Tyrosine kinase inhibitor herbimycin A reduces the stability of cyclin-dependent kinase Cdk6 protein in T-cells

The induction of macrophage-deactivating (interleukin-10 [IL-10] and transforming growth factor beta [TGF-beta] and macrophage-activating (IL-1, IL-6, and tumor necrosis factor alpha [TNF-alpha] cytokines by lipoarabinomannan (LAM) from pathogenic Mycobacterium tuberculosis Erdman and H37Rv strains (ManLAM) and nonpathogenic mycobacteria (AraLAM) in human blood monocytes was examined. ManLAM was significantly less potent in induction of TNF-alpha, IL-1, IL-6, and IL-10 protein and mRNA, whereas its ability to induce TGF-beta was similar to that of AraLAM. Differences in induction of TNF-alpha mRNA by the two LAM preparations only became apparent at late time points of culture (24 h). The induction of TNF-alpha and IL-1 by purified protein derivative of M. tuberculosis was significantly stronger than that by ManLAM. Pretreatment of monocytes with ManLAM did not, however, interfere with cytokine induction by lipopolysaccharide or AraLAM. The extensive mannosyl capping of arabinose termini of ManLAM may underlie the lack of ability to induce some cytokines (IL-1, TNF-alpha, and IL-10) and the retained ability to induce TGF-beta. The latter may have a role in shifting the cytokine milieu in favor of survival of M. tuberculosis.     

Expression of monocyte chemotactic protein-3 in human monocytes exposed to the mycobacterial cell wall component lipoarabinomannan

Monocyte chemotactic protein-3 (MCP-3) is a C-C chemokine which interacts with the CCR1, CCR2 (MCP-1) and CCR3 receptors and has a distinct spectrum of action. The present study was designed to assess whether mycobacterial components were able to induce expression and production of MCP-3 in human monocytes. Mycobacterial lipoarabinomannan (LAM) induced expression of MCP-3 mRNA in human peripheral blood mononuclear cells. The non-mannose-capped version of lipoarabinomannan (AraLAM) was considerably more potent than the mannose-capped version ManLAM or the simpler version phosphatidylinositol mannoside (PLM). Among mononuclear cells, monocytes were responsible for LAM-induced MCP-3 mRNA expression. Whole mycobacteria (Mycobacterium bovis BCG) strongly induced MCP-3 expression. Pretreatment with actinomycin D abolished LAM-induced MCP-3 expression, whereas cycloheximide only partially reduced the expression. LAM-induced MCP-3 expression was associated with the production of immunoreactive PTX3. Interleukin 10 (IL-10) and IL-13 inhibited the induction of MCP-3 by LAM. Thus mycobacterial cell wall components induced expression of MCP-3 in human monocytes. MCP-3, a chemokine active on mononuclear phagocytes, NK cells, T cells and dendritic cells, may be relevant to the induction and expression of immunity against mycobacteria.     

Cytokine networks with infection: mycobacterial infections, leishmaniasis, human immunodeficiency virus infection, and sepsis

Distinct cytokine profiles are clearly associated with and relate to the severity of several types of infections. Cytokine networks are apparent with selected human infectious diseases, such as mycobacterial infections (leprosy, tuberculosis), the parasitic infection leishmaniasis, human immunodeficiency virus (HIV) infection, and gram-negative sepsis. Cytokine profiles are determined to some extent by two functional subsets of T lymphocytes, Th1 and Th2. The Th1 cytokines (interferon gamma, interleukin-2 [IL-2], IL-12) enhance cell-mediated immunity, inhibit humoral immunity, and result in protective effect for pathogens that are removed primarily through cell-mediated immunity (Mycobacterium tuberculosis, Mycobacterium leprae, Leishmania). The Th2 cytokines (IL-4, IL-5, IL-10, IL-13) enhance humoral immunity and inhibit cell-mediated immunity, and result in protective effect for pathogens removed primarily through humoral mechanisms. Progression of HIV infection is associated with a switch from a Th1 to a Th2 profile. For sepsis, uncontrolled activation of proinflammatory cytokines (IL-1, tumor necrosis factor-alpha, interferon-gamma) may be a fundamental defect that promotes the detrimental aspects of inflammation, whereas Th2 cytokines may be beneficial in controlling inflammation. Knowledge of basic cytokine immunopharmacology, networks, and relationships with infectious processes will aid clinicians in determining treatment approaches that are likely to be effective.     

