Contribution of CD8+ T cells to innate immunity: IFN-gamma secretion induced by IL-12 and IL-18

The role of CD8+ T cells in adaptive immunity is well documented and involves numerous effector mechanisms including direct cytolysis of targets and secretion of cytokines. The role of CD8+ T cells in innate immunity has not been previously appreciated. Using J774 macrophages infected in vitro with the intracellular bacterium, Listeria monocytogenes (LM), we show that CD8+ T cells isolated from na?ve C57BL/6 (B6) mice respond rapidly by secreting IFN-gamma. CD8+ T cells secreting IFN-gamma can also be found in na?ve B6 mice 16 h after infection with LM. This rapid IFN-gamma response is TCR-independent and mediated through the actions of IL-12 and IL-18. Cell surface staining and cell sorting experiments indicate that these novel CD8+ T cells express memory markers. In vitro CFSE-labeling experiments show that IFN-gamma-secreting CD8+ T cells proliferate rapidly after 2 days in culture and after 4 days constitute the majority of the CD8+ T cell population. Together, these data suggest an important role for IFN-gamma-secreting CD8+ T cells in the innate response to bacterial pathogens.     

Role of IL-12 in macrophage activation during intracellular infection: IL-12 and mycobacteria synergistically release TNF-alpha and nitric oxide from macrophages via IFN-gamma induction

IL-12 is believed to play an important role in cell-mediated immunity against intracellular infection primarily by acting on T and NK cells. Recent evidence has suggested, however, that IL-12 has broader cellular targets than previously thought. In this study, we examined the role of IL-12 in macrophage TNF-alpha and nitric oxide (NO) release by using an in vitro model of intracellular infection. IL-12 alone released relatively little TNF-alpha and NO, whereas live mycobacteria alone released TNF-alpha markedly but little NO from murine alveolar macrophages. However, IL-12 and mycobacteria together enhanced TNF-alpha and NO release synergistically. Because IL-12 and mycobacteria also released IFN-gamma from macrophages synergistically, and exogenous IFN-gamma with mycobacteria enhanced TNF-alpha and NO release synergistically, we examined the role of endogenous IFN-gamma in IL-12/mycobacteria-stimulated macrophage activation. Using macrophages from mice deficient in IFN-gamma, we found that IL-12/mycobacteria-enhanced macrophage TNF-alpha and NO release was mediated through endogenous IFN-gamma. We further demonstrated that IFN-gamma and mycobacteria together had a selective effect on macrophage cytokine release because they released TNF-alpha synergistically but not macrophage chemotactic protein-1 (MCP-1). These findings reveal that IL-12 can activate macrophages potently during intracellular infection, and this activating effect is mediated primarily through its effect on macrophage IFN-gamma release.     

Regulation of murine macrophage function by IL-4: IL-4 and IFN-gamma differentially regulate macrophage tumoricidal activation.

To understand the differential role of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the process of macrophage tumoricidal activation, we investigated the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide in activated murine macrophages and the effects of those lymphokines on prostaglandin E2 (PGE2)-mediated down-regulation. IFN-gamma and IL-4 increased lipopolysaccharide (LPS)-induced TNF-alpha production by different mechanisms because IL-4, unlike IFN-gamma, failed to overcome the LPS-hyporesponsiveness in C3H/HeJ mice. Moreover, only IFN-gamma synergized with LPS to induce nitric oxide production and blocked eicosanoid-mediated down-regulation. These differential effects of IFN-gamma and IL-4 on the select efferent cytolytic activities may be the result of an altered or different signal transduction pathway. Because potentiation of protein kinase C (PKC) activity by IFN-gamma has been previously documented, we next studied the role of IFN-gamma and IL-4 in alteration of enzymatic activity of PKC. Two lymphokines caused translocation of PKC from cytosol to membrane with different levels, providing a biochemical basis for explaining how two lymphokines lead to different phenotypic responses. Although treatment of macrophages with IFN-gamma and IL-4 gave rise to a similar enhancing effect on macrophage TNF-alpha production, these two lymphokines appeared to differentially regulate the overall functional state of macrophages for tumour cell killing capability. Additionally, this differential regulation seems to be accomplished in part by different biochemical events.     

