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.     

Interferon-γ and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression

Interferon-γ (IFN-γ) and interleukin-4 (IL-4) are mutually antagonistic cytokines that stimulate CD4⁺ T cells to develop into either Th1 or Th2 cells. One feature of Th2 differentiation in mice is the loss of IL-12-induced Jak2 and Stat4 activation, which is accompanied by the inability to produce IFN-γ in response to IL-12. In this report, we show that freshly isolated human T cells activated with phytohemagglutinin (PHA) in the presence of IL-4 exhibit a greatly diminished response to IL-12, whereas the IL-12 response of T cells activated with PHA plus IFN-γ is enhanced. Radiolabeled IL-12 binding studies demonstrate that the impairment of T cell IL-12 responsiveness by IL-4 is associated with the down-regulation of high-affinity IL-12 receptor expression. In contrast, the enhancement of IL-12 responsiveness by IFN-γ is associated with the up-regulation of high-affinity IL-12 receptor expression. Through the use of a newly synthesized neutralizing antibody to the low-affinity IL-12 receptor β subunit (IL-12Rβ), we show that neither IL-4 nor IFN-γ affect the expression of IL-12Rβ, which we determine to be one of at least two low-affinity subunits required for high-affinity IL-12 binding. These findings suggest that IL-4 and IFN-γ exert opposite effects on T cell IL-12 responsiveness by differentially modulating the expression of low-affinity IL-12 receptor subunits that are distinct from IL-12Rβ and required, together with IL-12Rβ, for high-affinity IL-12 binding and IL-12 responsiveness. This provides a basis for understanding the interplay between different cytokines at the level of cytokine receptor expression, and offers insight into one of the mechanisms governing Th1 and Th2 development.     

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.     

Interleukin-12 but not interferon-γ production correlates with induction of T helper type-1 phenotype in murine candidiasis

By means of polymerase chain reaction-assisted mRNA amplification, we have monitored message levels of interleukin (IL)-12 in splenic macrophages and of interferon-γ (IFN-γ), IL-4, and IL-10 in CD4⁺ and CD8⁺ T cells using Candida albicans/host combinations that result either in a T helper type-1 (Th1)-associated self-limiting infection (“healer mice”) or in a Th2-associated progressive disease (“nonhealer mice”). The timing and pattern of message detection did not differ qualitatively by the expression of IFN-γ or IL-10 mRNA in CD4⁺ and CD8⁺ cells from healer (i.e. PCA-2 into CD2F1) vs. nonhealer (i.e. CA-6 into CD2F1 or PCA-2 into DBA/2) mice. In contrast, IL-4 mRNA was uniquely expressed by CD4⁺ cells from nonhealer animals. IL-12p40 was readily detected in macrophages from healer mice but was detected only early in infection in mice with progressive disease. Cytokine levels were measured in sera, and antigen-driven cytokine production by CD4⁺ and CD8⁺ cells was assessed in vitro, while IFN-γ-producing cells were enumerated in CD4^? CD8^? cell fractions. Overall, our results showed that (i) antigen-specific secretion of IFN-γ protein in vitro by CD4⁺ cells occurred only in healing infection; (ii) IL-4- and IL-10-producing CD4⁺ cells would expand in nonhealer mice in the face of high levels of circulating IFN-γ, likely released by CD4^? CD8^? lymphocytes; (iii) a finely regulated IFN-γ production correlated in the healer mice with IL-12 mRNA detection, and IL-12 was required in vitro for yeast-induced development of IFN-γ-producing CD4⁺ cells. Although the mutually exclusive production of IL-4/IL-10 and IFN-γ by early CD4⁺ cells may be the major discriminative factor of cure and noncure responses in candidiasis, IL-12 rather than IFN-γ production may be an indicator of Th1 differentiation.     