Determinant in human immunodeficiency virus type 1 for efficient replication under cytokine-induced CD4(+) T-helper 1 (Th1)- and Th2-type conditions

Cytokines are potent stimuli for CD4(+)-T-cell differentiation. Among them, interleukin-12 (IL-12) and IL-4 induce naive CD4(+) T cells to become T-helper 1 (Th1) or Th2 cells, respectively. In this study we found that macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains replicated more efficiently in IL-12-induced Th1-type cultures derived from normal CD4(+) T cells than did T-cell-line-tropic (T-tropic) strains. In contrast, T-tropic strains preferentially infected IL-4-induced Th2-type cultures derived from the same donor CD4(+) T cells. Additional studies using chimeric viruses demonstrated that the V3 region of HIV-1 gp120 was the principal determinant for efficiency of replication. Cell fusion analysis showed that cells expressing envelope protein from a T-tropic strain effectively fused with IL-4-induced Th2-type culture cells. Flow cytometric analysis showed that the level of CCR5 expression was higher on IL-12-induced Th1-type culture cells, whereas CXCR4 was highly expressed on IL-4-induced Th2-type culture cells, although a low level of CXCR4 expression was observed on IL-12-induced Th1-type culture cells. These results indicate that HIV-1 isolates exhibit differences in the ability to infect CD4(+)-T-cell subsets such as Th1 or Th2 cells and that this difference may partly correlate with the expression of particular chemokine receptors on these cells. The findings suggest that immunological conditions are one of the factors responsible for inducing selection of HIV-1 strains.     

Lipoarabinomannan of Mycobacterium tuberculosis promotes protein tyrosine dephosphorylation and inhibition of mitogen-activated protein kinase in human mononuclear phagocytes. Role of the Src homology 2 containing tyrosine phosphatase 1

Lipoarabinomannan (LAM) is a putative virulence factor of Mycobacterium tuberculosis that inhibits monocyte functions, and this may involve antagonism of cell signaling pathways. The effects of LAM on protein tyrosine phosphorylation in cells of the human monocytic cell line THP-1 were examined. LAM promoted tyrosine dephosphorylation of multiple cell proteins and attenuated phorbol 12-myristate 13-acetate-induced activation of mitogen-activated protein kinase. To examine whether these effects of LAM could be related to activation of a phosphatase, fractions from LAM-treated cells were analyzed for dephosphorylation of para-nitrophenol phosphate. The data show that LAM induced increased phosphatase activity associated with the membrane fraction. The Src homology 2 containing tyrosine phosphatase 1 (SHP-1) is important for signal termination and was examined as a potential target of LAM. Exposure of cells to LAM brought about (i) an increase in tyrosine phosphorylation of SHP-1, and (ii) translocation of the phosphatase to the membrane. Phosphatase assay of SHP-1 immunoprecipitated from LAM-treated cells, using phosphorylated mitogen-activated protein kinase as substrate, indicated that LAM promoted increased activity of SHP-1 in vivo. LAM also activated SHP-1 directly in vitro. Exposure of cells to LAM also attenuated the expression of tumor necrosis factor-alpha, interleukin-12, and major histocompatibility class II molecules. These results suggest that one mechanism by which LAM deactivates monocytes involves activation of SHP-1.     

Amelioration of collagen-induced arthritis and suppression of interferon-gamma, interleukin-12, and tumor necrosis factor alpha production by interferon-beta gene therapy

Human Vgamma9Vdelta2 T cells contribute to immunity against intracellular pathogens and recognize nonpeptidic antigens, such as the mycobacterial phosphoantigen TUBAg. HIV infection is associated with a polyclonal decrease of peripheral Vgamma9Vdelta2 T cells and we previously reported that the remaining cells show a proliferative anergy to stimulation with Mycobacterium tuberculosis in 60% of patients. Because of alterations in the Th1/Th2 cytokine balance reported in HIV infection, we analyzed, at the single-cell level, the influence of exogenous IL-4, IL-10, IL-12 and IL-15 on the response to mycobacterial phosphoantigens of gammadelta T cells from HIV-infected patients and healthy donors. We report that the strong gammadelta T cell response to TUBAg is characterized by the rapid and selective production of the Th1/proinflammatory cytokines IFN-gamma and TNF-alpha in responder HIV-infected donors. In addition, a positive regulation by IL-12 and IL-15 of the production of these cytokines by Vgamma9Vdelta2 T cells in response to nonpeptidic ligands was observed, whereas IL-4 and IL-10 had no effect. In contrast, Vgamma9Vdelta2 T cells from the anergic HIV-infected donors had lost the ability to produce Th1 cytokines and were not shifted towards a Th2 profile. Furthermore, neither IL-12 nor IL-15 could reverse this functional anergy. The consequences of these observations are discussed in the context of HIV pathogenesis.     

