Interferon-γ confers resistance to experimental allergic encephalomyelitis

In experimental allergic encephalomyelitis (EAE), T cells infiltrate the central nervous system (CNS) and induce inflammation. These CD4⁺ T cells secrete interferon (IFN)-γ, levels of which correlate with disease severity, and which is proposed to play a key role in disease induction. Many strains of mice are resistant to EAE. We have studied the effect of deletion of IFN-γ on the ability to induce EAE in resistant BALB/c-backcrossed mice. As expected, only 0–6 % of BALB/c or BALB/c-backcrossed mice developed EAE when immunized with myelin basic protein in adjuvant. Strikingly, abrogation of IFN-γ expression by targeted disruption of the IFN-γ gene (GKO mice) converted them to a susceptible phenotype. As many as 71 % of these IFN-γ-deficient mice developed EAE, a frequency comparable to that seen with the susceptible SJL/J strain. In addition, EAE was of unusually high severity in mice lacking IFN-γ. Immunological characteristics of disease in IFN-γ-deficient mice were comparable to those seen in susceptible (SJL/J) mice with EAE, including perivascular infiltration in the CNS and order-of-magnitude increases for both CD3 γ chain and TNF-α mRNA levels in the spinal cord. We thus demonstrate that lack of IFN-γ converts an otherwise EAE-resistant mouse strain to become susceptible to disease. Therefore, in BALB/c mice, IFN-γ confers resistance to EAE.     

Interferon-γ is essential for the development of cerebral malaria

Infection with Plasmodium berghei ANKA (PbA) causes fatal cerebral malaria (CM). While a pathogenic role for tumor necrosis factor (TNF) has been established, we asked whether a disruption of interferon-γ (IFN-γ) signaling would modulate CM. We demonstrate here that IFN-γR-deficient mice are completely protected from CM. PbA-induced release of TNF and up-regulation of endothelial intercellular adhesion molecule (ICAM)-1 expression, recruitment of mononuclear cells, and cerebral microvascular damage with vascular leakage occur only in wild-type mice. Protected mice die at a later time of severe anemia and overwhelming parasitemia. Resistance to CM in IFN-γR-deficient mice is associated with reduced serum TNF levels, reduced interleukin-12 expression in the brain and increased T-helper 2 cytokines. In conclusion, IFN-γ is apparently required for PbA-induced endothelial ICAM-1 up-regulation and subsequent microvascular pathology, resulting in fatal CM. In the absence of IFN-γ signaling, ICAM-1 and TNF up-regulation is reduced; hence, PbA infection fails to cause fatal CM.     

Modulation of interferon-γ receptor during human T lymphocyte alloactivation

Previous work has shown that neutralization of physiologically secreted interferon(IFN)-γ or blockade of its receptor during T lymphocyte activation inhibits both proliferation and cytotoxic T lymphocyte generation, suggesting that IFN-γ plays a crucial role in T lymphocyte induction and differentiation. In this study, the kinetics of the surface expression of the 90-kDa IFN-γ receptor (IFN-γR) was followed during human mixed lymphocyte reaction (MLR) to alloantigens. IFN-γR mRNA is constitutively expressed on resting peripheral blood lymphocytes emerging from nylon wood column (NW-PBL) and its expression increases two- to threefold on alloactivated NW-PBL. IFN-γR protein is poorly expressed on the membrane of resting CD3⁺ cells, but up-modulates after 3-day MLR and sharply down-modulates at day 6. Both the p55 and the p75 chains of interleukin-2 receptor (IL-2R) were shown to up-modulate in parallel with IFN-γR, whereas they were still highly expressed at day 6. After alloactivation, IFN-γ and IL-2 secretion starts at 24 h, peaks at day 3 and decreases just when IFN-γR and IL-2R begin to up-modulate. Proliferation peaks at day 6. Lastly, stimulation with distinct cell populations showed that the intensity of lymphocyte proliferation, IFN-γR membrane up-modulation, and IFN-γ and IL-2 secretion are regulated in a parallel manner, thus suggesting that they are interrelated. Taken as whole these results demonstrate that increased expression of IFN-γR on T lymphocytes can be a critical event during their activation, and strongly support the hypothesis that IFN-γ/IFN-γR interaction provides a signal for its progression.     

