokines IP-10, I-TAC, and MIG Are Differentially Regulated in Human Corneal Keratocytes

Chemokines responsible for recruiting lymphocytes such as activated T cells into the cornea have not been clearly defined. IP-10, I-TAC, and MIG are chemoattractants for these lymphocytes. The goal of this study was to determine whether human corneal keratocyte (HCKs) in culture synthesize these chemokines in response to proinflammatory mediators. HCKs grown in vitro were stimulated with IL-1alpha, TNF-alpha, or IFN-gamma. Induction of alpha-chemokine gene expression was quantitated by real-time PCR and ELISA. Activation of the transcriptional activator STAT1 by IFN-gamma receptors expressed on HCKs was determined by Western blot analysis. HCKs incubated with TNF-alpha, IL-1alpha, or IFN-gamma resulted in a >2000-fold increase in IP-10 protein secretion by 36 hours after stimulation. In contrast, stimulation with TNF-alpha, IL-1alpha, or IFN-gamma induced levels of MIG and I-TAC that were not significantly greater than constitutive levels. Treatment of HCKs with IFN-gamma activated STAT1 and, in combination with either TNF-alpha or IL-1alpha, enhanced MIG and I-TAC synthesis >20-fold. IP-10 synthesis is induced in HCKs by IL-1alpha, TNF-alpha, and IFN-gamma. In contrast, induction of I-TAC and MIG synthesis in HCKs requires costimulation with IFN-gamma and either IL-1alpha or TNF-alpha. The results suggest therefore, that the upregulation of I-TAC and MIG gene expression at sites of corneal inflammation are more tightly regulated than that of IP-10. A role for differential induction of the three alpha-chemokine genes in corneal inflammatory processes at the eye surface is discussed.

Signaling of short‐ and long‐term regulation of intestinal epithelial type 1 Na+/H+ exchanger by interferon‐γ

The present study evaluated the effect of interferon-gamma (IFN-gamma) on intestinal Na+/H+ exchange (NHE) activity and the intracellular signaling pathways set into motion after IFN-gamma receptor activation. Caco-2 cells express endogenous NHE1, NHE2 and NHE3 proteins, as detected by immunoblotting. Short- (0.5 h) and long- (24 h) term exposure of Caco-2 cells to IFN-gamma resulted in a concentration-dependent decrease in NHE activity. Inhibition of NHE activity by IFN-gamma was absent in cariporide-treated cells, but not in cells treated with S-3226. The long-term exposure to IFN-gamma was accompanied by a 20% increase in surface NHE1 abundance and no changes in total NHE1 abundance. Inhibition of Raf1, mitogen-activated protein kinase kinase (MAPKK/MEK) and p38 MAPK with, respectively, GW 5074, PD 98059 and SB 203580 and downregulation of protein kinase C (PKC) with phorbol-12,13-dibutyrate (100 nM for 24 h) prevented inhibition of NHE activity by IFN-gamma (0.5 and 24 h exposure). The signal transducer and activator transcription factor 1 (STAT1) inhibitor epigallocatechin-3-gallate (EGCG) prevented inhibition of NHE activity by long- but not the short-term treatment with IFN-gamma. Treatment with IFN-gamma activated phospho-p38 MAPK, this effect being detected as early as 1 h, persisting over 3 h and decreasing after 24 h. IFN-gamma produced a sustained action of phospho-STAT1 that was prevented by EGCG and partially attenuated by SB 203580 and insensitive to downregulation of PKC. In conclusion, short- and long-term inhibition of NHE1 activity by IFN-gamma involves a complex signaling pathway that includes PKC activation and STAT1 phosphorylation, respectively, but is not accompanied by downregulation of NHE1.

Immune responses in advanced colorectal cancer following repeated intradermal vaccination with the anti-CEA murine monoclonal antibody, PR1A3: results of a phase I study