The combination of soluble IL-18Ralpha and IL-18Rbeta chains inhibits IL-18-induced IFN-gamma.

Although the beta chain of interleukin-18 receptor (IL-18Rbeta) is required for signaling, the soluble (extracellular) form does not bind IL-18, and its role in inhibiting IL-18 is unclear. In the present study, both the soluble human IL-18 ligand binding alpha chain (sIL-18Ralpha) and the sIL-18Rbeta chain were investigated for inhibition of IL-18-induced interferon-gamma (IFN-gamma) production in human peripheral blood mononuclear cells (PBMC), whole blood, and KG-1 macrophage and natural killer (NK) cell lines. Neutralization of IL-18 by soluble receptors was compared with that of the IL-18 binding protein (IL-18BP). An equimolar concentration IL-18BP inhibited 90% of IL-18 activity, whereas a 4-fold molar excess of sIL-18Ralpha had no effect. A dimeric construct of sIL-18Ralpha linked to the Fc domain of IgG1 (sIL-18Ralpha:Fc) increased IL-18 activity 2.5-fold. In PBMC stimulated with lypopolysaccharide (LPS) or in whole blood stimulated with Staphylococcus epidermidis, 3 nM IL-18BP reduced IFN-gamma by 80%, whereas IL-18Ralpha:Fc had no effect. A construct of the sIL-18Rbeta linked to Fc (sIL-18Rbeta:Fc) did not affect IL-18-induced IFN-gamma even at 80-fold molar excess of IL-18. However, the combination of both soluble receptors reduced IFN-gamma by 80%. In KG-1 cells, a 50% reduction in IL-18 activity was observed using an 80-fold molar excess of sIL-18Ralpha:Fc but only in the presence of sIL-18Rbeta:Fc. Similarly, a 50% reduction was observed using sIL-18Rbeta:Fc in the presence of a molar excess of sIL-18Ralpha:Fc. Similar inhibition was observed in NK cells. These studies reveal that the combination of the ligand-binding and the nonligand-binding extracellular domains of IL-18R is needed to inhibit IL-18, whereas IL-18BP neutralizes at equimolar concentration.     

Regulation of LPS induced IL-12 production by IFN-gamma and IL-4 through intracellular glutathione status in human alveolar macrophages.

Interleukin-12 (IL-12) is secreted from monocytes and macrophages; it exerts pleiotropic effects on T cells and natural killer (NK) cells, and stimulates interferon-gamma (IFN-gamma) secretion. Glutathione tripeptide regulates the intracellular redox status and other aspects of cell physiology. We examined whether IFN-gamma and IL-4 affect the balance between intracellular reduced glutathione (GSH) and oxidized (GSSG) glutathione, as this may affect IL-12 production in human alveolar macrophages (AM). We used both AM from healthy non-smokers obtained by bronchoalveolar lavage and the monocytic THP-1 cell line in this study. Incubation of AM for 2 h with the GSH precursor N-acetylcysteine (NAC) increased the intracellular GSH/GSSG ratio, and enhanced lipopolysaccharide (LPS)-induced IL-12 secretion by AM. In THP-1 cells, NAC increased the GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA, whereas L-buthionine-[S,R]-sulphoximine (BSO) decreased these. NAC and BSO offset their own effects on the intracellular GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA. Furthermore, exposure of AM to the helper T cell type 1 (Th1) cytokine IFN-gamma or the helper T cell type 2 (Th2) cytokine IL-4 for 72 h increased and decreased the GSH/GSSG ratio, respectively. Lipopolysaccharide (LPS)-induced secretion of IL-12 in AM was enhanced by IFN-gamma but inhibited by IL-4. These results suggest that IFN-gamma and IL-4 oppositely affect the GSH/GSSG balance, which may regulate IL-12 secretion from AM in response to LPS.     

