Autocrine signaling by IL-10 mediates altered responsiveness of atopic sensitized airway smooth muscle

To elucidate the role and mechanism of action of interleukin (IL)-10 in regulating airway smooth muscle (ASM) responsiveness in the atopic asthmatic state, isolated rabbit tracheal ASM segments were passively sensitized with serum from atopic asthmatic patients or nonatopic nonasthmatic (control) subjects in both the absence and presence of an anti-IL-10 receptor blocking antibody (Ab). Relative to control ASM, atopic asthmatic serum-sensitized tissues exhibited enhanced isometric constrictor responses to administered acetylcholine and attenuated the relaxation responses to isoproterenol. These proasthmatic effects were prevented in atopic asthmatic serum-sensitized ASM that was pretreated with anti-IL-10 receptor Ab. In complementary experiments, exposure of cultured human ASM cells to atopic asthmatic serum induced upregulated expression of IL-10 mRNA. Moreover, extended studies demonstrated that 1) exogenous IL-10 administration to naive ASM elicited augmented contractility to acetylcholine and impaired relaxation to isoproterenol, 2) these effects of IL-10 were prevented by pretreating the tissues with an IL-5 receptor Ab, and 3) IL-10 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of atopic asthmatic serum-sensitized ASM is largely attributed to activation of an intrinsic T helper type 2-type autocrine mechanism involving IL-10-mediated release and the action of IL-5 in the sensitized ASM itself.     

Intrinsic ICAM-1/LFA-1 activation mediates altered responsiveness of atopic asthmatic airway smooth muscle

Cell adhesion molecules (CAMs) have been importantly implicated in the pathobiology of the airway responses in allergic asthma, including inflammatory cell recruitment into the lungs and altered bronchial responsiveness. To elucidate the mechanism of CAM-related mediation of altered airway responsiveness in the atopic asthmatic state, the expressions and actions of intercellular adhesion molecule-1 (ICAM-1) and its counterreceptor ligand lymphocyte function-associated antigen-1 (LFA-1; i.e., CD11a/CD18) were examined in isolated rabbit airway smooth muscle (ASM) tissues and cultured human ASM cells passively sensitized with sera from atopic asthmatic patients or nonatopic nonasthmatic (control) subjects. Relative to control tissues, the atopic asthmatic sensitized ASM exhibited significantly enhanced maximal contractility to acetylcholine and attenuated relaxation responses to isoproterenol. These proasthmatic changes in agonist responsiveness were ablated by pretreating the atopic sensitized tissues with a monoclonal blocking antibody (MAb) to either ICAM-1 or CD11a, whereas a MAb directed against the related beta(2)-integrin Mac-1 had no effect. Moreover, relative to control tissues, atopic asthmatic sensitized ASM cells displayed an autologously upregulated mRNA and cell surface expression of ICAM-1, whereas constitutive expression of CD11a was unaltered. Extended studies further demonstrated that 1) the enhanced expression and release of soluble ICAM-1 by atopic sensitized ASM cells was prevented when cells were pretreated with an interleukin (IL)-5-receptor-alpha blocking antibody and 2) administration of exogenous IL-5 to naive (nonsensitized) ASM cells induced a pronounced soluble ICAM-1 release from the cells. Collectively, these observations provide new evidence demonstrating that activation of the CAM counterreceptor ligands ICAM-1 and LFA-1, both of which are endogenously expressed in ASM cells, elicits autologously upregulated IL-5 release and associated changes in ICAM-1 expression and agonist responsiveness in atopic asthmatic sensitized ASM.     

Autocrine cytokine signaling mediates effects of rhinovirus on airway responsiveness