In vivo regulation of macrophage IL-12 production during type 1 and type 2 cytokine-mediated granuloma formation

IL-12 is a pivitol cytokine that promotes NK cell activity and Th1 (type 1)-mediated immune responses. This study analyzed the cytokines that regulate macrophage (M phi) IL-12 production in vitro and in vivo. IL-12 was produced by elicited but not resident peritoneal M phi stimulated with endotoxin. Addition of graded doses of cytokines (0.1 to 10 ng/ml) indicated that the Th1-related (type 1) cytokine, IFN-gamma, augmented endotoxin-stimulated IL-12 production by nearly sixfold in oil-elicited M phi. TNF-alpha also increased production but only at the 10 ng/ml concentration. In contrast, the Th2-related (type 2) cytokines, IL-4 and especially IL-10, were profoundly inhibitory. IL-1 beta and IL-2 had no effect. For in vivo analysis, type 1 and type 2 cytokine-mediated lung granulomas (GR) were induced in presensitized mice by embolization of beads coupled to purified protein derivative of Mycobacteria tuberculosis or soluble Ags derived from Schistosoma mansoni eggs. Analysis of M phi isolated from type 1, type 2, or control pulmonary GR revealed that M phi of type 2 GR develop impaired IL-12-producing capacity. Depletion studies using anti-IFN, anti-IL-12, anti-IL-10, and anti-IL-4 neutralizing polyclonal Abs corroborated the in vitro studies. Anti-IFN or anti-IL-12 reduced IL-12 production by M phi from type 1 GR (70 to 80%) as well as IFN and IL-12 production by draining lymph nodes (75 to 90%). Conversely, anti-IL-10 and anti-IL-4 reversed the impaired IL-12 production observed in type 2 GR M phi. These data indicate a positive feedback stimulation of IL-12 production by IFN that is regulated by IL-10 and IL-4 in vivo.     

Expression of the IL-12 receptor beta 1 and beta 2 subunits in human tuberculosis

To determine whether the Th1 response in tuberculosis correlated with IL-12R expression, we measured expression of the IL-12R beta 1 and IL-12R beta 2 subunits, as well as IL-12R beta 2 mRNA expression in tuberculosis patients and healthy tuberculin reactors. In tuberculosis patients, IFN-gamma production by Mycobacterium tuberculosis-stimulated PBMC was reduced, the percentages of T cells expressing IL-12R beta 1 and IL-12R beta 2 were significantly decreased, and IL-12R beta 2 mRNA expression was also markedly reduced. In contrast, in pleural fluid and lymph nodes at the site of disease in tuberculosis patients, in which IFN-gamma production is enhanced, IL-12R beta 2 mRNA expression was also increased. In M. tuberculosis-stimulated peripheral blood T cells from tuberculosis patients, anti-IL-10 and anti-TGF-beta enhanced IL-12R beta 1 and IL-12R beta 2 expression, and IFN-gamma production. In M. tuberculosis-stimulated peripheral blood T cells from healthy tuberculin reactors, recombinant IL-10 and TGF-beta reduced IL-12R beta 1 and IL-12R beta 2 expression, as well as IFN-gamma production. In combination with prior studies showing increased production of TGF-beta by blood monocytes from tuberculosis patients, this suggests that increased TGF-beta production is the underlying abnormality that reduces IL-12R beta 1 and IL-12R beta 2 expression in tuberculosis. Our findings provide evidence that IL-12R expression correlates well with IFN-gamma production in human tuberculosis, and that expression of IL-12R beta 1 and IL-12R beta 2 may play a central role in mediating a protective Th1 response.     