Inflammatory liver steatosis caused by IL-12 and IL-18.

Acute fatty degeneration in the liver is caused by various agents, such as aspirin, valproic acid, and ibuprofen, that directly inhibit mitochondrial beta-oxidation of fatty acid and oxidative phosphorylation. Endogenous molecules, such as cytokines and hormones, are also known to mediate microvesicular steatosis in liver failure. In this study, we examined how interleukin-12 (IL-12) and IL-18 cause steatosis in the liver. Administration of these cytokines in combination caused marked hepatosteatosis and weight loss in mice. There were marked increases in levels of interferon-gamma (IFN-gamma), nitrite (NO(2)/NO(3)), and fibrinogen in the circulation in these mice. On the other hand, the ATP concentration and blood flow in the liver were significantly reduced. These changes, except the production of IFN-gamma and NO, were partially inhibited by Z-VAD-fmk, a synthetic tripeptide inhibitor for NO-induced caspases. These results indicate that IL-12 and IL-18 may mediate inflammatory hepatosteatosis through impairment of the microcirculation, which leads to mitochondrial dysfunction in hepatocytes.     

IL-12 synergizes with IL-18 or IL-1beta for IFN-gamma production from human T cells

IL-18 is a proinflammatory cytokine that plays an important role in NK cell activation and T(h)1 response. IL-18 has a structural homology to IL-1, particularly IL-1beta. IL-18R, composed of IL-1R-related protein (IL-18Ralpha) and IL-1R accessory protein-like (IL-18Rbeta), belongs to the IL-1R family. Furthermore, IL-18R at least partly shares the signal transducing system with IL-1R. Thus, the IL-18-IL-18R system has a striking similarity to the IL-1-IL-1R system. For this reason, we regarded it important to investigate whether, like IL-18, IL-1beta synergizes with IL-12 in inducing IFN-gamma production from human T cells and plays an important role in the T(h)1 response. Here we show that IL-12 and IL-1beta synergistically induce T cells to proliferate and produce IFN-gamma without their TCR engagement. IL-12 stimulation induced an increase in the proportion of T cells positive for IL-18R. Then, IL-12-stimulated T cells responded to IL-18 or IL-1beta by their proliferation and IFN-gamma production, although levels of IL-1beta-induced responses were lower. CD4(+)CD45RA(+) T cells, although they constitutively expressed IL-18Rbeta mRNA, did not express IL-18Ralpha mRNA. Phytohemagglutinin (PHA) stimulation alone induced IL-18Ralpha mRNA without affecting the expression of IL-18Rbeta mRNA. T(h)1-inducing conditions (PHA, IL-12 and anti-IL-4) further increased this expression. We also show that T(h)1 cells but not T(h)2 cells have increased expression of IL-18R and IL-1R, and produce IFN-gamma in response to IL-18 and/or IL-1beta.     

The effect of IL-18 on IL-12-induced CD30 expression and IL-4 and IFN-gamma production by allergen and PPD specific T cells

CD30 is expressed on activated T cells that, as has been suggested, preferentially produce IFN-gamma. Interleukin 12 increases antigen-induced CD30 expression on T cells and IFN-gamma production. Synthesis of IFN-gamma can be augmented further by IL-18. The aim of our study was to investigate whether IL-18 affects the IL-12 induced CD30 expression and cytokine production by allergen or PPD specific T cells. Mononuclear cells of healthy or atopic volunteers were stimulated with PPD or allergen, respectively, to obtain specific T cell lines. T cells were restimulated with appropriate antigen and antigen-presenting cells in the presence of IL-12, IL-18 or a combination of these cytokines. After 3 days, expression of CD30 was investigated on CD4 and CD8 T cells and IFN-gamma and IL-4 cytokine production was estimated in the culture supernatants. Flow cytometric analyses showed no effect of IL-18 on CD30 expression during IL-12 co-stimulation. At the same time after the optimal stimulation for CD30 expression, the levels of IFN-gamma were high in PPD-stimulated cell lines but have not been up-regulated by IL-18. IFN-gamma levels were much lower in allergen-stimulated T cells and although they were up-regulated by IL-12 there was no additional or synergistic effect from IL-18. IL-18, however, increased production of IL-4 in allergen-stimulated cell lines. Our studies provide new information about IL-18 activity on human cells and question its exclusive role in Th1 mediated responses.     