The aim was to determine the toxicity, clinical and immune responses to the murine monoclonal anti-carcinoembryonic antigen (CEA) antibody, PR1A3, in patients with advanced colorectal cancer. Fifteen patients with advanced colorectal cancer received either 0.5-, 1.0- or 5.0-mg doses of PR1A3 mixed with 10% w/v Alum adjuvant (Superfos Biosector, Denmark) intradermally at 4-week intervals for 3 months. Patient serum was assessed for anti-idiotypic (Ab2), anti-anti-idiotypic (Ab3) and human anti-mouse antibody (HAMA) reactivity. Peripheral blood mononuclear cell (PBMC) proliferation with phytohaemagglutinin (PHA), CEA and PR1A3, stimulated IL-2, IL-4 and IFN-gamma levels and PR1A3-stimulated IL-2 receptor expression during immunotherapy were determined. Comparisons were made with 16 age-matched controls without malignant disease. Hyperimmune sera from 12 of the 15 patients showed Ab2 reactivity with no detectable Ab3 responses. Strong HAMA reactivity was recorded in 7 of the 15 cases with no adverse clinical effect. Delayed-type hypersensitivity (DTH) responses developed in 12 of the 15 patients. Pre-treatment PBMC proliferation with PHA was subnormal in each patient compared with controls, becoming normal (or supranormal) in all patients during immunisation (P<0.001). PBMC proliferation with CEA and PR1A3 increased during immunotherapy (P<0.001) along with stimulated production of IL-2, IFN-gamma and IL-2 receptor expression. Progressive disease was observed in 14 of the 15 patients with minimal toxicity. PR1A3 generated limited idiotypic responses but robust DTH reactivity in most patients. In vitro PBMC proliferation with mitogens and recall antigens is greatly increased during the course of immunisation, with a shift in stimulated cytokine profile.

Interferon-gamma is a potent inducer of catagen-like changes in cultured human anagen hair follicles

Interferon (IFN)-gamma appears to be an important hair cycle modulator in mice. It is unclear whether it has similar hair growth modulatory functions in human hair follicles. To study whether IFN-gamma can be exploited to modulate the growth, pigmentation and/or cycling of organ-cultured human anagen scalp hair follicles, as an in vitro indicator system for how IFN-gamma affects human hair growth in vivo. This was correlated with the hair follicle expression patterns of IFN-gamma receptors alpha and beta. In addition, we wanted to establish a new, simple tool for the rapid experimental induction of catagen in vitro. Normal human scalp hair follicles in the anagen VI stage of the hair cycle were cultured according to the method of Philpott et al., with or without IFN-gamma (50-1000 IU mL(-1)). Hair shaft elongation and pigmentation changes were measured, complemented by quantitative histomorphometry to assess changes in hair follicle cycling (hair cycle score), proliferation (Ki-67), melanogenesis (Masson-Fontana) and apoptosis (TUNEL). IFN-gamma receptors were also localized by immunofluorescence and EnVision technique. As transforming growth factor (TGF)-beta2 is a recognized key inducer of catagen in human hair follicles, TGF-beta2 expression was investigated by tyramide signal amplification and reverse transcription-polymerase chain reaction in anagen hair follicles treated with vehicle (phosphate-buffered saline) or IFN-gamma. IFN-gamma rapidly inhibited hair elongation in cultured human anagen hair follicles and induced morphological signs of catagen transformation after only 4 days of culture, i.e. faster than with other reported catagen-inducers (e.g. TGF-beta2). Proliferation was inhibited, apoptosis was increased and follicular melanogenesis was switched off in hair bulb keratinocytes treated in situ with IFN-gamma. Anagen hair follicles displayed strong IFN-gamma receptor alpha-like immunoreactivity, while the immunoreactivity for IFN-gamma receptor beta in the hair matrix was only weak. TGF-beta2 immunoreactivity and mRNA transcript levels were enhanced in hair follicles treated with IFN-gamma. These data suggest that IFN-gamma is a potent catagen inducer in normal human scalp hair follicles, which express cognate receptors, and show that IFN-gamma administration offers an excellent tool for experimental catagen induction in organ-cultured human hair follicles. This catagen induction probably occurs at least in part via upregulation of the recognized catagen-stimulatory growth factor TGF-beta2.

Cholera Toxin Potentiates Influences of IFN-γ Through Activation of NF-κB and Release of Tumor Necrosis Factor-α

Cholera toxin (Ctx) is a potent adjuvant in the mucosal immune system. Previous studies have indicated that Ctx induces intestinal interferon-gamma (IFN-gamma) production and that adjuvant properties require activation of the IFN-gamma receptor (IFNGR). Thus, we hypothesized that Ctx potentiates IFN-gamma responses in intestinal epithelia. Initial studies suggested that Ctx enhances IFN-gamma-mediated barrier disruption in cultured intestinal epithelia. This response was attributable to liberation of a soluble mediator into conditioned supernatants, subsequently identified as tumor necrosis factor-alpha (TNF-alpha). Extensions of these findings revealed that the Ctx A subunit induces transcriptional activation of proinflammatory genes in addition to TNF-alpha (interleukin-8 [IL- 8], intracellular adhesion molecule-1 [ICAM-1], and IL-6) and that such transactivation is mediated by the transcriptional regulator NF-kappaB. We conclude that Ctx elicits a proinflammatory phenotype in intestinal epithelia and that potentiation of IFN-gamma-mediated barrier disruption by TNF-alpha may contribute to the overall adjuvant properties of Ctx.