Human eosinophils produce biologically active IL-12: implications for control of T cell responses

The present study assessed the capacity of eosinophils (EOS) to synthesize the cytokine IL-12. Blood-derived, highly purified human EOS from six atopic patients and two nonatopic individuals were treated in culture with IL-4, IL-5, granulocyte-macrophage CSF, IFN-gamma, TNF-alpha, IL-1alpha, RANTES, and complement 5a, respectively. The expression of both IL-12 protein and mRNAs for the p35 and p40 IL-12 subunits was strongly induced in all donors by the Th2-like cytokines IL-4 and granulocyte-macrophage CSF and was also moderately induced by TNF-alpha and IL-1alpha. IL-5 treatment resulted in IL-12 synthesis in four atopic donors and one nonatopic donor, whereas IFN-gamma induced IL-12 synthesis in only two atopic donors. In contrast, RANTES exclusively induced mRNA for the p40 subunit without detectable protein release, and complement 5a had no effect on IL-12 mRNA or protein expression. EOS-derived IL-12 was biologically active, because supernatants derived from IL-4-treated EOS superinduced the Con A-induced expression of IFN-gamma by a human Th1-like T cell line. This activity was neutralized by anti-IL-12 Abs. In conclusion, EOS secrete biologically active IL-12 after treatment with selected cytokines, which mainly represent the Th2-like type. Consequently, EOS may promote a switch from Th2-like to Th1-like immune responses in atopic and parasitic diseases.     

Interferon gamma derived from CD4(+) T cells is sufficient to mediate T helper cell type 1 development

Interferon gamma (IFN-gamma) has been implicated in T helper type 1 (Th1) cell development through its ability to optimize interleukin 12 (IL-12) production from macrophages and IL-12 receptor expression on activated T cells. Various systems have suggested a role for IFN-gamma derived from the innate immune system, particularly natural killer (NK) cells, in mediating Th1 differentiation in vivo. We tested this requirement by reconstituting T cell and IFN-gamma doubly deficient mice with wild-type CD4(+) T cells and challenging the mice with pathogens that elicited either minimal or robust IL-12 in vivo (Leishmania major or Listeria monocytogenes, respectively). Th1 cells developed under both conditions, and this was unaffected by the presence or absence of IFN-gamma in non-T cells. Reconstitution with IFN-gamma-deficient CD4(+) T cells could not reestablish control over L. major, even in the presence of IFN-gamma from the NK compartment. These data demonstrate that activated T cells can maintain responsiveness to IL-12 through elaboration of endogenous IFN-gamma without requirement for an exogenous source of this cytokine.     

Lack of both types 1 and 2 cytokines, tissue inflammatory responses, and immune protection during pulmonary infection by Mycobacterium bovis bacille Calmette-Guérin in IL-12-deficient mice

Understanding of key cytokines and the nature of protective immune responses in pulmonary mycobacterial diseases remains a task of paramount importance. In this study, both wild-type (wt) and IL-12-deficient (IL-12(-/-)) mice were infected by airways inoculation of live Mycobacterium bovis bacille Calmette-Guérin (BCG). The type 1 cytokines IL-12, IFN-gamma, and TNF-alpha, but not the type 2 cytokines IL-4 and granulocyte macrophage (GM)-CSF, markedly increased in the lung and peripheral blood of wt mice postinfection, which resulted in the development of intense granulomatous responses and the effective control of mycobacterial infection in the lung. In contrast, IL-12(-/-) mice demonstrated a lack of both types 1 and 2 cytokines in the lung and blood and a severely impaired tissue immune-inflammatory response lacking not only macrophages and neutrophils but CD4 and CD8 T cells and NK cells in the lung throughout the entire course of study. Total lung mononuclear cells isolated from these mice, in contrast to wt mice, had an impaired recall immune response to Ag challenge in vitro. These impaired responses resulted in an uncontrolled local growth and systemic spread of bacilli. Our findings reveal that IL-12 plays an irreplaceable role in the initiation of Th1 responses, and the loss of its function cannot be compensated for by alternative mechanisms in the lung. This cytokine, together with IFN-gamma and TNF-alpha, and granulomatous inflammation are critically required for the effective control of pulmonary mycobacterial infection. Our results also indicate that the absence of type 1 cytokines does not necessarily favor a Th2 response.     