Interleukin-12 is required for interferon-γ production and lethality in lipopolysaccharide-induced shock in mice

Several cytokines, in particular tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), have been shown to be responsible for pathological reactions which may lead to shock and death observed in infection with Gram-negative bacteria and in response to endotoxins (lipopolysaccharides, LPS). Priming of mice with the avirulent Bacille Calmette Guérin (BCG) vaccine strain of Mycobacterium bovis increases the sensitivity of mice to the lethal effect of LPS and results in an efficient priming for cytokine production. In response to low doses (1 γg/mouse) of LPS, BCG-primed mice produce interleukin-12 (IL-12) which controls IFN-γ production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to suppress IFN-γ production. However, the concentration of the biologically active IL-12 p70 heterodimer is similar in the serum of both BCG-primed or unprimed mice, reaching levels of 1–3 ng/ml at 3–6 h after LPS injection, whereas IFN-γ production was observed only in BCG-primed mice. The priming effect of BCG on IFN-γ production appears to be mostly due to its ability to increase TNF-α production, which acts as cofactor with LPS-induced IL-12 in inducing IFN-γ production, as shown by the ability of injection of TNF-α and LPS (1 γg/mouse), but not LPS alone, to induce IFN-γ production. However, in addition to TNF-α, other LPS-induced cofactor(s) are required in cooperation with IL-12 to induce optimal IFN-γ production, because co-injection of TNF-α and IL-12, sufficient to induce serum concentrations of both cytokines higher and more persistent than those obtained by injection of LPS, was not sufficient to induce IFN-γ production in vivo. Neutralizing anti-IL-12 antibodies, in addition to inhibiting the in vivo LPS-induced IFN-γ production, also completely protect BCG-primed mice injected with up to 10 μg of LPS from shock-induced death. Thus, IL-12 is required for IFN-γ production and lethality in an endotoxic shock model in mice.     

The effects of a monoclonal antibody to interferon-γ on experimental autoimmune thyroiditis (EAT): prevention of disease and decrease of EAT-specific T cells

CBA/J mice immunized with thyreoglobulin (Tg) develop an experimental autoimmune thyroiditis (EAT) with lymphocytic infiltration of the thyroid glands, autoantibodies to Tg and occurrence of EAT-specific T cells. When these mice were treated for 4 weeks after immunization with 1 mg/week of a monoclonal antibody (mAb) that neutralizes the activity of interferon-γ (IFN) a beneficial effect on the onset of EAT was observed. Characteristic features of EAT were significantly reduced, including the lymphocytic infiltrations of the thyroid glands and the serum levels of autoantibodies to Tg. Moreover, in lymphoid organs, mAb to IFN-γ significantly reduced the percentages of Tg-specific CD8⁺ cells, labeled by the anti-clonotypic mAb AG7. These Tg-specific T cells seem responsible for thyroid damages and disease development, since EAT was simultaneously abrogated. These results show that IFN-γ plays an essential role in the pathophysiology of EAT and suggest the possibility to treat autoimmune thyroid diseases with mAb to IFN-γ or drugs able to antagonize the production and/or the action of this cytokine.     

Mercuric chloride down-regulates T cell interferon-γ Production in Brown Norway but not in Lewis rats; role of glutathione