The airway responses to allergen exposure in allergic asthma are qualitatively similar to those elicited by specific viral respiratory pathogens, most notably rhinovirus (RV), suggesting that the altered airway responsiveness seen in allergic asthma and that elicited by viral respiratory tract infection may share a common underlying mechanism. To the extent that T helper cell type 2 (Th2) cytokines have been implicated in the pathogenesis of allergic asthma, this study examined the potential role(s) of Th2-type cytokines in mediating pro-asthmatic-like changes in airway smooth muscle (ASM) responsiveness after inoculation of naive ASM with human RV. Isolated rabbit ASM tissues and cultured human ASM cells were exposed to RV (serotype 16) for 24 h in the absence and presence of monoclonal blocking antibodies (MAbs) or antagonists directed against either the Th2-type cytokines interleukin (IL)-4 and IL-5, intercellular adhesion molecule (ICAM)-1 (the endogenous host receptor for most RVs), or the pleiotropic proinflammatory cytokine IL-1beta. Relative to control (vehicle-treated) tissues, RV-exposed ASM exhibited significantly enhanced isometric contractility to acetylcholine and impaired relaxation to isoproterenol. These pro-asthmatic-like changes in ASM responsiveness were ablated by pretreating the RV-exposed tissues with either IL-5-receptor-alpha blocking antibody or human recombinant IL-1-receptor antagonist, whereas IL-4 neutralizing antibody had no effect. Extended studies further demonstrated that inoculation of ASM cells with RV elicited 1) an increased mRNA expression and release of IL-5 protein, which was inhibited in the presence of anti-ICAM-1 MAb, and 2) an enhanced release of IL-1beta protein, which was inhibited in the presence of IL-5 receptor-alpha antibody. Collectively, these observations provide new evidence demonstrating that RV-induced changes in ASM responsiveness are largely attributed to ICAM-1-dependent activation of a cooperative autocrine signaling mechanism involving upregulated IL-5-mediated release of IL-1beta by the RV-exposed ASM itself.     

Superantigen presentation by airway smooth muscle to CD4+ T lymphocytes elicits reciprocal proasthmatic changes in airway function

Microbial products serving as superantigens (SAgs) have been implicated in triggering various T cell-mediated chronic inflammatory disorders, including severe asthma. Given earlier evidence demonstrating that airway smooth muscle (ASM) cells express MHC class II molecules, we investigated whether ASM can present SAg to resting CD4(+) T cells, and further examined whether this action reciprocally elicits proasthmatic changes in ASM responsiveness. Coincubation of CD4(+) T cells with human ASM cells pulsed with the SAg, staphylococcal enterotoxin A (SEA), elicited adherence and clustering of class II and CD3 molecules at the ASM/T cell interface, indicative of immunological synapse formation, in association with T cell activation. This ASM/T cell interaction evoked up-regulated mRNA expression and pronounced release of the Th2-type cytokine, IL-13, into the coculture medium, which was MHC class II dependent. Moreover, when administering the conditioned medium from the SEA-stimulated ASM/T cell cocultures to isolated naive rabbit ASM tissues, the latter exhibited proasthmatic-like changes in their constrictor and relaxation responsiveness that were prevented by pretreating the tissues with an anti-IL-13 neutralizing Ab. Collectively, these observations are the first to demonstrate that ASM can present SAg to CD4(+) T cells, and that this MHC class II-mediated cooperative ASM/T cell interaction elicits release of IL-13 that, in turn, evokes proasthmatic changes in ASM constrictor and relaxant responsiveness. Thus, a new immuno-regulatory role for ASM is identified that potentially contributes to the pathogenesis of nonallergic (intrinsic) asthma and, accordingly, may underlie the reported association between microbial SAg exposure, T cell activation, and severe asthma.     

Mechanism of cooperative effects of rhinovirus and atopic sensitization on airway responsiveness

To elucidate the mechanistic interplay between rhinovirus (RV) exposure and atopic sensitization in regulating airway smooth muscle (ASM) responsiveness, isolated rabbit ASM tissue and cultured human ASM cells were passively sensitized with sera from atopic asthmatic or nonatopic nonasthmatic (control) subjects in the absence and presence of inoculation with RV serotype 16. Relative to control subjects, atopic asthmatic serum-sensitized and RV-inoculated ASM exhibited significantly increased contractility to acetylcholine, impaired relaxation to isoproterenol, and enhanced release of the proinflammatory cytokine interleukin-1beta. These effects were potentiated in atopic asthmatic serum-sensitized ASM concomitantly inoculated with RV and inhibited by pretreating the tissues with monoclonal blocking antibodies against intercellular adhesion molecule (ICAM)-1 (CD54), the host receptor for RV serotype 16, or lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18), the endogenous counterreceptor for ICAM-1. Moreover, RV inoculation was found to potentiate the induction of mRNA and surface protein expression of FcepsilonRII (CD23), the low-affinity receptor for IgE, in atopic asthmatic serum-sensitized ASM. Collectively, these observations provide new evidence demonstrating that 1) RV exposure and atopic sensitization act cooperatively to potentiate induction of proasthmatic changes in ASM responsiveness in association with upregulated proinflammatory cytokine release and FcepsilonRII expression and 2) the effects of RV exposure and atopic sensitization are mediated by cooperative ICAM-1-coupled LFA-1 signaling in the ASM itself.     