Lipoarabinomannan inhibits nonopsonic binding of Mycobacterium tuberculosis to murine macrophages

The initial phagocytic interaction between Mycobacterium tuberculosis and macrophages (M phi) in the lung is probably nonopsonic, which would mean that M phi receptors will bind directly to bacterial ligands without the involvement of serum opsonins. Lipoarabinomannan (LAM) is a major component of the cell wall of mycobacteria. The possibility that LAM is involved in the nonopsonic binding of M. tuberculosis to M phi was investigated by using competitive inhibition assays. LAM inhibited binding of M. tuberculosis to murine peritoneal M phi in a dose-dependent manner. LAM also inhibited the binding of Mycobacterium avium and Mycobacterium bovis BCG to M phi. Phosphatidylinositol mannoside and lipomannan have the same phosphatidylinositol (PI) moiety as LAM, but differ in their glycosylation patterns. Both molecules inhibited binding of M. tuberculosis to M phi. Deacylation of LAM abrogated its capacity to inhibit binding of M. tuberculosis to M phi. These observations indicated that it was the PI moiety of LAM that was important in mediating its inhibitory properties. In support of this hypothesis, commercial PI was shown to inhibit the binding of M. tuberculosis to M phi. Our results suggest that cellfree LAM is able to inhibit the binding of mycobacteria to M phi, but that it does not do so by competing with any interaction between M phi receptors and cell-associated LAM, because the PI end of the molecule is believed to be anchored in the bacterial plasma membrane, and therefore not available as a ligand on the cell surface. However, LAM that is released from M. tuberculosis in the course of its active replication during infection may be able to interfere with the phagocytic clearance of mycobacteria.     

Th1 cells induce and Th2 inhibit antigen-dependent IL-12 secretion by dendritic cells

Dendritic cells are the most relevant antigen-presenting cells (APC) for presentation of antigens administered in adjuvant to CD4+ T cells. Upon interaction with antigen-specific T cells, dendritic cells (DC) expressing appropriate peptide-MHC class II complexes secrete IL-12, a cytokine that drives Th1 cell development. To analyze the T cell-mediated regulation of IL-12 secretion by DC, we have examined their capacity to secrete IL-12 in response to stimulation by antigen-specific Th1 and Th2 DO11.10 TCR-transgenic cells. These cells do not differ either in TCR clonotype or CD40 ligand (CD40L) expression. Interaction with antigen-specific Th1, but not Th2 cells, induces IL-12 p40 and p75 secretion by DC. The induction of IL-12 production by Th1 cells does not depend on their IFN-gamma secretion, but requires direct cell-cell contact mediated by peptide/MHC class II-TCR and CD40-CD40L interactions. Th2 cells not only fail to induce IL-12 secretion, but they inhibit its induction by Th1 cells. Unlike stimulation by Th1, inhibition of IL-12 production by Th2 cells is mediated by soluble molecules, as demonstrated by transwell cultures. Among Th2-derived cytokines, IL-10, but not IL-4 inhibit Th1-driven IL-12 secretion. IL-10 produced by Th2 cells appears to be solely responsible for the inhibition of Th1 -induced IL-12 secretion, but it does not account for the failure of Th2 cells to induce IL-12 production by DC. Collectively, these results demonstrate that Th1 cells up-regulate IL-12 production by DC via IFN-gamma-independent cognate interaction, whereas this is inhibited by Th2-derived IL-10. The inhibition of Th1 -induced IL-12 production by Th2 cells with the same antigen specificity represents a novel mechanism driving the polarization of CD4+ T cell responses.     

Decreased drug accumulation in a mitoxantrone-resistant gastric carcinoma cell line in the absence of P-glycoprotein

The developmental commitment to a T helper 1 (Th1)- or Th2-type response can significantly influence host immunity to pathogens. Extinction of the IL-12 signaling pathway during early Th2 development provides a mechanism that allows stable phenotype commitment. In this report we demonstrate that extinction of IL-12 signaling in early Th2 cells results from a selective loss of IL-12 receptor (IL-12R) beta 2 subunit expression. To determine the basis for this selective loss, we examined IL-12R beta 2 subunit expression during Th cell development in response to T cell treatment with different cytokines. IL-12R beta 2 is not expressed by naive resting CD4+ T cells, but is induced upon antigen activation through the T cell receptor. Importantly, IL-4 and IFN-gamma were found to significantly modify IL-12 receptor beta 2 expression after T cell activation. IL-4 inhibited IL-12R beta 2 expression leading to the loss of IL-12 signaling, providing an important point of regulation to promote commitment to the Th2 pathway. IFN-gamma treatment of early developing Th2 cells maintained IL-12R beta 2 expression and restored the ability of these cells to functionally respond to IL-12, but did not directly inhibit IL-4 or induce IFN-gamma production. Thus, IFN-gamma may prevent early Th cells from premature commitment to the Th2 pathway. Controlling the expression of the IL-12R beta 2 subunit could be an important therapeutic target for the redirection of ongoing Th cell responses.     