Injection of a low dose of mercuric chloride into Brown Norway (BN) rats caused a marked decrease in the concanavalin A (ConA)-induced generation of interferon-γ-producing cells (IFN-γ pc) in spleen cell cultures prepared 1 h after mercury administration. A second injection 48 h later caused a further diminution of IFN-γ pc down to 30% of the number generated in splenocyte cultures of phosphate-buffered saline (PBS)-injected controls. Injection of Lewis rats with either one or two doses of HgCl₂ revealed no inhibitory effect on splenic IFN-γ production. The presence of the reduced form of glutathione (GSH) in the culture medium was found to be essential in these experiments. In the absence of GSH there was an overall 20-fold reduction of the number of IFN-γ pc in splenocyte cultures of normal or PBS-injected rats, which was further reduced to a 60- to 70-fold-lower level in cultures of rats exposed to HgCl2. This mercury-mediated extra reduction could be fully reversed with an excess (2 mM) of GSH in Lewis but not in BN splenocyte cultures. Since the bivalent Hg²⁺ ion is known to bind to and inactivate sulfydryl groups of proteins and low molecular weight thiols, most notably GSH, we investigated a possible role for thiols in IFN-γ production. It was found that the generation of IFN-y pc in normal BN and Lewis splenocyte cultures was strongly dependent on GSH or its precursor cysteine in the culture medium. Other thiol compounds were also effective but disulfides were completely inactive. Depletion of intracellular GSH in ConA-stimulated splenocytes by buthionine sulfoximide (BSO), an inhibitor of de novo GSH biosynthesis, strongly inhibited the generation of IFN-γ pc. The inhibitory effect of BSO was not abolished by the addition of interleukin-2 (IL-2), but was mimicked with antibodies directed to the IL-2 receptor. The data stress the importance of GSH in the enhancement of IL-2-mediated IFN-γ production and are most consistent with a model in which mercury interferes with T cell IFN-γ production by affecting the intracellular availability of GSH. The strain-specific susceptibility to mercury-mediated inhibition of IFN-γ production is discussed.     

Biphasic effect of interferon-γ in murine collagen-induced arthritis

Interferon-γ (IFN-γ) exerts both enhancing and suppressing influences on collagen-induced arthritis (CIA), depending on the route and protocol of administration. To study the role of IFN-γ on the autoimmune process of CIA, we treated DBA/1 mice with two different rat monoclonal antibodies (mAb) to murine IFN-γ. Treatments, given twice weekly for 4 weeks, consisted of intraperitoneal injections of either mAb. In early treatments, starting from the day of immunization with type II collagen (CII), the severity of arthritis was reduced in both groups of anti-IFN-γ-treated mice compared with control groups. Moreover, anti-CII antibody levels decreased in the sera of these mice. CIA was also down-regulated in mice treated from days 14 or 28 post immunization. In contrast, late treatments with anti-IFN-γ mAb either induced aggravating effects, or did not affect the course of the disease. On the other hand, administration of high doses (8 × 10⁴ U three times/week) of rat recombinant IFN-γ exerted a transient increase of CIA severity. These findings suggest that IFN-γ may play a critical role during both the induction and the course of CIA, first enhancing the immune response, and then regulating the arthritis process.     

Expression of pro-inflammatory cytokines by TCRαβ+ T and TCRγδ+ T cells in an experimental model of colitis

An inflammatory bowel disease (IBD) comparable to human ulcerative colitis is induced upon transfer of T cell-depleted wild-type (F1) bone marrow into syngeneic T cell-deficient (tgε26) mice (F1 → tgε26). Previously we have shown that activated CD4⁺ T cells predominate in transplanted tgε26 mice, and adoptive transfer experiments verified the potential of these cells to cause disease in immunodeficient recipient mice. Using flow cytometry for the detection of intracellular cytokine expression, we demonstrate in the present study that large numbers of CD4⁺ and CD8⁺ TCRαβ⁺ T cells from the intraepithelial region and lamina propria of the colon of diseased, but not from disease-free mice, produced interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Large numbers of T cells from peripheral lymphoid tissues of these animals also expressed IFN-α and TNF-α, but few expressed interleukin-4, demonstrating g strong bias towards Th1-type T cell responses in these animals. TCRγδ⁺ T cells, typically minor constituents of the inflammatory infiltrate of the colon in F1 → tgε26 mice, also expressed IFN-γ at a high frequency upon CD3 stimulation. In light of these findings we examined the potential involvement of TCRγδ⁺ T cells by testing their ability to induce colitis in tgε26 mice. We report here that tgε26 mice transplanted with T cell-depleted bone marrow from TCRα^null and TCRβ^null animals developed IBD. Furthermore, disease in these mice correlated with the development of peripheral and colonic TCRαδ⁺ T cells capable of IFN-γ production. These results suggest that IFN-γ may be a common mediator of IBD utilized by pathogenic T cells of distinct phenotype.     

