Type I IFNs inhibit human dendritic cell IL-12 production and Th1 cell development

We have investigated the role of type I IFNs (IFN-alpha and -beta) in human T cell differentiation using anti-CD3 mAb and allogeneic, in vitro-derived dendritic cells (DC) as APCs. DC were very efficient activators of naive CD4+ T cells, providing necessary costimulation and soluble factors to support Th1 differentiation and expansion. Addition of IFN-alphabeta to DC/T cell cultures resulted in induction of T cell IL-10 production and inhibition of IFN-gamma, TNF-alpha, and LT secretion. Diminished T cell IFN-gamma production correlated with IFN-alphabeta-mediated inhibition of the p40 chain of the IL-12 heterodimer secreted by DC. Suppression of p40 IL-12 and IFN-gamma was not due to increased levels of IL-10 in these cultures, and production of IFN-gamma could be restored by exogenous IL-12. These data indicate that type I IFNs inhibit DC p40 IL-12 expression, which is required for development of IFN-gamma-producing CD4+ T cells. Furthermore, when T cells were restimulated without IFN-beta, these cells induced less p40 IL-12 from DC, suggesting that the functional properties of T cells may regulate DC function. Thus, IFN-alphabeta inhibits both IL-12-dependent and independent Th1 cytokine production and provides a mechanism for inhibition of IL-12-mediated immunity in viral infections.     

In vitro prevention and reversal of lipopolysaccharide desensitization by IFN-gamma, IL-12, and granulocyte-macrophage colony-stimulating factor

LPS tolerance is characterized by a diminished monocytic synthesis of TNF-alpha and, interestingly, IL-10 after LPS restimulation. We wondered whether granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-12, and IFN-gamma can prevent or reverse this down-regulation of TNF-alpha and IL-10 production. The LPS-induced TNF-alpha amounts in desensitized PBMC treated with GM-CSF, IFN-gamma, or IL-12 and in naive, non-cytokine-primed cultures were similar, while much more TNF-alpha was induced in cytokine-primed naive cells. The effect of IL-12 was dependent on the presence of nonmonocytic cells and could be completely blocked with an IFN-gamma antiserum. Treatment of LPS-desensitized pure monocytes with IFN-gamma or GM-CSF resulted in a very high TNF-alpha expression and no difference to cytokine-primed naive monocytes was evident any longer. While IFN-gamma and IL-12 decreased IL-10 expression in naive PBMC, it was increased by both and by GM-CSF in LPS-tolerant cultures. Again, only IL-12 was dependent on the presence of nonmonocytic cells. For prevention of LPS tolerance, similar results were obtained. Recently, we have shown that IL-10 and TGF-beta mediate LPS desensitization in vitro and can be used to establish LPS hyporesponsiveness in the absence of LPS. IFN-gamma and GM-CSF prevented and reversed down-regulation of TNF-alpha and IL-10 synthesis also in the model of IL-10/TGF-beta1-induced LPS hyporesponsiveness, while IL-12 was ineffective because of its obvious inability to induce IFN-gamma. In summary, after LPS desensitization/hyporesponsiveness, IFN-gamma and GM-CSF tended to normalize pro- and anti-inflammatory monocytic behavior. Our results suggest that during LPS desensitization/hyporesponsiveness, monocytes acquire a hitherto unknown functional state with an altered reaction to biologic response modifiers.     

Antigen-driven but not lipopolysaccharide-driven IL-12 production in macrophages requires triggering of CD40

We demonstrated that two distinct pathways exist for the induction of IL-12 in APC. The first pathway for IL-12 production occurred during responses to T cell-dependent Ags such as OVA and required triggering of CD40 molecules on the APC. IL-12 production in this T cell-dependent system increased in direct proportion to Ag concentration and required TCR ligation but not CD28 costimulation. The second pathway occurred when bacterial products such as LPS or heat-killed Listeria monocytogenes were used to activate macrophages to produce IL-12 in the complete absence of T cells. In this second pathway, IL-12 production was completely independent of CD40 triggering. In both pathways, the presence of IFN-gamma was not required for induction of IL-12 synthesis when splenic adherent cells (SAC) from normal mice were used. However, addition of IFN-gamma to cultures of Th2 T cells and SAC increased IL-12 production two- to fivefold, and addition of rTNF-alpha with IFN-gamma further enhanced IL-12 production. The addition of TNF-alpha in the absence of IFN-gamma, however, had no effect on IL-12 production in the T cell-dependent pathway. Similarly, addition of TNF-alpha in the presence or the absence of IFN-gamma to cultures of LPS or heat-killed Listeria and SAC did not increase IL-12 production, but addition of IFN-gamma alone greatly enhanced IL-12 production, consistent with the idea that bacterial stimuli induce significant quantities of endogenous TNF-alpha production. These results indicate that the requirements for the induction of IL-12 production in T cell-dependent and T cell-independent responses differs mainly with regard to CD40 triggering. Furthermore, these results suggest that IL-12 production can be induced by bacterial products in patients with hyper-IgM syndrome who lack CD40 ligand expression and in those treated with soluble gp39 to interrupt CD40-CD40 ligand interactions.     