In vivo effects of T helper cell type 2 cytokines on macrophage antigen-presenting cell induction of T helper subsets

SJL mice provide an interesting paradigm to examine the role(s) of APC in the differential induction of Th1 and Th2 cells. Immunization of young male SJL mice results in the preferential induction of Th2 cells, whereas Th1 cells are induced in age-matched female or older male SJL mice. The absence of Th1 responses in young male mice is associated with in vivo IL-4 and IL-10 down-regulating Mac-3+ APC priming of Th1 cells. The present report examines the mechanism of this APC-dependent induction of Th subsets. Examination of the surface expression of MHC class II, adhesion molecules (CD11a, CD11b, CD48, CD54, and CD102) or costimulatory molecules (CD24, CD80, and CD86) showed no differences between male- and female-derived Mac-3+ APC populations. In addition, no differences were detected in IL-1alpha, IL-1beta, IL-18, TNF-alpha, or IL-12 p35 mRNA expression. However, reduced expression of both IL-10 and IL-12 p40 mRNA were found in Mac-3+ cells from male mice compared with those in Mac-3+ cells from female mice. Anti-IL-4 or anti-IL-10 mAb treatment of young male donor mice eliminated the reduction of both IL-10 and IL-12 p40 mRNA, suggesting that the Th2 inducer phenotype is related to a decreased IL-12 secretion. Consistent with this idea, fewer IL-12 p40-secreting Mac-3+ cells were found in male mice compared with female mice, and treatment with rIL-12 resulted in the priming of Th1 cells in male mice. These data suggest that increased Th2 cytokines in vivo before encounter with Ag inhibit APC expression of IL-12, resulting in the preferential induction of Th2 cells in male SJL mice.     

Meiotic crossing over between nonhomologous chromosomes affects chromosome segregation in yeast

This study addresses the nature of the pathogenic effector T cell in experimental autoimmune uveoretinitis and the effect of different cytokines on these cells in vitro. Lymph node cells of B10.RIII mice immunized with the uveitogenic peptide 161-180 of interphotoreceptor retinoid binding protein were cultured with the peptide with or without IL-12, IL-4, or anti-IL-4. An antigen-specific T cell line was subsequently derived from these cells. Primary cultures of immune lymph node cells stimulated with the peptide proliferated and produced IL-2 and some IL-4, but no IFN-gamma. The addition of recombinant IL-12 resulted in abundant production of IFN-gamma, which was blocked by the addition of IL-4 and was enhanced by anti-IL-4. Only those cultures that produced IFN-gamma in vitro were uveitogenic in vivo. A long-term uveitogenic T cell line, initially derived in the presence of IL-12, produced IFN-gamma and IL-2, but not IL-4, and was CD4+ (Th1-like). Antigen-specific proliferation and IFN-gamma production of the line were enhanced by exogenous IL-4, TGF-beta, IL-2, IL-6, IL-7, and IL-9 and were inhibited by IL-10 and TNF-alpha. Our results provide support for the hypothesis that the uveitogenic effector T cell has a Th1-like phenotype. Furthermore, the data suggest that the effects of the cytokine milieu on fully differentiated Th1 effectors may differ considerably from their effects on less mature stages of antigen-specific T cells.     

Interleukin-12 primes human CD4 and CD8 T cell clones for high production of both interferon-gamma and interleukin-10