Interferon-γ and interleukin-10 inhibit antigen presentation by Langerhans cells for T helper type 1 cells by suppressing their CD80 (B7-1) expression

CD80(B7-1) and CD86(B7-2) co-stimulatory molecules have been reported to activate Th1/Th2 development pathways differentially. It is well known that Langerhans cells (LC), potent antigen-presenting dendritic cells in the epidermis, express several co-stimulatory molecules and that this expression is modulated by several cytokines. Based on the recently reported effect of interferon (IFN)-γ and interleukin (IL)-10 on the expression of CD80 and CD86 by LC, we examined the effects of these cytokines on the expression of CD54 (intercellular adhesion molecule-1) and CD40 in addition to CD80 and CD86 on LC, and correlated the expression of each co-stimulatory molecule with antigen presentation for a Th1 clone by cultured LC (cLC) treated with these cytokines. LC cultured for 72 h significantly up-regulated MHC class II antigen expression and all the co-stimulatory molecules we examined. As previously reported, IL-10 or IFN-γ inhibited the up-regulation of CD80 expression. Granulocyte/macrophage-colony-stimulating factor (GM-CSF) partially restored the suppression of CD80 expression induced by IFN-γ on cultured LC, while it had virtually no effect on the inhibition induced by IL-10. Antigen presentation for the myoglobin-specific syngeneic Th1 clone by cLC, which were pre-incubated with these cytokines, correlated well with their CD80 expression. In addition, among the antibodies for CD80, CD86, CD28 or CD40, the suppression of the Th1 clone stimulation by LC was found to occur only with anti-CD80 and anti-CD28 antibodies. Finally, we studied the effects of IFN-γ and IL-10 on GM-CSF production by epidermal keratinocytes (KC). We could show that only IFN-γ, but not IL-10, suppressed GM-CSF production by KC. These findings suggest that both IFN-γ and IL-10 suppress antigen presentation by LC for Th1 cells by suppressing their CD80 expression. The inhibitory effect of IFN-γ on CD80 expression on LC appears to be partially mediated through the suppression of GM-CSF production by KC.     

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.     

T cells made deficient in interleukin-2 production by exposure to staphylococcal enterotoxin B in vivo are primed for interferon-γ and interleukin-10 secretion

The superantigen staphylococcal enterotoxin B (SEB) induces a defect in interleukin (IL)-2 production by T cells expressing specific T cell receptor Vβ domains. The present study was undertaken to determine the capacity of T cells, made deficient in IL-2 production by exposure to SEB in vivo, to secrete interferon (IFN)-γ and IL-10 and to induce pathology upon SEB rechallenge. For this purpose, BALB/c mice received two intraperitoneal injections of 100 μg SEB with a 48-h interval. First, we compared peak serum levels of IL-2, IFN-γ and IL-10 after SEB rechallenge with those measured after a single SEB injection in control mice. The expected defect in IL-2 production in SEB-pretreated mice was associated with a major increase in IL-10 and IFN-γ levels which were about fivefold higher than in controls. Experiments in mice depleted of CD4⁺ or CD8⁺ cells as well as studies in which purified T cell populations were rechallenged with SEB in vitro indicated that both CD4⁺ and CD8⁺ cells from SEB-pretreated mice were primed for IL-10 and IFN-γ production. Furthermore, SEB-pretreated mice were sensitized to the toxic effects of the superantigen as indicated by a 30-70% lethality rate (vs. 0% in naive mice) within 48 h after SEB rechallenge. IFN-γ was involved in the lethal syndrome as it could be prevented by injection of neutralizing anti-IFN-γ monoclonal antibody. We conclude that SEB-reactive T cells made deficient for the production of IL-2 by exposure to SEB in vivo are primed for IFN-γ and IL-10 production, and that IFN-γ up-regulation is involved in the shock syndrome occurring upon SEB rechallenge.     

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.     

Human recombinant interferon-β influences T helper subset differentiation by regulating cytokine secretion pattern and expression of homing receptors

Type I interferons (IFN) are important regulators of both innate and acquired immunity. We have used an in vitro system of human CD4⁺ T cell differentiation to determine how IFN-β influences development of T helper (Th) subsets and homing receptor expression. IFN-β promoted differentiation of CD4⁺ T cells that produce low levels of both IFN-γ and lymphotoxin compared to interleukin (IL)-12-derived Th1 CD4⁺ T cells. IFN-β inhibited production of Th2 cytokines (IL-5 and IL-13) and augmented IL-12-mediated IL-10 secretion. In addition, IFN-β significantly enhanced L-selectin expression on CD4⁺ T cells and synergized with IL-12 to induce expression of cutaneous lymphocyte-associated antigen (CLA). This Th1 L-selectin⁺, CLA⁺ phenotype is characteristic of T cells found in normal human skin and suggests a role for type I IFN in the regulation of Th subset differentiation and tissue-specific homing receptors.     