Requirement for both IL-12 and IFN-gamma signaling pathways in optimal IFN-gamma production by human T cells

Phytohemagglutinin (PHA)-derived T lymphoblasts or T cell clones from patients genetically deficient in IL-12R beta 1 (IL-12R beta 1(-/-)) or IFN-gamma R1 (IFN-gamma R1(-/-)) produced two- to threefold reduced IFN-gamma levels compared to the corresponding cells from healthy individuals after anti-CD3 and PMA stimulation. Moderate IFN-gamma production was observed in PHA-derived T lymphoblasts or T cell clones derived from healthy subjects in the presence of anti-IFN-gamma R1 or anti-IL-12 mAb, whereas it was negligible in the presence of both mAb. However, when anti-IFN-gamma R1 and/or anti-IL-12 mAb were added during restimulation, the cells produced normal levels of IFN-gamma, indicating that both IFN-gamma and IL-12 had an effect on the priming phase. Moderate production of IFN-gamma was partially enhanced only in IFN-gamma R1(-/-) T cell clones generated in the presence of IL-12, but was almost completely abolished when IL-12R beta 1(-/-) and IFN-gamma R1(-/-) T cell clones were generated in the presence of anti-IFN-gamma R1 or anti-IL-12 mAb, respectively. IL-4 production was enhanced in T cell clones from IL-12R beta 1(-/-),but not from IFN-gamma R1(-/-) patients, whereas IL-10 and IL-2 production did not differ significantly in polyclonal T cells or clones from healthy and deficient individuals. These results indicate that IL-12R beta 1- and IFN-gamma R1-dependent signals co-ordinately regulate IFN-gamma, but not IL-2 and IL-10 production, whereas only IL-12 negatively controls IL-4 production by in vitro-generated T cell clones. Thus, although IL-12 and IFN-gamma signals are each sufficient for moderate production of IFN-gamma by human T cells, both are needed for optimal IFN-gamma production, and in the absence of both IFN-gamma production is completely abrogated.     

Oligodeoxynucleotides containing CpG motifs induce IL-12, IL-18 and IFN-gamma production in cells from allergic individuals and inhibit IgE synthesis in vitro.

The effects of phosphorothioate oligonucleotides containing CpG motifs (CpG-ODN) on cultured cells from allergic patients and non-atopic individuals were investigated. In peripheral blood mononuclear cells (PBMC) CpG-ODN led to a significant increase of IFN-gamma. By intracellular cytokine staining, IFN-gamma production could be attributed to NK cells and inhibition experiments indicated an IL-12-dependent mechanism. Moreover, CpG-ODN increased mRNA expression of IL-12 and IL-18 in PBMC. In this respect, no significant difference between allergic and non-atopic individuals was observed. Monocyte-derived dendritic cells were identified as one IL-12- and IL-18-producing source. In addition, stimulation of PBMC derived from atopic patients with CpG-ODN led to a considerable increase of polyclonal IgG and IgM synthesis. In contrast, the production of total IgE was suppressed. CpG-ODN induced a significant rise of IgG and IgM specific for allergens to which the patients were sensitized, whereas allergen-specific IgE levels remained unchanged. Our data suggest that CpG-ODN display a strong influence on the ongoing immune response and might represent potential adjuvants for specific immunotherapy of type I allergy.     