Administration of anti-interleukin-2 receptor α antibody in vivo induces localized autoimmune disease

Neonatal thymectomy (Tx) of mice at day 3 after birth (Tx-3), but not day 7 (Tx-7), induces organ-localized autoimmune diseases such as oophoritis and gastritis. Lesions in Tx-3 mice can be prevented by injection of splenic CD4⁺ cells from syngeneic normal mice, and this CD4⁺ population with suppressor activity is activated extrathymically by self antigens. Since it is speculated that these CD4⁺ T suppressor cells (Ts) express the interleukin-2 receptor (IL-2R) as an activated T cell population, an attempt was made to eliminate these Ts from the developing immune system of Tx-7 mice and normal mice by i.p. injection of anti-IL-2Rα monoclonal antibodies. Interestingly, organ-localized autoimmune disease with quite similar characteristics to those observed after neonatal Tx developed in not only Tx-7 mice, but also normal mice. The results thus indicate that CD4⁺ cells expressing IL-2Rα play an important role, as Ts in the periphery, in maintaining immune tolerance.     

Interferon-γ and interleukin-4 reciprocally regulate the production of monocytes/macrophages and neutrophils through a direct effect on committed monopotential bone marrow progenitor cells

We studied the direct effects of interferon-γ (IFN-γ) in single cell colony assays of CD34⁺HLA-DR++ bone marrow progenitor cells stimulated by either granu-locyte-colony-stimulating factor (G-CSF), interleukin(IL)-3, granulocyte/macro-phage-colony-stimulating factor (GM-CSF), combinations of these CSF or medium conditioned by the 5637 human bladder carcinoma cell line. In this culture system IFN-γ stimulated monocytic colonies (CFU-M) no matter which CSF or CSF combination was used to support them and inhibited granulocytic colonies (CFU-G) if they were generated in the presence of G-CSF. IL-4 antagonized the myelopoietic effects of IFN-γ: the IFN-γ induced suppression of G-CSF-supported CFU-G, as well as the stimulation of CFU-M, were reversed by IL-4. In all cultures, IFN-γ had a limited, but statistically non-significant, inhibitory effect on CFU-GM, which was not affected by the presence of IL-4. These data show that IFN-γ and IL-4 reciprocally regulate the generation of myeloid cells involved in humoral (neutrophils) and cellular (macrophages) immune responses through a direct effect on monopotential myeloid progenitor cells.     

Chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice: enhancement by monoclonal antibodies against interferon-γ

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory and demyelinating disorder of the central nervous system. Depending on the experimental conditions, it takes an acute monophasic or a chronic relapsing-remitting course. We have previously reported that the incidence and severity of acute EAE in mice are reduced by administration of interferon (IFN)-γ and augmented by treatment with neutralizing antibodies against IFN-γ. Here, we investigated the role of IFN-γ in chronic relapsing models of EAE (CREAE) in SJL/J and Biozzi ABH mice. Spontaneous relapses in Biozzi mice as well as induced relapses in SJL/J mice were facilitated by administration of neutralizing monoclonal antibody (mAb) against IFN-γ in the disease-free interval. The enhancing effect of anti-IFN-γ mAb given before and during the primary attack did not carry over to the relapses. However, early administration of IFN-γ in Biozzi mice, which developed spontaneous relapses in a high proportion, provided partial protection not only against the first attack, but also against subsequent relapses. Administration of exogenous IFN-γ during the remission phase provided some protection against subsequent relapses. These results indicate that in both types of relapses, IFN-γ is produced and does provide a certain degree of protection against disease progression.     

Specific expression of surface interferon-γ on interferon-γ producing T cells from mouse and man

Interferon (IFN)-γ is a potent immunoregulatory protein secreted by CD4⁺ and CD8⁺ T cells and by natural killer cells. Here, we show that IFN-γ is specifically displayed at a low concentration on the cell surface of those activated T cells from mouse and man which express IFN-γ. It is transiently expressed on the cell surface with kinetics similar to those of intracellular IFN-γ expression. Detectable surface IFN-γ is not expressed by activated T helper (Th) cells producing other cytokines but which do not express IFN-γ. Thus, surface IFN-γ is the first available marker for live T lymphocytes expressing IFN-γ, e.g. Th1 cells.     

