Antigen-stimulated IL-4, IL-13 and IFN-γ production by human T cells at a single-cell level

To understand the intricate balance and the coordinate expression of the Th1 and Th2 cytokines following a natural mode of T cell triggering, antigen-stimulated IL-4, IL-13 and IFN-γ production was studied in primary peripheral blood mononuclear cell cultures at a single-cell level. Cells from filariasis patients who respond to parasite antigen by producing not only IFN-γ but also IL-4 and IL-13 were stimulated with Brugia malayi adult worm antigen and analyzed for co-expression of cytokines by intracellular staining. IL-4 and IL-13 were frequently co-expressed (54 % of IL-4⁺ cells stained for IL-13 and 29 % of IL-13⁺ cells expressed IL-4 at all time points), whereas IFN-γ expression was totally segregated from both IL-4 and IL-13. These data indicate that in human peripheral T cells the co-expression of the dominant Th1 and Th2 cytokines within a single cell is a rare event and that IL-13 is clearly more frequently associated with a Th2 than a Th1 type response in primary T cell cultures.     

Enhanced HIV expression during Th2-oriented responses explained by the opposite regulatory effect of IL-4 and IFN-γ on fusin/CXCR4

The human α-chemokine receptor fusin/CXCR4 is an important cofactor for entry of T lymphocyte-tropic HIV-1 strains. We investigated the possible regulatory role of T cell cytokine patterns on CXCR4 as well as HIV expression by using in vitro models of both secondary and primary immune responses. Antigen-specific memory CD4⁺ T cells infected with a T-tropic HIV-1 strain showed significantly higher CXCR4 and HIV-1 expression in Th0/2-oriented responses in comparison with Th1-oriented responses. Similarly, in naive CD4⁺ T cells activated in the presence of IL-4 or IL-12 and infected with the same T-tropic strain, IL-4 up-regulated whereas IL-12 down-regulated both CXCR4 and HIV-1 expression. The down-regulatory effect of IL-12 on CXCR4 expression was found to be dependent on its capacity to induce IFN-γ production. These observations can account for the higher risk of progression in HIV-1-infected individuals undergoing Th0/2-oriented immune responses.     

Anti-IL-12 antibody prevents the development and progression of collagen-induced arthritis in IFN-γ receptor-deficient mice

In several models of inflammation, including collagen-induced arthritis (CIA), the disease-promoting effect of IL-12 has been attributed to its well-known ability to produce IFN-γ. However, IFN-γ receptor knockout (IFN-γ R KO) mice of the DBA/1 strain have been reported to be more susceptible to CIA than corresponding wild-type mice, indicating the existence of an IFN-γ-mediated protective pathway in this model. In the present study the development of CIA was found to be completely prevented by pretreatment with a neutralizing anti-IL-12 antibody, not only in wild-type, but significantly also in IFN-γ R KO mice. In both strains of mice, the protective effect of anti-IL-12 was associated with lower production of anti-collagen type II antibodies. In vivo stimulation with anti-CD3 antibody in arthritic IFN-γ R KO mice resulted in production of higher levels of circulating IFN-γ, TNF and IL-2 than in corresponding control mice that had not received the arthritis-inducing immunization. This was not the case in arthritis-developing wild-type mice. Furthermore, the protective effect of anti-IL-12 antibody in mutant, but not in wild-type mice, was associated with lower circulating IFN-γ, TNF and IL-2 and higher IL-4 and IL-5 cytokine levels following an anti-CD3 challenge. The data indicate that IL-12 promotes the development of arthritis independently of its ability to induce or favor production of IFN-γ. In fact, any IFN-γ produced in the course of the disease process rather exerts a protective effect. Furthermore, our study suggests that, in the absence of a functional IFN-γ system, endogenous IL-12 exerts its disease-promoting effect by favoring production of other Th1-associated cytokines (IL-2 and TNF), by inhibiting development of IL-4- and IL-5-producing T cells and by stimulating production of anti-collagen autoantibodies.     

The Th1 and Th2 cytokines IFN-γ and IL-4 antagonize the inhibition of monocyte IL-1 receptor antagonist by glucocorticoids: involvement of IL-1