Expression of a dominant negative IFN‐γreceptor on mouse oligodendrocytes

The interferon-gamma (IFN-gamma) receptor is expressed by all nucleated cells, and binding of its cognate ligand, IFN-gamma, induces a wide variety of biological functions. Transgenic mice expressing a dominant negative IFN-gamma receptor 1 (IFN-gammaR1DeltaIC) on oligodendrocytes under control of the myelin proteolipid protein promoter are described. The mRNA encoding the transgene was only detected in the nervous system and protein expression was confirmed by immunohistochemistry. Transgenic receptor expression does not alter myelination and the mice exhibited no clinically apparent phenotype. Consistent with the restricted nervous system expression of the transgene, no alterations in peripheral immune responses were detected. Flow cytometric analysis demonstrated constitutive expression of both the IFN-gammaR1DeltaIC transgene and the endogenous IFN-gamma receptor 2 at high levels on oligodendrocytes derived from the transgenic mice. These oligodendrocytes also exhibited decreased STAT1 phosphorylation in response to IFN-gamma, confirming dominant negative transgene function. Transgenic mice in which oligodendrocytes have a diminished ability to respond to IFN-gamma showed delayed virus clearance from oligodendroglia compared with wild-type mice. This model will allow evaluation of oligodendrocyte responses to this critical cytokine during CNS inflammation.

Screening of retroviral cDNA libraries for factors involved in protein phosphorylation in signaling cascades

We report a novel approach that allows for the rapid identification of proteins mediating phosphorylation in signaling cascades after specific stimulation. As a proof of concept, we used the interferon- γ (IFN-γ)-induced phosphorylation of signal transducer and activator of transcription-1 (Stat1) in a human promonocytic cell line, which was previously shown to be deficient in this signaling pathway. By using retroviral cDNA expression libraries, transduced selector cells expressing single cDNAs were stimulated with IFN-γ, then fixed, permeabilized and stained intracellularly for phospho-Stat1 levels. Cells responding to the stimulation, which showed increased levels of phosphorylated Stat1, were enriched using fluorescence activated cell sorting (FACS). Genomic DNA was isolated from the enriched cell population and served as a template for cDNA amplification using PCR. After only one round of selection, a cDNA encoding the β-chain of the IFN-γ receptor (IFNGR2) was obtained and demonstrated to restore the selected phenotype. The approach now allows one to use phospho-events as reporters, alone or in tandem, for screening of signaling network states, overcoming a prior need to rely on the reporter genes that are often only indirect measures of phenotypes desired in a screen.

Defects in the interferon‐γ and interleukin‐12 pathways

The interferon-gamma (IFN-gamma)/interleukin-12 (IL-12) pathway is a pivotal player in the immune system and is central to controlling mycobacterial infections. We highlight the most recent and relevant advances in understanding this pathway and their repercussions on basic and clinical science. Human mutations in IFN-gamma receptor-1 (IFN-gammaR1), IFN-gammaR2, IL-12p40, IL-12 receptor-beta1, signal transducer and activator of transcription-1, and nuclear factor-kappaB essential modulator are analyzed in the context of genetic susceptibility to mycobacterial diseases. A diagnostic and therapeutic approach is described. The IFN-gamma/IL-12 pathway is central in immune control of both environmental and autochthonous challenges, as reflected in human mutations and animal models. Besides being crucial for mycobacterial control, the IFN-gamma/IL-12 pathway is also involved in the pathogenesis of autoimmune disease as well as tumor development and control. Genotype-phenotype correlations have been established for certain genes in this pathway, some of which have therapeutic implications.