Induction of macrophage nitric oxide production by interferon-γ and tumor necrosis factor-α is enhanced by interleukin-10

Interleukin-10 (IL-10) has been reported to inhibit nitric oxide (NO) synthesis and microbicidal activity of interferon-γ (IFN-γ)-stimulated macrophages (MΦ) by preventing the secretion of tumor necrosis factor-α (TNF-α) which serves as an autocrine activating signal. We have examined the effects of recombinant IL-10 on the capacity of IFN-γ together with exogenous TNF-α to induce NO synthesis by bone marrow-derived MΦ. Under these conditions and in contrast to its reported deactivating potential, IL-10 strongly enhanced NO synthesis measured as nitrite (NO) release (half maximal stimulation at approximately 10 U/ml). IL-10 further increased NO production by MΦ stimulated in the presence of optimal concentrations of prostaglandin E₂, a positive modulator of MΦ activation by IFN-γ/TNF-α. Increased steady state levels of NO synthase mRNA were observed in 4-h IFN-γ/TNF-α cultures and enhanced NO release was evident 24 h but not 48 h after stimulation. These results suggest that the effects of IL-10 on MΦ function are more complex than previously recognized.     

T helper type 1 development of naive CD4+ T cells requires the coordinate action of interleukin-12 and interferon-γ and is inhibited by transforming growth factor-β

It was observed in vitro and in vivo that both interferon (IFN)-γ and interleukin (IL)-12 can promote the development of T helper type 1 (T_H1) cells. Since IL-12 was shown to be a costimulator for the production of IFN-γ by T or natural killer (NK) cells, IL-12 might play only an indirect role in T_H1 differentiation by providing IFN-γ which represents the essential differentiation factor. Using anti-CD3 monoclonal antibody (mAb) for activation of naive CD4⁺ T cells in the absence of accessory cells we could demonstrate that costimulation by IFN-γ alone results only in marginal T_H1 development. Similarly, IL-12 in the absence of IFN-γ is only a poor costimulator for inducing differentiation towards the T_H1 phenotype. Our data indicate that both cytokines are required to allow optimal T_H1 development and that IL-12 has a dual role, it promotes differentiation by direct costimulation of the T cells and also enhances the production of IFN-γ which serves as a second costimulator by an autocrine mechanism. Another cytokine that was reported to favor T_H1 differentiation in certain experimental systems is transforming growth factor (TGF)-β. With naive CD4⁺ T cells employed in this study TGF-β strongly inhibited the production of IFN-γ triggered by IL-12 as well as the IL-12-induced T_H1 development. When TGF-β was combined with anti-IFN-γ mAb for neutralization of endogenous IFN-γ the T_H1-inducing capacity of IL-12 was completetly suppressed.     

Interferon-γ- and interleukin-4-producing T cells can be primed on dendritic cells in vivo and do not require the presence of B cells

The antigen-presenting cell (APC) requirements for the in vivo induction of Th1-and Th2-type responses were investigated using a severe combined immunodeficiency (SCID)mouse chimera model. SCID mice adoptively transferred with either T cells [SCID(T)] or T + B cells [SCID(T + B)] and immunized with antigen in adjuvant were able to generate antigen-specific T cells which could produce both interferon (IFN)-γ and interleukin (IL)-4 upon in vitro restimulation. This suggests that B cell APC are not necessary for the priming of either IFN-γ- or IL-4-producing T cells in vivo. The ability of different APC to activate Th2-dependent effector mechanisms was also investigated. SCID(T) and SCID(T + B) mice were infected with the nematode parasite Nippostrongylus brasiliensis and analyzed for the development of IL-5-dependent peripheral blood eosinophilia. Following infection both SCID(T) and SCID(T + B) mice generated similar numbers of peripheral blood eosilnophils, suggesting that similar amounts of IL-5 had been produced. Therefore, B cell APC are also not required for the in vivo activation of Th2 cells to lymphokine production. To establish more precisely which APC prime T cells to produce IFN-γ and IL-4, normal mice were immunized by injection of syngeneic splenic dendritic cells which had been pulsed with antigen in vitro. T cells from these immunized mice were able to produce good IFN-γ and IL-4 responses upon in vitro restimulation with specific antigen; therefore, dendritic cells appear to be sufficient APC for the in vivo priming of both IFN-γ- and IL-4-producing T cells.