Heparin inhibits the antiparasitic and immune modulatory effects of human recombinant interferon-γ

Interferon-γ (IFN-γ) is a potent immune regulatory cytokine and is, in addition, involved in the induction of antiparasitic effector mechanisms in different cell types. The first step of IFN-γ action is its binding to a specific receptor. Furthermore, it has been shown that IFN-γ binds with a great affinity to the heparin-like structure of heparan sulfate, which is localized in basement membranes and on cell surfaces. In this study, we analyze the effect of heparin and heparan sulfate on three different IFN-γ-mediated activities inducible in human glioblastoma cells (87HG31 and 86HG39). We find firstly that heparin is able to inhibit IFN-γ-mediated induction of major histocompatibility complex (MHC) class II antigen expression on 87HG31 cells, an effect which can be abrogated by protamine. Secondly, we show that heparin inhibits the IFN-γ-induced toxoplasmostasis within 86HG39 cells in a dose-dependent fashion, and thirdly that heparin inhibits the IFN-γ-mediated induction of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase. In contrast to IFN-γ-induced effects, the activity of other cytokines, such as interleukin (IL)-1, IL-2 and IL-6, is not influenced by heparin. The possible mechanism of heparin-induced inhibition of IFN-γ is discussed.     

A third interferon-γ-induced subunit exchange in the 20S proteasome

The 20S proteasome is a protease complex of functional importance for antigen processing. Two of the 14 proteasome subunits, δ and MB1, can be replaced by the major histocompatibility complex (MHC)-encoded and interferon-γ (IFN–γ)-inducible subunits LMP2 and LMP7, respectively. LMP2 and LMP7 alter the cleavage site specificity of the 20S proteasome and are required for the efficient generation of T cell epitopes from a number of viral proteins and for optimal MHC class I cell surface expression. We compared the 20S proteasome subunit pattern from IFN-γ-induced and non-induced mouse fibroblasts on two-dimensional gels and identified a third subunit exchange by microsequencing: the non-MHC-encoded subunit MECL-1 is induced by IFN-γ and replaces a so-far barely characterized β subunit designated ‘MC14’. In analogy to LMP2 and LMP7, MECL-1 may be functional in MHC class I-restricted antigen presentation.     

Interleukin-12 induces interferon-γ-dependent switching of IgG alloantibody subclass

Interleukin-12 (IL-12) is a key immunoregulatory cytokine which promotes cell-mediated immunity. Its influence on the humoral immune response is less clearly defined. In this study, the effect of systemic IL-12 treatment on the T cell-dependent humoral immune response in the rat was examined using an experimental system in which PVG-RT1^u congenic rats were immunized with class I A^a alloantigen in the form of a blood transfusion from the recombinant PVG.R8 rat strain. Administration of IL-12 following allo-immunization augmented the humoral immune response as determined by increased levels of cytotoxic anti-class I major histocompatibility complex antibodies. However, the effect of IL-12 on individual IgG isotypes was highly selective. Levels of allospecific antibodies of the IgG2b and IgG2c subclasses were markedly increased, whereas IgG1 alloantibody levels were profoundly reduced. The observed alterations in alloantibody response were dependent, in large part, on the stimulatory effect of IL-12 on interferon (IFN)-γ production by T lymphocytes and natural killer cells, since they were abrogated by co-administration of neutralizing anti-IFN-γ monoclonal antibody following alloantigen immunization.     