Human macrophages induced in vitro by macrophage colony-stimulating factor are deficient in IL-12 production

IL-12 is important for Th1 differentiation. Myeloid-derived antigen-presenting cells (APC) such as monocytes, macrophages (Mphi) and dendritic cells (DC) are believed to be major sources of IL-12 in vivo. We have compared IL-12 production of fresh monocytes with Mphi differentiated in vitro using macrophage colony-stimulating factor (M-CSF) or human plasma, and in vitro generated dendritic cells, since these differentiated cell types represent APC at sites of antigen challenge. Macrophages stimulated with lipopolysaccharide (LPS) or heat-killed Listeria monocytogenes in the presence or absence of IFN-gamma produced minimal IL-12 p70 by comparison with DC or monocytes, despite comparable production of TNF-alpha. M-CSF-induced Mphi produced low levels of IL-10 constitutively and high levels after stimulation with LPS, but neutralization of IL-10 did not augment Mphi IL-12 production. Exposure of Mphi to TNF-alpha, granulocyte-macrophage CSF or IFN-gamma did not substantially up-regulate IL-12. Therefore M-CSF induces a differentiated Mphi phenotype in which IL-12 production is down-regulated, perhaps irreversibly. This may be the default pathway for monocyte-Mphi development in the absence of inflammation.     

Inhibition of IL-10 expression by IFN-gamma up-regulates transcription of TNF-alpha in human monocytes

Stimulation of human monocytes with LPS induces expression of multiple cytokines, including TNF-alpha, IL-1 beta, IL-6, and IL-10, IL-10 expression is delayed relative to that of TNF-alpha, IL-1 beta, and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF-alpha, IL-1 beta, and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. The Th1-type lymphokine, IFN-gamma, markedly up-regulates TNF-alpha production in monocytes. However, the precise mechanism by which IFN-gamma mediates this effect is unknown. We examined the effects of IFN-gamma on IL-10 expression in LPS-stimulated monocytes, and the relationship between IL-10 and TNF-alpha production in these cells. LPS stimulation induced rapid, ordered expression of multiple cytokines. Steady-state mRNA levels for TNF-alpha increased rapidly, reached maximal levels by 2 to 3 h poststimulation, and then declined sharply. IL-1 beta and IL-6 mRNA levels also increased markedly following stimulation with LPS, but decreased more slowly than did TNF-alpha. Down-regulation of mRNA for TNF-alpha, IL-1 beta, and IL-6 coincided with a delayed and more gradual increase in IL-10 mRNA levels. Furthermore, neutralization of IL-10 with anti-IL-10 Abs prolonged TNF-alpha mRNA expression, and significantly increased net TNF-alpha production. IFN-gamma suppressed expression of IL-10 mRNA and protein in a dose-dependent manner. Moreover, inhibition of IL-10 production correlated with a marked increase in both the magnitude and duration of TNF-alpha expression. Thus, potentiation of TNF-alpha production by IFN-gamma in monocytes is coupled to inhibition of endogenous IL-10 expression.     