Involvement of interferon-? and its receptor in the activation of astrocytes in the mouse hippocampus following entorhinal deafferentation

The activation of glial cells has been thought to be a universal and important reaction to trauma and pathology in the mammalian central nervous system. The mechanism of glial activation is not completely clear to date, but numerous cytokines have been demonstrated to effectively influence the process in vitro and in vivo. Here we reported the axotomy-induced upregulation of interferon-gamma (IFN-gamma) receptor mRNA in the mouse hippocampus following transections of the entorhinal afferents. Northern blot analysis showed that the transcripts of IFN-gamma receptor were upregulated in a transient manner in the deafferented mouse hippocampus. In situ hybridization confirmed the temporal upregulation of IFN-gamma receptor mRNA specifically in the denervated areas of the mouse hippocampus, which showed that the expression of IFN-gamma receptor mRNA rose slightly at 2 days postlesion, increased remarkably at 3 days postlesion, nearly reached the maximum at 7 days postlesion, and almost returned to control levels at 15 days postlesion. Double labeling further proved that the upregulated IFN-gamma receptor mRNA was confined to reactive astrocytes. At 2 and 3 days postlesion, we also observed the expression of IFN-gamma mRNA by a small number of cells in the denervated areas. We noted that the upregulation of both IFN-gamma and its receptor expression coincided spatiotemporally with astroglial activation, suggesting the potential involvement of IFN-gamma and its receptor in the activation process of astrocytes in the hippocampus following entorhinal deafferentation.

Endometriotic cells are resistant to interferon-γ-induced cell growth inhibition and apoptosis: a possible mechanism involved in the pathogenesis of endometriosis

In order to evaluate the involvement of cell proliferation and apoptosis in the pathogenesis of endometriosis, we investigated the effects of interferon-gamma (IFN-gamma) on cell growth inhibition and apoptosis of cultured ovarian endometriotic cyst stromal cells (ECSC), eutopic endometrial stromal cells with endometriosis (ESCwE) and normal endometrial stromal cells (NESC) by modified methylthiazoletetrazolium assay, 5-bromo-2'-deoxyuridine incorporation assay and internucleosomal DNA fragmentation assay. The expression of apoptosis-related molecules and IFN-gamma receptor 1 was also examined in ECSC, ESCwE and NESC using western blot analysis. IFN-gamma significantly inhibited cell proliferation and DNA synthesis of ESCwE and NESC, and induced apoptosis of these cells. In contrast, IFN-gamma did not show apparent effects on the viable cell number, DNA synthesis, or apoptosis of ECSC. An up-regulated expression of Bcl-2 and Bcl-X(L) proteins was observed in ECSC in comparison with ESCwE and NESC, whereas the levels of Bax, Bad, Fas and Fas ligand proteins in ECSC were similar to those in ESCwE and NESC. IFN-gamma receptor 1 expression was detected in ECSC, ESCwE and NESC. Enhanced expression of anti-apoptotic molecules in the ectopic endometrial cells may contribute to the development of endometriosis by conferring resistance to cytokine-induced apoptosis and increasing the chance that these cells will survive and implant outside the uterus. Further investigations on the regulation of cell proliferation in both the endometriotic and the normal endometrium may be important for the elucidation of the pathogenesis of endometriosis.

Defective CD4+CD25+ regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis, counter-regulated by endogenous IFN-γ

Mice with a deficiency in IFN-γ or IFN-γ receptor (IFN-γR) are more susceptible to collagen-induced arthritis (CIA), an experimental autoimmune disease that relies on the use of complete Freund's adjuvant (CFA). Here we report that the heightened susceptibility of IFN-γR knock-out (KO) mice is associated with a functional impairment of CD4+CD25+ Treg cells. Treatment of wild-type mice with depleting anti-CD25 antibody after CFA-assisted immunisation with collagen type II (CII) significantly accelerated the onset of arthritis and increased the severity of CIA. This is an indication of a role of Treg cells in the effector phase of CIA. IFN-γR deficiency did not affect the number of CD4+CD25+ T cells in the central and peripheral lymphoid tissues. In addition, CD4+CD25+ T cells isolated from naive IFN-γR KO mice had a normal potential to suppress T cell proliferation in vitro. However, after immunisation with CII in CFA, the suppressive activity of CD4+CD25+ T cells became significantly more impaired in IFN-γR-deficient mice. Moreover, expression of the mRNA for Foxp3, a highly specific marker for Treg cells, was lower. We further demonstrated that the effect of endogenous IFN-γ, which accounts for more suppressive activity in wild-type mice, concerns both Treg cells and accessory cells. Our results demonstrate that the decrease in Treg cell activity in CIA is counter-regulated by endogenous IFN-γ.