Monocytes express IL-1 and IL-1 receptor antagonist (IL-1Ra) in response to lipopolysaccharide (LPS). IL-1 self-induction contributes to the increase in IL-1 following LPS stimulation. LPS-stimulated IL-1 and IL-1Ra production are inhibited by glucocorticoids. In the present work we examined the regulation of IL-1Ra by Th1 cytokine IFN-γ, Th2 cytokine IL-4, glucocorticoids and IL-1 in human monocytes. We demonstrate that IL-1 contributes to LPS-induced IL-1Ra expression as shown by IL-1 blockade in LPS-stimulated monocytes using a specific anti-IL-1β antibody or recombinant IL-1Ra. Glucocorticoids inhibited IL-1β-stimulated IL-1Ra mRNA expression and protein production. Glucocorticoids inhibited both IL-1-mediated and non-mediated LPS stimulation of IL-1Ra expression. Both IFN-γ and IL-4 reversed the inhibitory effect of glucocorticoids on IL-1Ra expression and secretion. The effect of IFN-γ was blocked by pretreatment of monocytes with an anti-IL-1β blocking antibody, whereas the effect of IL-4 could not be blocked, demonstrating that IFN-γ acts through a mechanism dependent on endogenous IL-1 production, whereas IL-4 acts through an IL-1-independent one. Consistent with this finding, IFN-γ (but not IL-4) failed to reverse the inhibitory effect of glucocorticoids when stimulated by IL-1, and only IL-4 combined with IL-1 showed synergism resulting in an increase in IL-1Ra production. The differential regulation and involvement of IL-1 in the expression of IL-1Ra by IFN-γ, IL-4 and glucocorticoids sets the level of monocyte responsiveness during the Th1 or Th2 responses.     

IFN-γ-induced TNF-α is a prerequisite for in vitro production of nitric oxide generated in murine peritoneal macrophages by IFN-γ

The effect of IFN-γ to stimulate formation of nitric oxide (NO) by normal murine peritoneal macrophages (Mϕ) has been found to be completely dependent on the ability of IFN-γ to activate secretion of TNF-α. The NO-stimulatory effect of IFN-γ was abolished by anti-TNF-α antibodies, the inhibitory intervention of which could be fully reversed by exogenously supplied TNF-α. Accordingly, the failure of Mϕ from C3H/HeJ mice to secrete TNF-α upon stimulation with IFN-γ was associated with their complete incapability to generate NO, unless they were simultaneously treated with IFN-γ + TNF-α. Collectively, the data document that similar to the NO up-regulatory action of other cytokines, the effect of IFN-γ is not independent, but depends on a synergistic cooperation with the self-produced TNF-α. The findings thus indicate that a widespread opinion claiming that IFN-γ per se is able to stimulate biosynthesis of NO needs revision.     

Sequential production of IL-2, IFN-γ and IL-10 by individual staphylococcal enterotoxin B-activated T helper lymphocytes

Upon primary activation, T helper (Th) cell populations express different cytokines transiently and with different kinetics. Stimulation of naive murine splenic Th cells with the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) in vitro results in expression of IL-2, IFN-γ and IL-10 with fast, intermediate and slow kinetics, respectively. This first report of a functional analysis of cells separated alive according to cytokine expression shows that these cytokines are not produced by different Th cell subpopulations, but can be expressed sequentially by individual Th cells. Th cells, activated with SEB for 1 day and isolated according to expression of IL-2, using the cellular affinity matrix technology, upon continued stimulation with SEB later secrete most of the IFN-γ and IL-10. Likewise, after 2 days of SEB culture, cells expressing IFN-γ, separated according to specific surface-associated IFN-γ as detected by magnetofluorescent liposomes, 1 day later secrete IL-10. Thus, individual Th1 cells can contribute to the control of their own IFN-γ expression by sequential expression of first IL-2, supporting their proliferation, and later IL-10, down-regulating the production of IFN-γ-inducing monokines and limiting the pro-inflammatory effects of IFN-γ.     

Interferon-γ maintains the binding and functional capacity of receptors for IL-8 on cultured human T cells

The neutrophil chemotactic cytokine, IL-8, has been reported to also chemoattract T lymphocytes in vitro and in vivo. Previously we showed that freshly isolated T cells migrated in response to IL-8, but incubation of T cells at 37 °C resulted in progressively decreased levels of IL-8 binding sites on T cells in association with reduced chemotactic responses. However, this reduced binding and migration of cultured T cells in response to IL-8 can be prevented by the presence of mononuclear cells in the culture. In order to define the factor(s) responsible for the restoration of T cell binding and migration in response to IL-8, we examined the effects of various cytokines. Addition of IFN-γ in cultured T cells maintained both the CXC chemokine receptor CXCR1 and CXCR2 binding sites for IL-8 on these cells to the level comparable to that expressed on freshly purified T cells accompanied by an almost complete restoration of their chemotactic response to IL-8. The results suggest that Th1 cytokine, IFN-γ, produced by mononuclear cells stimulated by proinflammatory signals may play an important role in regulating IL-8 receptor expression on T cells and in sustaining the function of these cells in response to IL-8.     