Effects of interferon-α on cytokine profile,T cell receptor repertoire and peptide reactivity of human allergen-specific T cells

A large panel of T cell clones (TCC) specific for the recombinant form of Poa pratensis allergen (rKBG7.2 or Poa p9) were established from the peripheral blood of a grass pollen-sensitive donor in the absence or presence of recombinant interferon-α (IFN-α) in bulk culture and their pattern of cytokine secretion, peptide reactivity and TCR Vβ repertoire was examined. The majority of allergen-specific TCC derived in absence of IFN-α produced high amounts of interleukin-4 (IL-4) and IL-5 but not IFN-γ (Th2 cells), while most of TCC derived in presence of IFN-α produced IFN-γ but not, or limited amounts of, IL-4 and IL-5 (Th1 or Th0 cells). Of 24 TCC established in the presence of IFN-α, 22 were able to recognize a single allergen peptide, p26, while none of the clones established in the absence of IFN-α showed a similar specificity. The majority of both clones expressed the Vβ2 element regardless of whether they were established in the presence of IFN-α, but the presence of IFN-α favored the expansion of Vβ2⁺, Vβ17⁺ and Vβ22⁺ Poa p9-specific T cells, whereas in the absence of IFN-α, other TCR Vβ-bearing T cells (Vβ5, Vβ6.7 and Vβ14) were expanded in addition to Vβ2⁺ T cells. None of Vβ2⁺ clones established in the absence of IFN-α reacted with p26, whereas all the Vβ2⁺ clones established in its presence responded to this peptide. IFN-α also shifted the TCR Vβ repertoire of both Poa p9- and Lolium perenne group 1 (Lol p1)-specific T cell lines generated from the same patient and from a different grass-sensitive individual. These data demonstrate that IFN-α modulates the development of allergen-specific T cells in vitro, and suggest that IFN-α may represent an useful tool for novel immunotherapeutic approaches in allergic disorders.     

Interleukin-12 activates human γδ T cells: synergistic effect of tumor necrosis factor-α

γδ T cell populations are known to expand in response to intracellular bacterial infectious agents regardless of previous priming. We have shown previously that soluble factor(s) produced by Mycobacterium-stimulated monocytes activate cord blood γδ T cells to proliferate. In this study, we investigated whether cytokines produced by monocytes are responsible for γδ T cell activation in vitro: interleukin (IL)-1β, IL-6, IL-8, IL-12, tumor necrosis factor (TNF)-α and granulocyte/macrophage colony-stimulating factor were examined. Recombinant human IL-12 stimulated γδ T cells, but not αβ T cells in peripheral blood mononuclear cells, to express CD25 on their surfaces, and to expand in number in vitro. IL-12-primed γδ T cell numbers increased to a greater extent in the culture to which exogenous IL-2 (5 U/ml) was added. Anti-TNF-α monoclonal antibody inhibited IL-12-induced up-regulation of CD25 on γδ T cells, suggesting that endogenous TNF-α may play a role in IL-12-induced activation of γδ T cells. Recombinant TNF-α synergistically augmented IL-12-induced activation of γδ T cells. Furthermore, IL-12 up-regulated TNF receptors on γδ T cells in vitro: TNF-α binding to its receptor induced CD25 expression on the γδ T cells in an autocrine or paracrine fashion, or perhaps both. It also became evident that both IL-12 and TNF-α were produced by mycobacterial lysate-stimulated monocytes. Taken together, these results suggest that upon confrontation with mycobacterial organisms, γδ T cells can be quickly and antigen-nonspecifically activated by soluble factors including IL-12 and TNF-α, both of which are produced by mononuclear phagocytes in response to mycobacterial organisms.     