Role of Tyrosine 441 of Interferon-γ Receptor Subunit 1 in SOCS-1-mediated Attenuation of STAT1 Activation

Suppressor of cytokine signaling (SOCS)-1, the key negative regulator of interferon (IFN)-gamma-dependent signaling, is induced in response to IFNgamma. SOCS-1 binds to and inhibits the IFNgamma receptor-associated kinase Janus-activated kinase (JAK) 2 and inhibits its function in vitro, but the mechanism by which SOCS-1 inhibits IFNgamma-dependent signaling in vivo is not clear. Upon stimulation, mouse IFNgamma receptor subunit 1 (IFNGR1) is phosphorylated on several cytoplasmic tyrosine residues, and Tyr(419) is required for signal transducer and activator of transcription (STAT) 1 activation in mouse embryo fibroblasts. However, the functions of the other three cytoplasmic tyrosine residues are not known. Here we show that Tyr(441) is required to attenuate STAT1 activation in response to IFNgamma. Several tyrosine to phenylalanine mutants of IFNGR1, expressed at normal levels in stable pools of IFNGR1-null cells, were analyzed for the phosphorylation of STAT1 during a 48-h period, and antiviral activity in response to IFNgamma was also measured. Stronger activation of STAT1 was observed in cells expressing all IFNGR1 variants mutated at Tyr(441), and, consistently, stronger antiviral activity was also observed in these cells. Furthermore, constitutive overexpression of SOCS-1 inhibited IFNgamma-dependent signaling only in cells expressing IFNGR1 variants that included the Tyr(441) mutation. Mutation of Tyr(441) also blocked the ability of SOCS-1 to bind to IFNGR1 and JAK2 in response to IFNgamma and the normal down-regulation of STAT1 activation and antiviral activity. These results, together with data from the literature, suggest a model in which, in response to IFNgamma, phosphorylation of Tyr(441) creates a docking site for SOCS-1, which then binds to JAK2 within the receptor-JAK complex to partially inhibit JAK2 phosphorylation. Furthermore, the virtually complete blockade of STAT1 phosphorylation by overexpressed SOCS-1 in this experiment suggests that the binding of SOCS-1 to Tyr(441) also blocks the access of STAT1 to Tyr(419) and that this effect may be the principal mechanism of inhibition of downstream signaling.

Experimental reproduction of proliferative enteropathy and the role of IFN-gamma in protective immunity against Lawsonia intracellularis in mice

Proliferative enteropathy was reproduced in IFN-gamma receptor knockout (IFN-gamma R-) mice by experimental infection with Lawsonia intracellularis (L. intracellularis). The cecum and the colon of the infected mice were evidently enlarged 2 weeks post infection. The presence of L. intracellularis was identified in the stool and the cecum of the mice after infection. However, high levels of IFN-gamma were detected in the sera of the infected mice 2 weeks PI. These data indicated that the IFN-gamma produced in the infected mice should have been utilized by it's receptor to elicit protective immune responses against L. intracellularis infections.

Omega‐3 Polyunsaturated Fatty Acids Impair In Vivo Interferon‐γ Responsiveness via Diminished Receptor Signaling

A high intake of omega-3 polyunsaturated fatty acids (n-3 PUFAs) in mice causes impaired host resistance to Listeria monocytogenes. We wished to determine the role of interferon (IFN)-gamma signaling in this increased disease susceptibility. A feeding trial was conducted with mice unable to produce IFN-gamma (IFN-gamma KO); we provided exogenous recombinant IFN-gamma during L. monocytogenes challenge. The experimental diets were nutritionally complete and differed only in fat source: lard (devoid of n-3 PUFAs) or menhaden fish oil (rich in n-3 PUFAs). The administration of IFN-gamma significantly enhanced bacterial clearance in IFN-gamma KO mice fed a diet devoid of n-3 PUFAs but had no effect in mice fed a diet rich in n-3 PUFAs. Ex vivo analysis of immune cells showed that n-3 PUFAs did not affect IFN-gamma receptor expression on immune cells. However, on IFN-gamma treatment, the phosphorylation of signal transducer and activator of transcription 1 was significantly reduced in peritoneal macrophages isolated from mice fed n-3 PUFAs. These data suggest that diminished IFN-gamma signaling in murine macrophages is one mechanism by which n-3 PUFAs impair host resistance to L. monocytogenes. To our knowledge, this is the first report of a nutrient affecting IFN-gamma signaling and in vivo responsiveness to this cytokine.