Human and rodent interferon-γ as a growth factor for Trypanosoma brucei

An example for the bidirectional exchange of activating signals between a pathogen and immunocompetent cells in the host is presented. Trypanosoma brucei, which include subspecies that cause African sleeping sickness, secrete a molecule that triggers lymphocytes to produce interferon (IFN)-γ. We now report that proliferation of T. brucei is stimulated in axenic cultures by IFN-γ. The growth-enhancing effect on the pathogen is inhibited by anti-IFN-γ receptor (R) antibodies and does not occur after exposure to other cytokines, i.e. IFN-α, IFN-β and tumor necrosis factor (TNF)-α. While rodent-pathogenic T. brucei strains are stimulated by rat IFN-γ, human pathogenic strains are more potently stimulated by human IFN-γ. Rat and human IFN-γ can partially block each others effects. Mice with disrupted IFN-γ genes have reduced parasitemia and prolonged survival, while the outcome is reversed in mice that lack the IFN-γR gene.     

Nitric oxide-mediated suppression of T cell responses during Trypanosoma brucei infection: soluble trypanosome products and interferon-γ are synergistic inducers of nitric oxide synthase

African trypanosome infections result in lymphocyte unresponsiveness and anemia in the mammalian host. In murine infections, these effects are mediated by suppressor macrophages releasing nitric oxide (NO). We investigated the mechanism of activation of macrophages to produce NO during trypanosomiasis in vitro. A soluble component of trypanosome lysates induced NO synthesis in peritoneal macrophage cultures only when the macrophages were co-stimulated with interferon-gamma (IFN-γ). The macrophage-activating factor was also released in a soluble form by live bloodstream-form trypanosomes, but not procyclic trypanosomes. When splenocyte cultures were exposed to IFN-γ and trypanosomes, an NO-dependent suppression of T cell proliferation occurred. This is similar to the suppression observed in the spleens of trypanosome-infected mice, suggesting that a combination of trypanosome-released macrophage-activating factors and IFN-γ are a trigger of immune dysfunction in trypanosomiasis.     

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.     

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.     

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.     

Effect of interferon-γ on antigen processing in human monocytes

The effect of interferon-γ (IFN-γ) on the ability of human monocytic cells to process exogenous (major histocompability complex class II) antigens was investigated. The processing (i.e. protein degradation) of antigens that were internalized via Fcγ receptor (FcγR) was followed for various times after treatment of cells with IFN-γ. THP-1 cells that had been treated with IFN-γ for 4 h degraded antigen, internalized as an immune complex, at an enhanced rate. After 24 h of IFN-γ treatment the rate of processing was similar to untreated cells. Unexpectedly, in cells which had been treated for 48–72 h there was a significant decrease in the rate of processing of the exogenous antigen. These effects were not due to changes in the rate of internalization of immune complex. The inhibition of the rate of processing was independent of the type of antigen, was dependent on the dose of IFN-γ, and also occurred with normal human peripheral monocytes. Analysis of the degraded peptides by high-pressure liquid chromatography indicated that some of the peptides generated in the IFN-γ-treated cells were both quantitatively and qualitatively different from the peptides generated in untreated cells. These data suggest that IFN-γ modulates the way in which antigens, internalized through Fc receptors as immune complexes, are processed. Additionally, the results imply that decreases in the rate of antigen processing may lead to more efficient antigen presentation.     

Apoptosis of cultured mouse luteal cells induced by tumor necrosis factor-α and interferon-γ