Effects of interferons on tumour necrosis factor alpha production from human keratinocytes

Interferons (IFNs) have been reported to have pleiotrophic effects including the ability to induce the production of other cytokines in several cell types. Tumour necrosis factor alpha (TNF-alpha) is pro-inflammatory cytokine a known to be produced by a variety of cells including human keratinocytes. In the present study, we sought to determine the effects of IFNs on TNF-alpha production from human keratinocytes. IFN-gamma (50-100 ng/ml) induced TNF-alpha production dose dependently, but no induction of TNF-alpha was observed with IFN-alpha or IFN-beta. Since in the epidermis cytokines often work with in a cascade fashion and keratinocytes are a source of primary cytokine, IL-1 alpha, whether combined treatment with IFN-gamma and IL-1 alpha had a synergistic effect on TNF-alpha production was examined. Combined treatment with IFN-gamma (100 ng/ml) and IL-1 alpha (10 ng/ml) induced 2-3-fold higher level of TNF-alpha than IL-1 alpha alone. These results suggest that IFN-gamma is a positive regulator for the production of TNF-alpha from human keratinocytes and likely to increase skin inflammation.     

Lipopolysaccharide-induced IL-12 expression in the central nervous system and cultured astrocytes and microglia

We examined whether the cytokine IL-12 could be induced locally in the brain or in glial cell cultures following LPS treatment. In the brain, expression of IL-12 p35 mRNA was constitutive and did not alter following i.p. injection of LPS. In contrast, IL-12 p40 mRNA was only detectable in the brain of mice given two staggered injections of LPS. Dual labeling in situ analysis revealed IL-12 p40 RNA-positive cells scattered throughout the brain parenchyma, with a small number of these cells being identified as astrocytes, while the majority of IL-12 p40 RNA-expressing cells appeared to be microglia. In cultured microglia or astrocytes, LPS and to a much lesser degree IL-1beta, but not IFN-gamma or TNF-alpha, induced the expression of IL-12 p40 mRNA. Numerous glial fibrillary acidic protein-immunopositive cells colabeled for IL-12 p40 RNA; indicating that LPS-stimulated astrocytes expressed IL-12 in vitro. Immunoblot analysis of lysates from LPS-treated astrocytes revealed the presence of multiple species of 40, 43, 75, and 120 kDa containing the IL-12 p40 protein. Finally, secretion of the IL-12 p75 heterodimer was detectable by ELISA from astrocytes treated with LPS plus IFN-gamma, but not with LPS alone. The findings indicate that IL-12 gene expression can be activated in the brain, with the resident glial cells being a prodigious source of this cytokine. The localized production of IL-12 may have a significant impact on the development of cell-mediated immune responses within the central nervous system.     

Fas/APO-1 (CD95)-mediated apoptosis is activated by interferon-gamma and interferon- in interleukin-6 (IL-6)-dependent and IL-6-independent multiple myeloma cell lines

A poor response to Fas-induced apoptosis is evident in some multiple myeloma (MM) cell lines and primary cells. In this study, we have examined the possibility to increase the sensitivity to Fas-induced apoptosis by pretreatment of MM cells with interferon-gamma (IFN-gamma) or interferon-alpha (IFN-alpha). Both IFN-gamma and IFN-alpha markedly increased the Fas-induced apoptosis in all cell lines tested (U-266-1970, U-266-1984, and U-1958). In the U-266-1970 and U-1958 cell lines, pretreatment with either IFN-gamma or IFN-alpha also inhibited proliferation in a dose-dependent manner. In contrast, IFN-gamma activation of the Fas death pathway in the U-266-1984 cells was not accompanied by growth inhibition. Incubation with the IFNs increased the Fas antigen expression in one of three cell lines but did not alter the expression of Bcl-2 or Bax. The IFNs are important regulators of growth and survival in MM cells. Our results suggest that activation of Fas-mediated apoptosis is a novel mechanism by which the IFNs exert inhibitory effects on MM cells.     

Interleukin 12 unmasks HLA class I differences during mixed lymphocyte reaction induced interferon gamma production

We investigated the genetic control of IFN-gamma release during MLR and its relationship with TNF-alpha and IL-12. Blocking experiments demonstrated the IFN-gamma dependence of TNF-alpha production and the significant contribution of IL-12 to IFN-gamma secretion. We studied informative pairs allowing the evaluation of the relative importance of HLA class I and class II antigens. Maximal IFN-gamma secretion allowing discrimination between fully HLA different and identical subjects required 5 days. In class I different but DRB1 identical pairs, a moderate but discriminant IFN-gamma release was found. Exogenous IL-12 addition after 24 hours of preactivation by MLR resulted in a marked enhancement of IFN-gamma production at day 2. In pairs differing only by class I antigens, the discriminating capacity was significantly increased as compared to values obtained in absence of IL-12 at day 2 (p < 0.004) and at day 5 (p < 0.004). The crucial role of class I antigens on IFN-gamma release was further substantiated by the blocking action of the W6/32 mAb directed against a monomorphic epitope common to all HLA-A, -B, and -C antigens. We conclude that IFN-gamma production during MLR is under the control of class I antigens. Furthermore, exogenous IL-12 strongly amplifies their influence.     