IL-13 may mediate allergen-induced hyperresponsiveness independently of IL-5 or eotaxin by effects on airway smooth muscle

IL-13 is a mediator of allergen-induced airway hyperresponsiveness (AHR). The aim of this study was to evaluate whether eotaxin and IL-5 were implicated in the effects of IL-13 on allergen-induced AHR or whether IL-13 may exert its effects through direct actions on airway smooth muscle (ASM). To study this question airway inflammation and AHR were induced in mice by sensitization and subsequent challenge on three successive days with ovalbumin. A monoclonal anti-IL-13 antibody administered before each challenge significantly reduced AHR without affecting airway eosinophilia. No changes of mRNA in BAL and lung tissues or protein levels in BAL of IL-5 or eotaxin were found following anti-IL-13 treatment. Combined injection of monoclonal anti-IL-5 and antieotaxin antibodies before each antigen challenge blocked airway eosinophilia but failed to reduce AHR. IL-13 induced calcium transients in cultured murine ASM cells and augmented the calcium and contractile responses of these cells to leukotriene D4. These results suggest that IL-13 plays an important role in allergen-induced AHR and is important in the early phases of the inflammatory process. Its effects on AHR are mediated independently of IL-5 and eotaxin and may involve a direct effect on ASM to augment its responsiveness.     

IL-4 suppression of in vivo T cell activation and antibody production

Injection of mice with a foreign anti-IgD Ab stimulates B and T cell activation that results in large cytokine and Ab responses. Because most anti-IgD-activated B cells die before they can be stimulated by activated T cells, and because IL-4 prolongs the survival of B cells cultured with anti-Ig, we hypothesized that treatment with IL-4 at the time of anti-IgD Ab injection would decrease B cell death and enhance anti-IgD-induced Ab responses. Instead, IL-4 treatment before or along with anti-IgD Ab suppressed IgE and IgG1 responses, whereas IL-4 injected after anti-IgD enhanced IgE responses. The suppressive effect of early IL-4 treatment on the Ab response to anti-IgD was associated with a rapid, short-lived increase in IFN-gamma gene expression but decreased CD4+ T cell activation and decreased or delayed T cell production of other cytokines. We examined the possibilities that IL-4 stimulation of IFN-gamma production, suppression of IL-1 or IL-2 production, or induction of TNF-alpha or Fas-mediated apoptosis could account for IL-4's suppressive effect. The suppressive effect of IL-4 was not reversed by IL-1, IL-2, or anti-TNF-alpha or anti-IFN-gamma mAb treatment, or mimicked by treatment with anti-IL-2Ralpha (CD25) and anti-IL-2Rbeta (CD122) mAbs. Early IL-4 treatment failed to inhibit anti-IgD-induced Ab production in Fas-defective lpr mice; however, the poor responsiveness of lpr mice to anti-IgD made this result difficult to interpret. These observations indicate that exposure to IL-4, while T cells are first being activated by Ag presentation, can inhibit T cells activation or promote deletion of responding CD4+ T cells.     

Sevoflurane Inhibits Nuclear Factor-κB Activation in Lipopolysaccharide-Induced Acute Inflammatory Lung Injury via Toll-Like Receptor 4 Signaling

Background

Infection is a common cause of acute lung injury (ALI). This study was aimed to explore whether Toll-like receptors 4 (TLR₄) of airway smooth muscle cells (ASMCs) play a role in lipopolysaccharide (LPS)-induced airway hyperresponsiveness and potential mechanisms.

Methods

In vivo: A sensitizing dose of LPS (50 µg) was administered i.p. to female mice before anesthesia with either 3% sevoflurane or phenobarbital i.p. After stabilization, the mice were challenged with 5 µg of intratracheal LPS to mimic inflammatory attack. The effects of sevoflurane were assessed by measurement of airway responsiveness to methacholine, histological examination, and IL-1, IL-6, TNF-α levels in bronchoalveolar lavage fluid (BALF). Protein and gene expression of TLR₄ and NF-κB were also assessed. In vitro: After pre-sensitization of ASMCs and ASM segments for 24h, levels of TLR₄ and NF-κB proteins in cultured ASMCs were measured after continuous LPS exposure for 1, 3, 5, 12 and 24h in presence or absence of sevoflurane. Constrictor and relaxant responsiveness of ASM was measured 24 h afterwards.