Placental Expression of Interferon-γ (IFN-γ) and Its Receptor IFN-γR2 Fail to Switch from Early Hypoxic to Late Normotensive Development in Preeclampsia

The inability of the mother to switch from T helper cell type 1 (Th1) to Th2 cytokine profiles at the fetal-maternal interface has been proposed as one of the primary causes of miscarriage, intrauterine growth restriction (IUGR), and preeclampsia (PE). The Th1 [interferon-gamma (IFN-gamma), TNF-alpha, and IL-12] and Th2 (IL-4 and IL-10) cytokines have opposite effects on human pregnancy. Leukemia inhibitory factor (LIF) promotes embryo implantation and sustains pregnancy, whereas IFN-gamma and TNF-alpha are detrimental to pregnancy. Both IFN-gamma and LIF are produced by maternal cells and tissues at the fetal-maternal interface, whereas the IFN-gamma receptors (IFN-gamma R1 and IFN-gamma R2) and LIF receptor are abundantly expressed on the surface of placental trophoblasts. The effect of IFN-gamma on T lymphocyte activation is influenced by the relative membrane density of its two receptors, particularly IFN-gamma R2. In this study we report that in PE (25-40 wk gestation) and PE complicated by IUGR, IFN-gamma R2 protein expression is severely down-regulated and is similar to that observed in early placenta (7-10 wk gestation) developing under low O(2) tension. IFN-gamma production was found to be inversely related to the IFN-gamma R2 protein expression, and LIF receptor protein expression in PE mimicked that in early placental development. These results show that in PE, placental trophoblasts fail to establish an early to late switch with respect to IFN-gamma and IFN-gamma R2 expression. This supports the hypothesis that trophoblasts control the polarization of maternal immune effectors and cytokine profiles at the fetal-maternal interface that could be subject to oxidative stress in PE.

Acquisition of multidrug resistance by L1210 leukemia cells decreases their tumorigenicity and enhances their susceptibility to the host immune response.

The use of antineoplastic drugs for cancer treatment is frequently associated with the acquisition of a multidrug-resistant (MDR) phenotype that renders tumoural cells insensitive to antineoplastics. It remains elusive whether the acquisition of the MDR phenotype alters immunological parameters that could influence the cell sensitivity to an eventual host immune response. We report that immunisation of syngeneic mice with gamma-irradiated L1210S (parental line) and L1210R (MDR phenotype) cells results in a significant rejection of subsequently implanted L1210R-based tumours, but not of the L1210S ones. Notably, L1210R tumours display a twofold reduction in vivo proliferative capacity and are less aggressive in terms of mouse survival than their sensitive counterparts. Also, analysis of surface expression of molecules involved in antigen presentation and cytokine activity revealed a slight increase in IFN-gamma receptor expression, a decrease of Fas molecule, and a fourfold up-regulation of MHC class I molecules in L1210R cells. Nonetheless, both cell lines were able to induce a cytotoxic response in syngeneic mice and were equally susceptible to cytotoxicity by splenic cells. Together, these findings indicate that acquisition of drug resistance by L1210 cells is accompanied by pleiotropic changes that result in reduced tumour proliferative capacity and tumorigenicity in syngeneic mice. Hence, immunological studies of MDR tumours may assist in the design of specific therapeutic strategies that complement current chemotherapy treatments.

Re-examination of the Role of Suppressor of Cytokine Signaling 1 (SOCS1) in the Regulation of Toll-like Receptor Signaling

Suppressor of cytokine signaling 1 (SOCS1) is an obligate negative regulator of cytokine signaling and most importantly in vivo, signaling via the interferon-gamma (IFN-gamma) receptor. SOCS1, via its Src homology 2 domain, binds to phosphotyrosine residues in its targets, reducing the amplitude of signaling from cytokine receptors. SOCS1 is also implicated in blocking Toll-like receptor (TLR) signaling in macrophages activated by TLR agonists such as lipopolysaccharide (LPS), thus regulating multiple steps in the activation of innate immune responses. To rigorously test this, we isolated macrophages from Socs1-/- mice on multiple genetic backgrounds. We found no evidence that SOCS1 blocked TLR-activated pathways, endotoxin tolerance, or nitric oxide production. However, Socs1-/-;IFN-gamma-/- mice were extremely susceptible to LPS challenge, confirming previous findings. Because LPS induces IFN-beta production from macrophages, we tested whether SOCS1 regulates IFN-alpha/beta receptor signaling. We find that SOCS1 is required to inhibit IFN-alpha/beta receptor signaling in vitro. Furthermore, the absence of a single allele encoding TYK2, a JAK (Janus kinase) family member essential IFN-alpha/beta receptor signaling, rescued Socs1-/- mice from early lethality, even in the presence of IFN-gamma. We conclude that previous reports linking SOCS1 to TLR signaling are most likely due to effects on IFN-alpha/beta receptor signaling.