Role of IL-18 induced Amphiregulin expression on virus induced ocular lesions

This report deals with the possible mechanism by which IL-18 can contribute to the control and resolution of inflammatory lesions in the cornea caused by herpes simplex virus infection. Our results demonstrate that the expression of the IL-18R by both regulatory T cells (Treg) and effector T cells was a pivotal event that influenced lesion pathogenesis. The engagement of IL-18R on Treg with its cytokine ligand resulted in Amphiregulin expression a molecule associated with tissue repair. In support of this scheme of events, lesion severity became more severe in animals unable to express the IL-18R because of gene knockout and was reduced in severity when IL-18 was overexpressed in the cornea. These changes in lesion severity correlated with the frequency and number of both Treg and Teff that expressed Amphiregulin. Additional experiments indicated that IL-12 and IL-18 acted synergistically to enhance Amphiregulin expression in Treg, an event partly dependent on P38 MAPK activity. Finally, sub-conjunctival administration of Amphiregulin resulted in resolution of both developing and developed lesions. Thus, overall our results imply that IL-18 may participate in controlling the severity of SK and contribute to tissue repair by converting both Treg and effector T cells into those that produce Amphiregulin.     

Gamma interferon (IFN-gamma) and IFN-gamma-inducing cytokines interleukin-12 (IL-12) and IL-18 do not augment infection-stimulated bone resorption in vivo

Periapical granulomas are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. In previous studies we have reported that the bone resorption in this model is primarily mediated by macrophage-expressed interleukin-1 (IL-1). The expression and activity of IL-1 is in turn modulated by a network of Th1 and Th2 regulatory cytokines. In the present study, the functional roles of the Th1 cytokine gamma interferon (IFN-gamma) and IFN-gamma-inducing cytokines IL-12 and IL-18 were determined in a murine model of periapical bone destruction. IL-12-/-, IL-18-/-, and IFN-gamma-/- mice were subjected to surgical pulp exposure and infection with a mixture of four endodontic pathogens, and bone destruction was determined by microcomputed tomography on day 21. The results indicated that all IL-12-/-, IL-18-/-, and IFN-gamma-/- mice had similar infection-stimulated bone resorption in vivo as wild-type control mice. Mice infused with recombinant IL-12 also had resorption similar to controls. IFN-gamma-/- mice exhibited significant elevations in IL-6, IL-10, IL-12, and tumor necrosis factor alpha in lesions compared to wild-type mice, but these modulations had no net effect on IL-1alpha levels. Recombinant IL-12, IL-18, and IFN-gamma individually failed to consistently modulate macrophage IL-1alpha production in vitro. We conclude that, at least individually, endogenous IL-12, IL-18, and IFN-gamma do not have a significant effect on the pathogenesis of infection-stimulated bone resorption in vivo, suggesting possible functional redundancy in proinflammatory pathways.     

Constitutive expression of IL-18 and IL-18R in differentiated IEC-6 cells: effect of TNF-alpha and IFN-gamma treatment.

The multifunctional cytokine interleukin-18 (IL-18) is an important mediator in intestinal inflammatory processes. The aim of this study was to evaluate the constitutive expression of IL-18 and its receptors (IL-18Ralpha and IL-18Rbeta) in intestinal epithelial cells (IEC) stimulated by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In addition, cellular proliferation and evaluation of brush border enzymes as differentiation markers were studied. Nontransformed rat intestinal epithelial IEC-6 cells were grown on an extracellular matrix (ECM) in medium with or without TNF-alpha, IFN-gamma, or a combination of both. Gene expression of IL-18, its receptors and apoptotic markers was evaluated using real-time PCR. Expression of IL-18Ralpha protein was demonstrated by flow cytometry and Western blot. Enzymatic activities of brush border enzymes and caspase-1 were determined. The constitutive expression of IL-18, IL-18Ralpha and IL-18Rbeta mRNAs and proteins were detected in IEC-6 cells. The biologically active form of IL-18 was released in response to TNF-alpha and IFN-gamma treatment. Exogenous IL-18 had no effect on cellular proliferation, brush border enzyme activities, and gene expression of apoptotic markers. However, the addition of IL-18 stimulated production and release of the chemokine IL-8. These data suggest that IEC-6 cells may be not only a source of IL-18 but also a target for its action.     