Regulation of interleukin-12 receptor β1 chain expression and interleukin-12 binding by human peripheral blood mononuclear cells

The interleukin-12 receptor (IL-12R)β1 chain is an essential component of the functional IL-12R on both human T and natural killer cells. In this report it is shown that activation of human peripheral blood mononuclear cells (PBMC) with anti-CD3 monoclonal antibody (mAb) or phytohemagglutinin resulted in the up-regulation of IL-12Rβ1 expression and IL-12 binding. Kinetic studies revealed that maximum expression of IL-12Rβ1 and IL-12 binding occurred on days 3–4. Anti-CD3-induced expression of IL-12Rβ1 chain and IL-12 binding by PBMC was augmented by anti-CD28 mAb, indicating that the potentiating effect of anti-CD28 on T cell responses to IL-12 could be mediated, at least in part, by the enhancement of IL-12R expression. Among 16 cytokines tested, IL-2, IL-7 and IL-15 markedly induced IL-12Rβ1 expression and IL-12 binding on resting PBMC, whereas IL-1α and tumor necrosis factor-α had a minimal enhancing effect. In contrast, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, interferon (IFN)-α, IFN-γ, granulocyte/macrophage colony-stimulating factor and transforming growth factor (TGF)-β2 had no detectable enhancing effect. Anti-CD3-induced expression of IL-12Rβ1 and of low-affinity IL-12 binding sites was partially inhibited by TGF-β2, IL-10 and IL-4; however, TGF-β2 and IL-10 completely abolished anti-CD3-induced expression of high-affinity IL-12 binding sites. Consistent with the reduction of high affinity IL-12 binding sites, PBMC activated with anti-CD3 mAb in the presence of TGF-β2 or IL-10 failed to produce IFN-γ or to proliferate in response to IL-12. These results suggest that Th2 cell-derived cytokines can inhibit IL-12-induced biological functions by inhibiting IL-12R expression and that expression of a second subunit of the IL-12R (IL-12Rβ2), required for the formation of high-affinity IL-12 binding sites, may be more highly regulated by TGF-β2 and IL-10 than is expression of IL-12Rβ1.     

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.     

Number of interleukin-4- and interferon-γ-secreting human T cells reactive with tetanus toxoid and the mycobacterial antigen PPD or phytohemagglutinin: distinct response profiles depending on the type of antigen used for activation

The enzyme-linked immunospot (ELISPOT) assay has been proven to be an efficient and sensitive method for the enumeration of single cells secreting antibodies or cytokines. Here we have used this method to determine the number of interleukin-4 (IL-4)- and interferon-γ (IFN-γ)-producing cells in in vitro secondary responses to tetanus toxoid (TT) and the mycobacterial antigen (purified protein derivative; PPD) or the mitogen phytohemagglutinin (PHA). PHA-induced IL-4 and IFN-γ secretion was well correlated suggesting polyclonal activation of cells. This was not the case with the specific antigens, where PPD preferentially induced IFN-γ- and very few IL-4-producing cells, while TT-induced both IL-4 and IFN-γ. These differences are probably a reflection of the types of immunity the two antigens induce, mycobacteria preferentially inducing a cell-mediated T helper type 1 (Th 1) type of immunity, while immunity to tetanus is an antibody-dependent, Th 2 type of response. In individuals recently boosted with TT, a significant increase in both IL-4- and IFN-γ-producing cells in response to TT was seen at day 7 after boost, followed by decline. This was in contrast to what was seen in response to PPD where an increase of IFN-γ-producing cells after the TT boost at day 7 persisted for at least 14 days. These results suggest that after an in vivo boost both antigen-specific and nonspecific T cells are activated and that antigen-specific cells home to other organs and therefore may be difficult to demonstrate in the circulation. Our data show that the ELISPOT assay is a powerful tool for determining the frequency of cells secreting cytokines. The assay has several advantages over other assays since it is sensitive, measures the number of actually secreting cells, and avoids the problems of binding of cytokines to their cell-bound or soluble receptors.