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.     

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.     

Recombinant IFN-γ abrogates allograft tolerance induced by donor-specific blood transfusion by restoring alloantibody production

Donor-specific tolerance to heart allograft was induced in adult Lewis rats by pregraft donor-specific blood transfusion (DST). We previously showed that this tolerant state is characterized by a dramatic inhibition of T cell and macrophage activation. In addition, tolerant animals could not mount an efficient anti-donor humoral response whereas transfer of sera from rejecting animals triggered rejection in tolerant animals. This tolerance can be abrogated by daily post-graft administration of recombinant IFN-γ (rIFN-γ). To elucidate the mechanisms of action of rIFN-γ, T cell, macrophage and B cell functions were assessed in allograft recipients. IFN-γ did not restore the expression of Th1-related cytokine mRNA or the activated macrophage product inducible nitric oxide synthase in allografts. Importantly, rIFN-γ treatment promptly restored the anti-donor humoral response in DST-treated recipients. We conclude that rIFN-γ treatment in DST-treated allograft recipients cannot reverse the unresponsive state of Th1 cells and macrophages infiltrating the graft, but can provide B cell help for IgG alloantibody production which is lacking in these animals.     

Experimental autoimmune thyroiditis (EAT) in mice lacking the IFN-γ receptor gene

To investigate the role of interferon-γ (IFN-γ) in experimental autoimmune thyroidits (EAT), H-2k mice with a disrupted IFN-γ receptor (IFN-γ R) gene were immunized with porcine thyroglobulin (pTg). We observed that EAT occurred on day 19 and remitted on day 35 in IFN-γ R- deficient (IFN-γR0/0) mice, whereas in wild-type mice, EAT occurred on day 21 and remitted on day 42 – 49. Moreover, EAT in the mutant mice was attenuated and accompanied by diminished Tg-specific cytotoxic and proliferative responses and decreased titers of anti-Tg antibodies, notably of the IgG2a and IgG2b isotypes. In contrast, Tg-specific IgG1 was increased in the IFN-γ R0/0 mice. In supernatants from T cells further stimulated in vitro by Tg, IFN-γ levels were higher in IFN-γ R0/0 than in wild-type mice throughout the course of the dis ease, whereas interleukin-10 was transiently increased prior to EAT onset in both groups of mice. Finally, using IFN-γ R0/0 mice, we demonstrate that induction of EAT does not require an intact IFN-γ system, while progression to full-blown disease depends on the action of IFN-γ.     

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.     

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.     

Regulation of Fcγ receptors and immunoglobulin G-mediated phagocytosis in mouse microglia

As resident macrophages in the CNS, microglia can transform from a surveillance state to an activated phenotype in response to brain injury. During this transition microglia become highly capable phagocytic cells. Invading pathogens undergo opsonization with immunoglobulins and microglia recognize these opsonized pathogens through interaction with their cognate F_c receptors. In mice, both FcγRI and FcγRIIb receptors are involved in IgG-mediated phagocytosis of opsonzied pathogens. At sites of inflammation, microglial activity is regulated by T-cell derived cytokines. Here we first investigated the effects of IFN-γ, IL-4, IL-13 and GM-CSF on expression of FcγRI and FcγRIIb mRNA levels in both primary microglia and microglial cell line N9. Using quantitative real-time PCR we show that IFN-γ induced a 4-fold increase in the mRNA level of FcγRI but did not induce changes in FcγRIIb expression. IL-4 and IL-13 induced approximately 2-fold increases in expression of FcγRIIb mRNA, but had no effect on FcγRI expression. GM-CSF increased both FcγRI and FcγRIIb mRNA expression. We then characterized the ability of these same cytokines to regulate phagocytosis of immune complexes composed of IgG and the bacteria Staphylococcus aureus. IFN-γ and GM-CSF both induced approximately 2-fold increases in IgG-mediated phagocytosis whereas IL-4 and IL-13 both decreased IgG-mediated phagocytosis by about one-third. None of the cytokines influenced basal levels of phagocytosis. These findings demonstrate a highly selective cytokine-induced regulation of both phagocytosis-related Fcγ receptor subtypes and IgG-mediated phagocytosis itself in microglia. This selective regulation has implications for our understanding of the pathophysiology of CNS infection and autoimmune disease.     

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.     

IL-4-producing NK T cells are biased towards IFN-γ production by IL-12. Influence of the microenvironment on the functional capacities of NK T cells

NK T cells are an unusual T lymphocyte subset capable of promptly producing several cytokines after stimulation, in particular IL-4, thus suggesting their influence in Th2 lineage commitment. In this study we demonstrate that, according to the cytokines present in the micro environment, NK T lymphocytes can preferentially produce either IL-4 or IFN-γ. In agreement with our previous reports showing that their IL-4-producing capacity is strikingly dependent on IL-7, CD4⁻ CD8⁻ TCRα β⁺ NK T lymphocytes, obtained after expansion with IL-1 plus granulocyte-macrophage colony-stimulating factor, produced almost undetectable amounts of IL-4 or IFN-γ in response to TCR/CD3 cross-linking. However, the capacity of these T cells to produce IFN-γ is strikingly enhanced when IL-12 is added either during their expansion or the anti-CD3 stimulation, while IL-4 secretion is always absent. A similar effect of IL-12 on IFN-γ production was observed when NK T lymphocytes were obtained after expansion with IL-7. It is noteworthy that whatever cytokines are used for their expansion, IL-12 stimulation, in the absence of TCR/CD3 cross-linking, promotes consistent IFN-γ secretion by NK T cells without detectable IL-4 production. Experiments in vivo demonstrated a significant up-regulation of the capacity of NK T cells to produce IFN-γ after anti-CD3 mAb injection when mice were previously treated with IL-12. In conclusion, we provide evidence that the functional capacities of NK T cells, which ultimately will determine their physiological roles, are strikingly dependent on the cytokines present in their microenvironment.     