Results

The mRNA and protein levels of NF-κB and TLR₄ in ASM were increased and maintained at high level after LPS challenge throughout 24h observation period, both in vivo and in vitro. Sevoflurane reduced LPS-induced airway hyperresponsiveness, lung inflammatory cell infiltration and proinflammatory cytokines release in BALF as well as maximal isometric contractile force of ASM segments to acetylcholine, but it increased maximal relaxation response to isoproterenol. Treatment with specific NF-κB inhibitor produced similar protections as sevoflurane, including decreased expressions of TLR₄ and NF-κB in cultured ASMCs and improved pharmacodynamic responsiveness of ASM to ACh and isoproterenol.

Conclusions

This study demonstrates the crucial role of TLR₄ activation in ASMCs during ALI in response to LPS. Sevoflurane exerts direct relaxant and anti-inflammatory effects in vivo and in vitro via inhibition of TLR₄/NF-κB pathway.     

Bi-directional activation between human airway smooth muscle cells and T lymphocytes: role in induction of altered airway responsiveness

Because both T lymphocyte and airway smooth muscle (ASM) cell activation are events fundamentally implicated in the pathobiology of asthma, this study tested the hypothesis that cooperative intercellular signaling between activated T cells and ASM cells mediates proasthmatic changes in ASM responsiveness. Contrasting the lack of effect of resting human T cells, anti-CD3-activated T cells were found to adhere to the surface of naive human ASM cells, increase ASM CD25 cell surface expression, and induce increased constrictor responsiveness to acetylcholine and impaired relaxation responsiveness to isoproterenol in isolated rabbit ASM tissues. Comparably, exposure of resting T cells to ASM cells prestimulated with IgE immune complexes reciprocally elicited T cell adhesion to ASM cells and up-regulated T cell expression of CD25. Extended studies demonstrated that: 1) ASM cells express mRNAs and proteins for the cell adhesion molecules (CAMs)/costimulatory molecules, CD40, CD40L, CD80, CD86, ICAM-1 (CD54), and LFA-1 (CD11a/CD18); 2) apart from LFA-1, ASM cell surface expression of the latter molecules is up-regulated in the presence of activated T cells; and 3) pretreatment of ASM cells and tissues with mAbs directed either against CD11a or the combination of CD40 and CD86 completely abrogated both the activated T cell-induced changes in expression of the above CAMs/costimulatory molecules in ASM cells and altered ASM tissue responsiveness. Collectively, these observations identify the presence of bi-directional cross-talk between activated T cells and ASM cells that involves coligation of specific CAMs/costimulatory molecules, and this cooperative intercellular signaling mediates the induction of proasthmatic-like changes in ASM responsiveness.     

Interleukin-2 enhances the growth of human melanoma cells derived form primary but not from metastatic tumours

Previously, we demonstrated that in vitro treatment of B16F10 murine melanoma cells with interleukin-2 (IL-2) enhances proliferation and metastasis. To further investigate the role played by IL-2 in human melanomas, we studied the expression of IL-2/IL-2 receptor and the effect of IL-2 on the proliferation of melanoma cell lines derived from primary (A375 and RMS cell lines) and metastatic (Hs294T cell line) tumours. We found a constitutive expression of cytoplasmic IL-2 and alpha, beta and gamma-subunits of the IL-2R on the surface of the three melanoma cell lines. The presence of IL-2 in the culture increased the proliferation rate in A375 and RMS cell lines, but no effect was observed in Hs294T metastatic cells. Biologically active IL-2 could be found in the supernatant of the three melanoma cell lines, particularly in A375 and RMS cells, in which an inhibition of the proliferation rate was observed when IL-2 was blocked. Moreover, the combination of anti-IL-2R beta and anti-IL-2R gamma blocking antibodies induced a significant down-regulation of cell proliferation in the three melanoma cell lines, and the combination of anti-IL-2R alpha, anti-IL-2R beta and anti-IL-2R gamma blocking antibodies inhibited IL-2-mediated growth stimulation in A375 and Hs294T cell lines. In RMS cells, a more significant effect was observed when only IL-2R gamma was blocked. Finally, exogenous IL-2 modulated the IL-2 endogenously produced by melanoma cells. These data show that IL-2 may modulate the growth of melanoma cells through autocrine or/and paracrine mechanisms.     