Reduced IL-12 production by monocytes upon ultraviolet-B irradiation selectively limits activation of T helper-1 cells

The capacity of APC to stimulate the proliferation of human peripheral blood T cells decreases upon ultraviolet-B (UVB) irradiation. The aim of this study was to investigate whether all T cell subsets are equally sensitive to this reduced APC function. Established human Th1, Th2, and Th0 clones were stimulated with monocytes in a soluble CD3 mAb-mediated assay that is dependent on the presence of APC. Monocytes were exposed to low nonlethal doses of UVB radiation before coculture with T cells. UVB irradiation inhibited the capacity of monocytes to stimulate the proliferation and IFN-gamma production of Th1 cells in a dose-related fashion. In contrast, UVB-treated monocytes induced normal proliferation and IL-4 production in Th2 cells. Stimulation of Th0 cell proliferation by UVB-irradiated monocytes was normal, but a preferential suppression of IFN-gamma production was observed, thus leading to a more Th2-like cytokine response. The loss of Th1 proliferation upon stimulation with UVB-irradiated monocytes could be overcome by rIL-2; however, IFN-gamma production remained suppressed. IFN-gamma production could be completely restored by rIL-12, whereas the addition of IL-1 beta, TNF-alpha, or indomethacin had no such effect, nor did the addition of mAb to CD28, added to compensate for the reduced B7 expression of UVB-irradiated monocytes. Monocytes exposed to UVB radiation exhibited reduced expression of mRNA for the IL-1 2 subunits p35 and p40 and suppressed production of the IL-12 p70 protein. Our results thus indicate that UVB irradiation of APC selectively impairs Th1-like responses, a phenomenon caused by the UVB-induced suppression of monocyte IL-12 production.     

Interleukin-8 differentially modulates interleukin-4- and interleukin-2-induced human B cell growth

The effect of interleukin-8 (IL)-8 on human B cell growth, as determined by thymidine uptake and viable cell numbers was studied. IL-8 inhibited IL-4-induced growth of B cells costimulated with anti-mu antibodies (Ab) or Staphylococcus aureus Cowan strain I (SAC) in a dose-dependent fashion. In contrast, IL-8 did not inhibit IL-2-induced growth of B cells. The IL-8-mediated inhibition was specific, since it was blocked by anti-IL-8 mAb but not by control IgG1. Moreover, anti-tumor necrosis factor-alpha (anti-TNF-alpha) Ab blocked IL-8-mediated inhibition. On the other hand, TNF-alpha, but not other cytokines including IL-1 beta, IL-3, IL-5, IL-6, interferon-alpha (IFN-alpha) or IFN-gamma, inhibited IL-4-mediated growth, and inhibition by TNF-alpha was blocked by anti-TNF-alpha Ab but not by control IgG. IL-4 had no effect on TNF-alpha binding by B cells while it decreased TNF-alpha production by B cells. IL-8 had no effect in binding of IL-4, IL-2 or TNF-alpha by B cells, however, it enhanced TNF-alpha production by B cells. These results indicate that IL-8 inhibited IL-4-induced human B cell growth by enhancement of endogenous TNF-alpha production.     

Involvement of the IL-2 receptor gamma-chain (gammac) in the control by IL-4 of human monocyte and macrophage proinflammatory mediator production

IL-4 has potent anti-inflammatory properties on monocytes and suppresses both IL-1beta and TNF-alpha production. Well-characterized components of the IL-4 receptor on monocytes include the 140-kDa alpha-chain and the IL-2R gamma-chain, gammac, which normally dimerize 1:1 for signaling from the receptor. However, mRNA levels for gammac were very low in 7-day-cultured monocytes. As mRNA levels for gammac declined with culture, so too did the ability of IL-4 to down-regulate LPS-induced TNF-alpha production. In contrast, IL-4 consistently down-regulated IL-1beta production by cultured monocytes. Immunoprecipitation and Western blot analyses demonstrated that 7-day-cultured monocytes do not express the functionally active 64-kDa gammac protein. This was associated with decreased STAT6 activation by IL-4. Studies with Abs to gammac and an IL-4 mutant that is unable to bind to gammac showed that IL-4 can suppress IL-1beta but not TNF-alpha production by LPS-stimulated monocytes in the presence of little or no functioning gammac. IL-4 also suppressed IL-1beta but not TNF-alpha production by Mono Mac 6 cells, which express minimal levels of gammac. For gammac-expressing LPS/PMA-activated U937 cells, IL-4 decreased both TNF-alpha and IL-1beta production. These results suggest that functional gammac is not present on in vitro-derived macrophages, and that while some anti-inflammatory responses to IL-4 are lost with this down-regulation of functional gammac, others are retained. We conclude that different functional responses to IL-4 by human monocytes and macrophages are regulated by different IL-4 receptor configurations.     