The costimulatory effect of IL-18 on the induction of antigen-specific IFN-gamma production by resting T cells is IL-12 dependent and is mediated by up-regulation of the IL-12 receptor beta2 subunit

IL-18 was originally described as a cytokine which induced IFN-gamma production by established Th1 cells in an IL-12-independent manner. However, subsequent studies demonstrated that exogenous IL-18 in the absence of IL-12 failed to drive Th1 differentiation of naive cells and induced IFN-gamma from established Th1 cells only in combination with IL-12. We have examined the role of endogenous IL-18 in controlling Th1 lineage commitment. When naive TCR-transgenic T cells were stimulated with antigen, anti-IL-18 antibodies resulted in partial inhibition of IFN-gamma production, but did not inhibit Th1 differentiation. To distinguish whether the inhibitory effect of anti-IL-18 antibodies was mediated directly by blocking IFN-gamma production or indirectly by blocking IL-12Rbeta2 up-regulation, naive T cells from IL-12 - / - mice were stimulated with anti-CD3 and IL-18. These cells failed to produce IFN-gamma, but markedly up-regulated IL-12Rbeta2 expression. We propose that the major effect of IL-18 on Th1 development is mediated by up-regulation of IL-12Rbeta2 expression, thereby enhancing IL-12-mediated signaling. The enhancement of IL-12Rbeta2 expression by IL-18 may be particularly important for the differentiation of foreign antigen- or autoantigen-specific Th1 cells when the stimulatory concentration of IL-12 in the microenvironment is just below the threshold required for Th1 development.     

IL-12、IL-18基因对HIV-1外膜蛋白基因疫苗诱导的免疫应答的影响

目的 研究IL-12、IL-18基因对HIV-1外膜蛋白基因疫苗诱导免疫应答的影响，以探求HIV-1核酸疫苗的新策略。方法 pVAX1GP120联合IL-12、IL-18基因或者pVAX1GP120单独免疫BALB／c小鼠，采用ELISA检测免疫小鼠的特异性抗体和IFN-γ水平，以NIT比色法检测免疫小鼠脾淋巴细脯增殖，用乳酸脱氢酶（LDH）试验检测小鼠特异性细胞毒性T淋巴细胞（CIL）的应答。结果 与pVAX1GP120免疫组比较，pVAX1GP120联合IL-12、IL-18基因免疫组小鼠血清的抗HIV-1 gp120抗体滴度升高，IFN-γ升高，小鼠的脾淋巴细胞增殖实验刺激指数（SI）以及特异性CTL活性均高，差异均有统计学意义（P〈0．01）。结论 IL-12、IL-18基因联合HIV-1核酸疫苗免疫小鼠，有可能增强特异性TH1细胞和CTL反应，IL-12、IL-18基因对体液免疫可能有抑制作用。因此，IL-12、IL-18基因对于治疗性HIV-1核酸疫苗可能是具有较好应用前景的免疫佐剂。     

Fucoidan选择性抑制LPS诱导巨噬细胞分泌IL-12

目的探讨配体fucoidan活化清道夫受体后能否抑制LPS刺激巨噬细胞分泌IL-12。方法不同剂量的fucoidan（以多价阴离子chondroitin为阴性对照）与RAW264．7作用10min后,加人等量的LPS刺激,检测上清中IL-12p70、IL-6和TNF-α;同时观察不同剂量fucoidan对RAW264．7贴壁的影响。结果不同剂量的fucoidan能不同程度促进LPS诱导RAW264．7分泌炎症因子IL-6和TNF-α,但却能明显地抑制IL-12的分泌;随着所加fucoidan量的增加,RAW264．7细胞的贴壁能力逐渐下降。结论Fucoidan可能通过与清道夫受体SR-A（scavenger receptor A）结合发挥其特异抑制LPS诱导巨噬细胞分泌IL-12的作用。