Progesterone-induced blocking factor activates STAT6 via binding to a novel IL-4 receptor

Progesterone-induced blocking factor (PIBF) induces Th2-dominant cytokine production. Western blotting and EMSA revealed phosphorylation as well as nuclear translocation of STAT6 and inhibition of STAT4 phosphorylation in PIBF-treated cells. The silencing of STAT6 by small interfering RNA reduced the cytokine effects. Because the activation of the STAT6 pathway depends on the ligation of IL-4R, we tested the involvement of IL-4R in PIBF-induced STAT6 activation. Although PIBF does not bind to IL-4R, the blocking of the latter with an Ab abolished PIBF-induced STAT6 activation, whereas the blocking of the IL-13R had no effect. PIBF activated suppressor of cytokine signaling-3 and inhibited IL-12-induced suppressor of cytokine signaling-1 activation. The blocking of IL-4R counteracted all the described effects, suggesting that the PIBF receptor interacts with IL-4R alpha-chain, allowing PIBF to activate the STAT6 pathway. PIBF did not phosphorylate Jak3, suggesting that the gamma-chain is not needed for PIBF signaling. Confocal microscopic analysis revealed a colocalization and at 37 degrees C a cocapping of the FITC PIBF-activated PIBF receptor and PE anti-IL-4R-labeled IL-4R. After the digestion of the cells with phosphatidylinositol-specific phospholipase C, the STAT6-activating effect of PIBF was lost, whereas that of IL-4 remained unaltered. These data suggest the existence of a novel type of IL-4R composed of the IL-4R alpha-chain and the GPI-anchored PIBF receptor.     

Evaluation of TNF-alpha and IL-1 blockade in collagen-induced arthritis and comparison with combined anti-TNF-alpha/anti-CD4 therapy

We have evaluated the effects of anti-TNF-alpha, anti-IL-1, and combined anti-TNF-alpha/anti-CD4 therapy in collagen-induced arthritis. Blockade of TNF-alpha or IL-1 before disease onset delayed, but did not prevent, the induction of arthritis. When treatment was initiated after onset of arthritis, anti-TNF-alpha, anti-IL-1beta, and anti-IL-1R (which blocks IL-1alpha and IL-1beta) were all found to be effective in reducing the severity of arthritis, with anti-IL-1R and anti-IL-1beta showing greater efficacy than anti-TNF-alpha. Anti-IL-1beta was equally as effective as anti-IL-1R, indicating that IL-1beta plays a more prominent role than IL-1alpha in collagen-induced arthritis. An additive effect was observed between anti-TNF-alpha and anti-IL-1R in the prevention of joint erosion and in normalization of the levels of serum amyloid P. Combined anti-TNF-alpha/anti-CD4 therapy also caused normalization of serum amyloid P levels. The therapeutic effect of anti-TNF-alpha plus anti-CD4 was comparable to that of anti-TNF-alpha plus anti-IL-1R, suggesting that combined anti-TNF-alpha/anti-CD4 therapy prevents both TNF-alpha- and IL-1-mediated pathology. Anti-TNF-alpha treatment reduced IL-1beta expression in the joint and, conversely, anti-IL-1beta treatment reduced TNF-alpha expression. Combined anti-TNF-alpha/anti-CD4 treatment almost completely blocked the expression of IL-1beta, thereby confirming the ability of this form of combination therapy to prevent IL-1ss-mediated pathology.     

Differential regulation of human eosinophil IL-3, IL-5, and GM-CSF receptor alpha-chain expression by cytokines: IL-3, IL-5, and GM-CSF down-regulate IL-5 receptor alpha expression with loss of IL-5 responsiveness,but up-regulate IL-3 receptor alpha expression