Reprogramming of lipopolysaccharide-primed macrophages is controlled by a counterbalanced production of IL-10 and IL-12

We studied the potential role of a cytokine regulatory mechanism(s) in LPS-dependent reprogramming and modulation of TNF-alpha and nitric oxide (NO) responses in mouse peritoneal macrophages. Reciprocal regulation of TNF-alpha and NO production by LPS-primed and LPS-stimulated macrophages was found to be dependent on the presence of soluble secretory products released by the cells during the initial LPS priming interaction. Pretreatment of naive macrophages with different mouse recombinant cytokines such as rIL-10, rIL-12, and rIFN-gamma dose dependently and differentially regulated subsequent LPS-induced production of TNF-alpha, IL-6, and NO by cytokine-primed cells. Analysis of IL-12 and IL-10 levels present in culture supernatants of LPS-primed and LPS-stimulated macrophages revealed a high degree of correlation between the profiles of TNF-alpha and IL-12 as well as NO and IL-10. Furthermore, LPS priming of macrophages in the presence of anti-IL-12-neutralizing mAb attenuated TNF-alpha responses while at the same time up-regulated NO production. In contrast, neutralization of endogenous IL-10 with anti-IL-10 mAb resulted in considerable TNF-alpha response at LPS priming doses under conditions that would otherwise strongly inhibit TNF-alpha production. We also found that the initial LPS priming of naive macrophages differentially and dose dependently regulates expression of mRNAs for IL-10, IL-12, and IFN-gamma in LPS-primed macrophages. Collectively, our data provide experimental support for the hypothesis that a cytokine regulatory network, most probably autocrine, tightly controls the reciprocal modulation of TNF-alpha and NO responses in LPS-primed macrophages.     

IL-18 accounts for both TNF-alpha- and Fas ligand-mediated hepatotoxic pathways in endotoxin-induced liver injury in mice

When LPS is administered to heat-killed Propionibacterium acnes-primed BALB/c nude mice, they develop endotoxin-induced liver injury. As previously reported, this liver injury can be prevented by treatment with an Ab against IL-18, a novel cytokine with the ability to induce IFN-gamma production and up-regulate functional Fas ligand (FasL) expression. To identify the pathologic role of IL-18 in this liver injury, we investigated the hepatic cytokine network and FasL induction after LPS challenge. After LPS challenge to BALB/c nude mice, their livers expressed IL-12 mRNA, followed by the induction of IFN-gamma and FasL mRNA and then by the late elevation of TNF-alpha mRNA, but stably expressed IL-18 mRNA. The TNF-alpha induction curve had two peaks. The first peak was the result of the direct reaction to LPS, and the late peak might have been induced, since P. acnes-elicited Kupffer cells showed one-peak TNF-alpha kinetics in response to LPS stimulation in vitro. LPS-activated P. acnes-elicited Kupffer cells secreted both IL-12 and IL-18, as determined by ELISA and bioassay, respectively. The in vivo administration of anti-IL-18 just before an LPS challenge suppressed not only the induction of IFN-gamma and the late TNF-alpha elevation, but also the FasL induction, resulting in the total prevention of liver injury, whereas such an anti-IL-12 treatment did not. Anti-IFN-gamma treatment reduced the late increase in TNF-alpha, but not FasL, resulting in a partial prevention of the liver injury. The administration of anti-TNF-alpha just before elevation of the late TNF-alpha peak also markedly, but incompletely, suppressed the LPS-induced liver injury. These data suggested that IL-18 activates both TNF-alpha- and FasL-mediated hepatocytotoxic pathways in endotoxin-induced liver injury.