Background: Macrophages and T lymphocytes have been identified in the regressing corpus luteum, and they are thought to participate in structural luteolysis (destruction and removal of luteal cells). Since these cells produce cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), we investigated the effects of these two cytokines on death of luteal cells in vitro. Methods: Mouse luteal cells were cultured in serum-free medium with TNF-α at 0,500,1,000,3,000, or 5,000 U/ml in the presence or absence of IFN-γ at 1,000 U/ml for 3 or 6 days. Then, for estimation of the actions of these cytokines on induction of luteal cell death, we determined the number of viable cells, the percentage of fragmented DNA in total DNA extracted from cultured cells, and the percentage of cells with fragmented DNA in their nuclei by the trypan blue exclusion test, the sensitive micromethod for DNA assay, and the in situ DNA 3′ end labeling method, respectively. DNA fragmentation was also analysed by agarose gel electrophoresis, and cultured cells were examined by electron microscopy. Results:On day 3 of culture, IFN-γ alone at 1,000 U/ml or TNF-α alone at 500–5,000 U/ml did not decrease the number of viable cells, but a combination of IFN-γ (1,000 U/ml) and TNF-α (5,000 U/ml) did. On day 6, IFN-γ alone at 1,000 U/ml or TNF-α alone at 500, 1,000 and 3,000 U/ml did not decrease the number of viable cells, whereas TNF-α alone at 5,000 U/ml did, and combinations of IFN-γ and TNF-α at 1,000, 3,000, and 5,000 U/ml decreased the number of viable cells in proportion to the concentration of TNF-α. On days 3–6 of culture, combinations of IFN-γ and TNF-α that decreased the number of viable cells also increased the percentages of fragmented DNA in total DNA of cultured luteal cells and the percentages of luteal cells with fragmented DNA in their nuclei. Agarose gel electrophoresis of fragmented DNA showed a ladder-like pattern, and electron microscopic examination showed luteal cells with the characteristics of apoptosis. Conclusions: The presence of IFN-γ modulates the ability of TNF-α to induce a reduction in the number of viable cells, although TNF-α alone at high concentrations can induce a reduction in the number of viable cells. © 1995 Wiley-Liss, Inc.     

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.     

Comparison of the effects of interleukin-1α, interleukin-lβ and interferon-γ-inducing factor on the production of interferon-γ by natural killer

Interferon-γ inducing factor (IGIF) is a recently identified cytokine which stimulates the production of interferon-γ (IFN-γ) by T cells and enhances natural killer (NK) cell cytolytic activity. Protein fold recognition, structure prediction and comparative modeling have revealed that IGIF is a member of the interleukin (IL)-1 cytokine family and has prompted the designation IL-1γ. Here we report functional similarities between members of the IL-1 family by comparing the effects of IL-1α, IL-1β and IGIF on NK cell production of IFN-γ. All three IL-1 types enhanced NK cell production of IFN-γ when induced by IL-2 or IL-12, although at high concentrations (>10 ng/ml), IGIF was five- to tenfold more potent than IL-1α or IL-1β. This effect correlated with enhanced levels of mRNA for IFN-γ when NK cells were stimulated with IGIF plus IL-12. In contrast to IL-12 and IL-2, the ability of IGIF to stimulate NK cell production of IFN-γ was not increased by IL-1α or IL-1β. The ability of IGIF to enhance IFN-γ production was independent of the type I and type II IL-1 receptors or the IL-1R accessory protein. Together, these results identify IGIF as a potent stimulator of NK cell production of IFN-γ and demonstrate that the effect of IGIF on NK cell production of IFN-γ is similar to that of IL-1α and IL-1β but distinct from that of IL-12.     

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.     

Growth requirements for avian γδ T cells include exogenous cytokines,receptor ligation and in vivo priming

These studies analyze growth requirements for the normal γδ T cell population in peripheral lymphoid tissues. Avian γδ T cells can respond well to T cell mitogens in the presence of αβ T cells, but our studies indicate that they do not grow well alone. Exogenous growth factors were required in order for γδ T cells to proliferate in response to receptor ligation by anti-T cell receptor antibodies or other T cell mitogens. Interleukin-2 was implicated as one of the necessary growth factors that the γδ cells cannot produce adequately on their own. The response to dual stimulation (receptor ligation plus exogenous T cell factors) was attributable to a discrete subpopulation of γδ T cells that could be identified by their cell surface CD8, major histocompatibility complex class II expression and relative increase in cell size. Conversely, non-responsive γδ T cells did not exhibit these activation markers. These observations suggest a physiological basis for the relatively late appearance of γδ T cells in inflammatory responses and their failure as a population to match the growth potential of αβ T cells. More importantly, the results imply that the biological role of γδ T cells must be understood within the context of their interaction with αβ T cells.