Bacille Calmette Guérin and interleukin-12 down-modulate interleukin-4-producing CD4+ NK1+ T lymphocytes

Early production of interleukin-12 (IL-12) by macrophages and of IL-4 from CD4⁺ NK1⁺ T cells influence development of the acquired immune response against infectious agents, namely differentiation of interferon-γ-secreting T helper 1 (Th1) cells against intracellular pathogens and of IL-4-producing Th2 cells against helminths. Evidence has been presented for transient convertibility of Th1 and Th2 cells in the presence of the polarizing cytokines IL-4 or IL-12, respectively. Moreover, it is likely that IL-4 dominates over IL-12, suggesting that Th2 cell development is preferred in the presence of both cytokines. Mycobacterium bovis Bacille Calmette Guérin (BCG) and IL-12 are potent inducers of Th1 responses. Here we show that BCG and IL-12 down-modulate IL-4-producing CD4⁺ NK1⁺ TCRα/β^intermediate liver lymphocytes. Our data provide further insights into the mechanisms by which BCG and IL-12 may promote unrestricted development of Th1 responses in vivo: BCG and IL-12 not only provide the positive stimuli for Th1 cell differentiation, but also interfere with antagonizing signals.     

IFN-γ inhibits the production of latent transforming growth factor-β1 by mouse inflammatory macrophages

Transforming growth factor (TGF)-β is a multifunctional cytokine, which in mammals exists in three isoforms (TGF-β1, 2 and 3). It is synthesized by a variety of cells including macrophages, and exerts potent immunoregulatory effects such as the inhibition of Th1 development and the suppression or reversal of IFN-γ-induced macrophage activation. In this study we analyzed the effect of IFN-γ on the production of TGF-β1 by thioglycolate-elicited mouse peritoneal macrophages under serum-free conditions. Untreated macrophages released TGF-β1 in its latent form, which became detectable in a capture ELISA specific for active TGF-β1 after acid activation of the culture supernatants. Treatment with IFN-γ reduced the amount of latent TGF-β1 in the culture supernatants in a dose-dependent fashion. The effect of IFN-γ was confirmed by a newly developed Western blot system for the detection of mouse TGF-β1 protein. IFN-γ only weakly (16 – 24 %) reduced the levels TGF-β1 mRNA at early and late time points of stimulation, and no evidence was obtained that IFN-γ suppresses the secretion of latent TGF-β1. Thus, inhibition of TGF-β1 production by IFN-γ is most likely due to decreased synthesis and/or stability of the TGF-β1 protein, and might be important for the generation of fully activated macrophages and a Th1 response.     

γδ T cells are secondary participants in acute graft-versus-host reactions initiated by CD4+ αβT cells

To examine the role of T cell subpopulations in an acute graft-versus-host (GVH) reaction, γδ T cells and αβ T cells expressing one of the two prototypic Vβ gene families were negatively isolated from adult blood samples and injected into allogeneic chick embryos. CD4⁺ αβ T cells expressing either Vβ1 or Vβ2 receptors were equally capable of inducing acute GVH reactions, consistent with the idea that αβ T cell alloreactivity is determined by CDR3 variability. By themselves, the γδ T cells were incapable of inducing GVH reactions. However, host γδ T cells were recruited into the donor αβ T cell-initiated lesions, where they were activated and induced to proliferate. The data suggest that γβ T cells may play a secondary role in GVH reactions.     

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 IFN-γ production in granulocyte-macrophage colony-stimulating factor-deficient mice

Granulocyte-macrophage colony-stimulating factor-deficient (GM-CSF−/−) mice produce far lower serum levels of IFN-γ in response to LPS than GM-CSF+/+ mice. CD4⁺ and CD8⁺ T cells from LPS-injected GM-CSF−/− mice showed a deficiency in IFN-γ production and proliferative activity in response to IL-2 and IL-12, whereas IFN-γ production by NK cells was not compromised. These defects of T cells were reversed by administration of GM-CSF in vivo, but not by supplementation with GM-CSF in vitro. GM-CSF−/− mice do not have an intrinsic defect in IFN-γ production, because IL-12 injection induces the same high levels of IFN-γ in GM-CSF−/− and GM-CSF+/+ mice. To investigate the inhibitory effect of LPS on GM-CSF−/− T cells and the indirect restorative activity of GM-CSF, we tested the action of supernatants from cultured dendritic cells (DC). A factor or factors in the DC supernatant normalized serum IFN-γ levels and T cell responses in LPS-injected GM-CSF−/− mice. IL-18 reproduced some but not all of these in vivo and in vitro effects of DC supernatants. Our results indicate that GM-CSF is important in protecting T cells from inhibitory signals generated during immunization or exposure to LPS, and that this effect of GM-CSF is indirect and mediated by factors produced by DC.     