Our recent data suggested that tissue eosinophils may be relatively insensitive to anti-IL-5 treatment. We examined cross-regulation and functional consequences of modulation of eosinophil cytokine receptor expression by IL-3, IL-5 GM-CSF, and eotaxin. Incubation of eosinophils with IL-3, IL-5, or GM-CSF led to reduced expression of IL-5R alpha, which was sustained for up to 5 days. Eosinophils incubated with IL-5 or IL-3 showed diminished respiratory burst and mitogen-activated protein kinase kinase phosphorylation in response to further IL-5 stimulation. In contrast to these findings, eosinophil expression of IL-3R alpha was increased by IL-3, IL-5, and GM-CSF, whereas GM-CSF receptor alpha was down-regulated by GM-CSF, but was not affected by IL-3 or IL-5. CCR3 expression was down-regulated by IL-3 and was transiently reduced by IL-5 and GM-CSF, but rapidly returned toward baseline. Eotaxin had no effect on receptor expression for IL-3, IL-5, or GM-CSF. Up-regulation of IL-3R alpha by cytokines was prevented by a phosphoinositol 3-kinase inhibitor, whereas this and other signaling inhibitors had no effect on IL-5R alpha down-regulation. These data suggest dynamic and differential regulation of eosinophil receptors for IL-3, IL-5, and GM-CSF by the cytokine ligands. Since these cytokines are thought to be involved in eosinophil development and mobilization from the bone marrow and are present at sites of allergic inflammation, tissue eosinophils may have reduced IL-5R expression and responsiveness, and this may explain the disappointing effect of anti-IL-5 therapy in reducing airway eosinophilia in asthma.     

The relative contribution of IL-4 receptor signaling and IL-10 to susceptibility to Leishmania major

The roles of IL-10 and IL-4 receptor signaling were evaluated in a murine model of Leishmania major infection. In previous studies the L. major substrain LV39 caused progressive, nonhealing lesions in BALB/c mice deficient for IL-4R alpha-chain (IL-4R alpha), while substrain IR173 was highly controlled. To explore whether IL-10 is responsible for inducing susceptibility to LV39, wild-type and IL-4R alpha(-/-) mice were treated with anti-IL-10R mAb, and in a genetic approach, the IL-4R alpha(-/-) mice were crossed with BALB/c IL-10(-/-) mice. In contrast to the lack of resistance conferred by IL-4R alpha gene deletion, partial resistance to LV39 was conferred by IL-10 gene deletion or treatment of BALB/c mice with anti-IL-10R mAb. Lesion sizes and LV39 parasite numbers were further and dramatically reduced in both anti-IL-10R-treated IL-4R alpha(-/-) mice and IL-4R alpha x IL-10 double knockouts. Anti-IL-10R mAb treatment further suppressed parasite growth in IL-4R alpha(-/-) mice infected with L. major IR173. Production of IFN-gamma was only increased relative to wild-type or littermate controls in IL-4R alpha(-/-) mice with complementary defects in IL-10. Comparisons of IFN-gamma-treated infected macrophages in vitro indicated that LV39 required 25- to 500-fold greater concentrations of IFN-gamma than IR173-infected macrophages to achieve a similar efficiency of parasite killing. These studies suggest that regardless of parasite substrain, IL-10 is as important as IL-4/IL-13 in promoting susceptibility to L. major and even more so for those substrains that are relatively resistant to IFN-gamma mediated killing.     

An absolute requirement for STAT4 and a role for IFN-gamma as an amplifying factor in IL-12 induction of the functional IL-18 receptor complex

IL-12 and IL-18 are both proinflammatory cytokines that contribute to promoting Th1 development and IFN-gamma expression. However, neither IL-12R nor IL-18R is expressed as a functional complex on most resting T cells. This study investigated the molecular mechanisms underlying the induction of an IL-18R complex in T cells. Resting T cells expressed IL-18Ralpha chains but did not exhibit IL-18 binding sites as detected by incubation with rIL-18 followed by anti-IL-18 Ab, suggesting a lack of IL-18Rbeta expression in resting T cells. Although they also failed to express IL-12R, stimulation with anti-CD3 plus anti-CD28 generated IL-12R. Exposure of these cells to IL-12 led not only to up-regulation of IL-18Ralpha expression but also to induction of IL-18R binding sites on both CD4(+) and CD8(+) T cells concomitant with IL-18Rbeta mRNA expression. The IL-18 binding site represented a functional IL-18R complex capable of exhibiting IL-18 responsiveness. IL-12 induction of an IL-18R complex and IL-18Rbeta mRNA expression was not observed in STAT4-deficient (STAT4(-/-)) T cells and was substantially decreased in IFN-gamma(-/-) T cells. However, the failure of STAT4(-/-) T cells to induce an IL-18R complex was not corrected by IFN-gamma. These results indicate that STAT4 and IFN-gamma play an indispensable role and a role as an amplifying factor, respectively, in IL-12 induction of the functional IL-18R complex.