Superoxide generation by alveolar macrophages from aged rats: improvement by in vitro treatment with IFN-γ

Alveolar macrophages (AM) from aged rats show an impaired oxidative response, but it is unclear whether or not this is due to the inability of these cells to be activated. To elucidate this, we investigated the capacity of AM from young (16-week-old) and aged (100-week-old) rats to become primed with recombinant rat interferon-γ (IFN-γ) for increased phorbol myristate acetate (PMA)-elicited O₂⁻ production, utilizing an MCLA-dependent chemiluminescent assay. We also compared concanavalin A- or Bacillus Calmette Guerin (BCG)-induced IFN-γ production by the spleen cells of young and aged animals. The data indicated that AM freshly harvested from non-sensitized aged rats produced less O₂⁻ than those from young animals. A similar result was obtained in BCG-sensitized rats. However, AM from aged rats were primed with in vitro treatment with IFN-γ for increased rate of O₂⁻ production to an equivalent level of that by AM from young animals. In addition, the ability of spleen cells to produce IFN-γ was well maintained in aged rats. These results suggest that AM function is suppressed in the lungs of aged animals. Our observation that the decreased AM function in aged rats can be reversed is important because it suggests that appropriate treatment may reduce the incidence and mortality of respiratory infections in the elderly.     

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.     

Fatal hematophagic histiocytosis after granulocyte-macrophage colony-stimulating factor and chemotherapy for high-grade malignant lymphoma

Fatal hematophagic histiocytosis occurred in two patients after they had received granulocyte-macrophage colony-stimulating factor (GM-CSF) in addition to chemotherapy for malignant non-Hodgkin's lymphoma. In one patient GM-CSF promoted the activity of subclinical hematophagic histiocytosis, resulting in severe pancytopenia and multiorgan failure. In the other patient the syndrome that caused persistent bone marrow failure began after the institution of GM-CSF therapy. Exogenous GM-CSF appears to upregulate preexisting hematophagic histiocytosis and may even contribute to its de novo initiation. It is therefore conceivable that endogenous GM-CSF also plays an essential role in the pathogenesis of this syndrome.Abbreviations CMV cytomegalovirus - G-CSF granulocyte colony-stimulating factor - GM-CSF granulocyte-macrophage colony-stimulating factor Correspondence to: A. Schaffner     

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.     

IL-1β and IFN-γ induce the regenerative epidermal phenotype of psoriasis in the transwell skin organ culture system. IFN-γ up-regulates the expression of keratin 17 and keratinocyte transglutaminase via endogenous IL-1 production

Skin biopsies from healthy human skin and non-lesional skin from patients with psoriasis were cultured for 24h and stimulated with interleukin-1β(IL-1β) and interferon-γ (IFN-γ) in a skin organ culture model and the induction of the psoriasiform regenerative epidermal phenotype was analysed using immunostaining. In the presence of IL-1β, the psoriasiform regenerative epidermal phenotype was clearly induced. This involved strong up-regulation of the expression of keratin 16, keratin 17, and keratinocyte transglutaminase (TGk) in the suprabasal layers, strong up-regulation and a shift of the expression of keratin 5 and integrin β₁from the basal to suprabasal keratinocytes, and induction of the expression of ICAM-1 and HLA-DR on basal keratinocytes. The effects of IL-1β in the organ cultures of normal skin could be completely neutralized by anti-IL-1 polyclonal antibodies. The effects of IFN-γ in healthy and non-lesional psoriatic skin were qualitatively similar to those of IL-1β. The IFN-γ-induced epidermal expression of keratin 17 and TGk could be completely blocked by culturing the biopsies in the presence of IL-1ra or anti-IL-1 antibodies, while the induction of HLA-DR and ICAM-1 was not inhibited. The induction of the psoriasiform regenerative epidermal phenotype by IFN-γ is partially mediated via endogenous epidermal IL-1. Copyright © 1999 John Wiley & Sons, Ltd.     

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.     

粒细胞-巨噬细胞集落刺激因子对HBV宫内感染婴儿的免疫调节作用

目的 观察粒细胞-巨噬细胞集落刺激因子(GM-CSF)对HBV宫内感染免疫失败婴儿的免疫调节作用.方法 宫内感染婴儿分为治疗组和对照组(均为20例),都给予乙肝疫苗10μg免疫用量,同一部位三角肌注射(疫苗注射时间按0、1、6方案),乙肝免疫球蛋白200IU臀部肌肉注射,20天1次,共3次.治疗组注射乙肝疫苗3天后,同一部位皮内注射GM-CSF 10μg/Kg.1岁时抽外周静脉血检测T淋巴细胞亚群的水平、血清干扰素-γ(IFN-γ)和白细胞介素4(IL-4)的水平.结果 (1)治疗组CD4+T细胞百分数及CD4 +/CD8+比值高于对照组(t=11.67,P＜0.05;t=13.06,P＜0.05),CD8+百分数低于对照组(t=6.25,P＜0.05),两组比较差异均有显著意义.(2)患者外周血IFN-γ水平治疗组高于对照组(t=13.92,P＜0.05);IL-4水平与对照组相比,差异无统计学意义(t=1.30,P＞0.05).结论 GM-CSF可提高HBV宫内HBV感染免疫失败婴儿CD4 +/CD8+比值,并且调节Th1/Th2细胞因子比例平衡.     

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.