Inborn errors of IL-12/23- and IFN-γ-mediated immunity: molecular, cellular, and clinical features

Mendelian susceptibility to mycobacterial diseases confers predisposition to clinical disease caused by weakly virulent mycobacterial species in otherwise healthy individuals. Since 1996, disease-causing mutations have been found in five autosomal genes (IFNGR1, IFNGR2, STAT1, IL12B, IL12BR1) and one X-linked gene (NEMO). These genes display a high degree of allelic heterogeneity, defining at least 13 disorders. Although genetically different, these conditions are immunologically related, as all result in impaired IL-12/23-IFN-γ-mediated immunity. These disorders were initially thought to be rare, but have now been diagnosed in over 220 patients from over 43 countries worldwide. We review here the molecular, cellular, and clinical features of patients with inborn errors of the IL-12/23-IFN-γ circuit.     

Cyclosporin A increases IFN-γ production by T cells when co-stimulated through CD28

Despite its calcineurin-inhibiting properties, cyclosporin A (CsA) can not inhibit IL-2 production when T cells are co-stimulated by CD80/CD86 on the antigen-presenting cells. We studied the in vitro effect of CsA on IFN-γ production. Anti-CD3 monoclonal antibody (mAb) was used as the primary stimulus for activation of purified human T cells. A stimulating anti-CD28 mAb, or CD80 or CD86 on stably transfected P815 cells, provided the co-stimulatory signal. IL-2 production was hardly affected by CsA under these stimulating conditions, while IFN-γ (at the protein and mRNA level) was markedly stimulated by CsA. The use of anti-CD3 or phorbol 12-myristate 13-acetate with ionomycin as the primary stimulus, together with co-stimulation through either CD28 or CD2 using transfectants with the appropriate ligands, allowed us to demonstrate that the resistance of IFN-γ production to inhibition by CsA required both CD3 and CD28 triggering. Inhibition of IL-10 production, and to a lesser degree of IL-4 production, by CD4⁺ cells was responsible for the enhancement of IFN-γ production in the presence of CsA. In conclusion, IFN-γ production by CD28-co-stimulated CD4⁺ T cells is resistant to inhibition by CsA and can even be facilitated by CsA as a result of removing a negative regulatory signal which is mainly IL-10 mediated. This finding might have implications for immunosuppressive strategies based upon the use of CsA.     

Mycobacteria-reactive γδ T cells in HIV-infected individuals: lack of Vγ9 cell responsiveness is due to deficiency of antigen-specific CD4 T helper type 1 cells

Human γδ T lymphocytes expressing the variable T cell receptor elements Vγδ paired with Vδ2 are activated by antigen derived from Mycobacterium tuberculosis (M. tb.) and presented by antigen-presenting cells (APC). The subsequent proliferation is strictly dependent on the presence of CD4⁺TCRαβ⁺ T helper type 1 (Th1) cells producing interleukin-2 (IL-2). In this study, we report that the reactivity of Vγ9 cells to M. tb. stimulation in vitro was drastically decreased or absent in the majority of the analyzed HIV-1-infected individuals (CDC stages III and IV). We show that the failure of Vγ9 cells frim HIV^? individuals to proliferate following M. tb. stimulation was not due to an intrinsic qualitative or quantitative defect of γδ T cells but rather to a deficiency of M. tb.-reactive CD4 Th1 cells. Thus, Vγ9 responsiveness could be restored if cultures of M. tb.-stimulated T cells from HIV⁺ donors were reconstituted with one of the following: (i) exogenous IL-2; (ii) purified CD4T cells from allogeneic donors; or (iii) T cell-depleted APC from allogeneic donors. In the majority of HIV⁺ patients, the defective Th1 activity of M. tb.-stimulated CD4 T cells could be increased neither by cytokines known to favor Th1 development (IL-12, interferon-γ) nor by neutralization of the Th1-suppressing Th2 cytokine IL-10. We suggest that measurement of Vγ9 cell expansion within M. tb.-stimulated peripheral blood mononuclear cells provides a sensitive assay for the functional capacity of antigen (M. tb.)-specific CD4 Th1 cells in HIV-infected individuals.     

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.     

Interleukin-4-producing CD4+ NK1.1+ TCRα/βintermediate liver lymphocytes are down-regulated by Listeria monocytogenes

Experimental infection of mice with the intracellular bacterium, Listeria monocytogenes, provides a paragon model for immune defence dominated by T helper type 1 (Th1) responses. Potent production of interleukin (IL)-12 by infected macrophages is considered the determining factor in Th1 cell development. In contrast, it is assumed that IL-4 producers remain virtually unstimulated in listeriosis. In the liver, the major target organ of listeriosis, an unusual T lymphocyte population exists with the intriguing phenotype CD4⁺NK1.1⁺ TCRα/β^intermediate (TCRα/β^int). Here we show that IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes are down-regulated early in listeriosis. We assume that curtailment of IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes promotes unconstrained development of Th1 cells which are central to protection against intracellular bacteria.