Interleukin-12 (IL-12) induces differentiation of T helper 1 (Th1) cells, primarily through its ability to prime T cells for high interferon-gamma (IFN-gamma) production. We now report that the presence of IL-12 during the first several days of in vitro clonal expansion in limiting dilution cultures of polyclonally stimulated human peripheral blood CD4+ and CD8+ T cells also induces stable priming for high IL-10 production. This effect was demonstrated with T cells from both healthy donors and HIV+ patients. Priming for IL-4 production, which requires IL-4, was maximum in cultures containing both IL-12 and IL-4. IL-4 modestly inhibited the IL-12-induced priming for IFN-gamma, but almost completely suppressed the priming for IL-10 production. A proportion of the clones generated from memory CD45RO+ cells, but not those generated from naive CD45RO- CD4+ T cells, produced some combinations of IFN-gamma, IL-10, and IL-4 even in the absence of IL-12 and IL-4, suggesting in vivo cytokine priming; virtually all CD4+ clones generated from either CD45RO(-) or (+) cells, however, produced high levels of both IFN-gamma and IL-10 when IL-12 was present during expansion. These results indicate that each Th1-type (IFN-gamma) and Th2-type (IL-4 and IL-10) cytokine gene is independently regulated in human T cells and that the dichotomy between T cells with the cytokine production pattern of Th1 and Th2 cells is not due to a direct differentiation-inducing effect of immunoregulatory cytokines, but rather to secondary selective mechanisms. Particular combinations of cytokines induce a predominant generation of T cell clones with anomalous patterns of cytokine production (e.g., IFN-gamma and IL-4 or IFN-gamma and IL-10) that can also be found in a proportion of fresh peripheral blood T cells with "memory" phenotype or clones generated from them and that may identify novel Th subsets with immunoregulatory functions.     

Interferon-gamma and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression

Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) are mutually antagonistic cytokines that stimulate CD4+ T cells to develop into either Th1 or Th2 cells. One feature of Th2 differentiation in mice is the loss of IL-12-induced Jak2 and Stat4 activation, which is accompanied by the inability to produce IFN-gamma in response to IL-12. In this report, we show that freshly isolated human T cells activated with phytohemagglutinin (PHA) in the presence of IL-4 exhibit a greatly diminished response to IL-12, whereas the IL-12 response of T cells activated with PHA plus IFN-gamma is enhanced. Radiolabeled IL-12 binding studies demonstrate that the impairment of T cell IL-12 responsiveness by IL-4 is associated with the down-regulation of high-affinity IL-12 receptor expression. In contrast, the enhancement of IL-12 responsiveness by IFN-gamma is associated with the upregulation of high-affinity IL-12 receptor expression. Through the use of a newly synthesized neutralizing antibody to the low-affinity IL-12 receptor beta subunit (IL-12Rbeta), we show that neither IL-4 nor IFN-gamma affect the expression of IL-12Rbeta, which we determine to be one of at least two low-affinity subunits required for high-affinity IL-12 binding. These findings suggest that IL-4 and IFN-gamma exert opposite effects on T cell IL-12 responsiveness by differentially modulating the expression of low-affinity IL-12 receptor subunits that are distinct from IL-12Rbeta and required, together with IL-12Rbeta, for high-affinity IL-12 binding and IL-12 responsiveness. This provides a basis for understanding the interplay between different cytokines at the level of cytokine receptor expression, and offers insight into one of the mechanisms governing Th1 and Th2 development.     

IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN-gamma production

IL-18 is a product of macrophages and with IL-12 strikingly induces IFN-gamma production from T, B, and NK cells. Furthermore, IL-18 and 1L-12 synergize for IFN-gamma production from Th1 cells, although this combination fails to affect Th2 cells. In this study, we show that IL-12 and IL-18 promptly and synergistically induce T and B cells to develop into IFN-gamma-producing cells without engaging their Ag receptors. We also studied the mechanism underlying differences in IL-18 responsiveness between Th1 and Th2 cells. Pretreatment of T or B cells with IL-12 rendered them responsive to IL-18, which induces cell proliferation and IFN-gamma production. These IL-12-stimulated cells had both high and low affinity IL-18R and an increased IL-18R mRNA expression. In particular, IL-12-stimulated T cells strongly and continuously expressed IL-18R mRNA. However, when T cells developed into Th1 cells after stimulation with anti-CD3 and IL-12, they lowered this IL-12-induced-IL-18R mRNA expression. Then, such T cells showed a dominant response to anti-CD3 by IFN-gamma production when they were subsequently stimulated with anti-CD3 and IL-18. In contrast, Th2 cells did not express IL-18R mRNA and failed to produce IFN-gamma in response to anti-CD3 and IL-18, although they produced a substantial amount of IFN-gamma in response to anti-CD3 and IL-12. However, when Th1 and Th2 cells were stimulated with anti-CD3, IL-12, and IL-18, only the Th1 cells markedly augmented IFN-gamma production in response to IL-18, suggesting that IL-18 responsiveness between Th1 and Th2 cells resulted from their differential expression of IL-18R.