Michio Tomura

Synergy of IL-12 and IL-18 for IFN-γ Gene Expression: IL-12-Induced STAT4 Contributes to IFN-γ Promoter Activation by Up-Regulating the Binding Activity of IL-18-Induced Activator Protein 1

IL-12 and IL-18 synergistically enhance IFN-γ mRNA transcription by activating STAT4 and AP-1, respectively. However, it is still unknown how STAT4/AP-1 elicit IFN-γ promoter activation. Using an IL-12/IL-18-responsive T cell clone, we investigated the mechanisms underlying synergistic enhancement of IFN-γ mRNA expression induced by these two cytokines. Synergy was observed in a reporter gene assay using an IFN-γ promoter fragment that binds AP-1, but not STAT4. An increase in c-Jun, a component of AP-1, in the nuclear compartment was elicited by stimulation with either IL-12 or IL-18, but accumulation of serine-phosphorylated c-Jun was induced only by IL-18 capable of activating c-Jun N-terminal kinase. The binding of AP-1 to the relevant promoter sequence depended on the presence of STAT4. STAT4 bound with c-Jun, and a phosphorylated c-Jun-STAT4 complex most efficiently interacted with the AP-1-relevant promoter sequence. Enhanced cobinding of STAT4 and c-Jun to the AP-1 sequence was also observed when activated lymph node T cells were exposed to IL-12 plus IL-18. These results show that STAT4 up-regulates AP-1-mediated IFN-γ promoter activation without directly binding to the promoter sequence, providing a mechanistic explanation for IL-12/IL-18-induced synergistic enhancement of IFN-γ gene expression.

Granulocyte/Macrophage Colony-Stimulating Factor Inhibits IL-12 production of Mouse Langerhans Cells

We investigated the capacity of mouse Langerhans cells (LC) to produce IL-12, a central cytokine in a Th1 type of immune responses. We prepared purified LC (>95%) from BALB/c mouse skin by the panning method using anti-I-Ad mAb. An ELISA showed that purified LC spontaneously produced IL-12 p40, and that its production was up-regulated following simultaneous stimulation with anti-CD40 mAb and IFN-γ. Surprisingly, GM-CSF strikingly inhibited IL-12 p40 production by anti-CD40/IFN-γ-stimulated LC (% inhibition = 97.0 ± 0.9% at 1 ng/ml GM-CSF). Supernatants of 48-h cultured keratinocytes (KC) also caused the inhibition of LC IL-12 p40 secretion, and this effect was neutralized by anti-GM-CSF mAb. IL-1α (1 ng/ml)-stimulated KC produced much more GM-CSF than unstimulated KC (60.9 ± 0.2 pg/ml vs 20.9 ± 1.7 pg/ml), and IL-1α-stimulated KC supernatants strongly inhibited IL-12 p40 production by anti-CD40/IFN-γ-stimulated LC (% inhibition = 89.4 ± 1.4%). A bioassay using an IL-12-dependent T cell line demonstrated the correlation of the level of IL-12 p40 with the bioactivity of IL-12. These results provide important implications for the pathogenesis of atopic dermatitis, which involves the participation of LC and KC with the capacity to produce IL-12 and GM-CSF, respectively.

An Absolute Requirement for STAT4 and a Role for IFN-γ 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-γ 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-18Rα 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-18Rβ 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-18Rα expression but also to induction of IL-18R binding sites on both CD4+ and CD8+ T cells concomitant with IL-18Rβ 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-18Rβ mRNA expression was not observed in STAT4-deficient (STAT4−/−) T cells and was substantially decreased in IFN-γ−/− T cells. However, the failure of STAT4−/− T cells to induce an IL-18R complex was not corrected by IFN-γ. These results indicate that STAT4 and IFN-γ play an indispensable role and a role as an amplifying factor, respectively, in IL-12 induction of the functional IL-18R complex.

IL-12 as well as IL-2 upregulates CCR5 expression on T cell receptor-triggered human CD4+ and CD8+ T cells.

The expression of chemokine receptors on leukocytes is related to their activation state. However, the exact mechanism underlying the induction of each chemokine receptor is poorly understood. Here, we investigated how CCR5, a chemokine receptor implicated in T cell trafficking and HIV infection, is induced in human T cells. CCR5 was marginally detected on a freshly prepared human peripheral blood mononuclear cell (PBMC) population. Long-term (8-day) stimulation of PBMC with IL-2 resulted in high levels of CCR5 expression on T cells. IL-12 failed to induce CCR5 on T cells in such a directly stimulated PBMC population. Stimulation of PBMC T cells with anti-CD3 plus anti-CD28 induced detectable albeit very low levels of CCR5 along with the induction of IL-12 receptor. However, these TCR-triggered T cells expressed much higher levels of CCR5 when stimulated with IL-12. Although IL-2 also induced CCR5 expression, CCR5 expression was more potent in IL-12 than IL-2 stimulation. These results indicate that, in addition to IL-2, IL-12 plays an important role in the induction of CCR5 expression on T cells, particularly TCR-triggered T cells.

A critical role for IL-12 in CCR5 induction on T cell receptor-triggered mouse CD4(+) and CD8(+) T cells.

Despite increasing evidence for the role of the chemokine system in leukocyte trafficking, the mechanism underlying the induction of chemokine receptors is poorly understood. Here, we investigated how CCR5, a chemokine receptor implicated in T cell migration to inflammatory sites, is induced in the T cell. CCR5 mRNA was hardly detected in resting T cells and marginally induced following T cell receptor (TCR) stimulation. However, TCR‐triggered T cells expressed IL‐12 receptor, and stimulation with recombinant IL‐12 resulted in high levels of CCR5 expression on both CD4+ and CD8+ T cells. In contrast, IL‐2 failed to up‐regulate CCR5 expression. The effect of IL‐12 was selective to CCR5 because IL‐12 did not up‐regulate CXCR3 expression. Surface expression of CCR5 was shown by staining with anti‐CCR5 monoclonal antibody. Stimulation of these CCR5‐positive T cells with the relevant chemokine MIP‐1α elicited Ca2+ influx, showing that IL‐12‐induced CCR5 is functional. These results indicate a critical role for IL‐12 in the induction of CCR5 on TCR‐triggered T cells.

ESTABLISHMENT OF AN IL-12-RESPONSIVE T CELL CLONE : ITS CHARACTERIZATION AND UTILIZATION IN THE QUANTITATION OF IL-12 ACTIVITY

We previously demonstrated that proliferation of terminally differentiated Thl clones depends primarily on an interleukin‐12 (IL‐12)‐paracrme mechanism mediated by their interactions with antigen‐presenting cells (APC) rather than on an IL‐2‐autocrine mechanism. Such a Thl clone (4‐86, C57BL/6 origin) was cultured with recombinant IL‐12 (rIL‐12) in the absence of either antigen or APC. Some cells survived for several passages of culture with only rIL‐12, and by limiting dilution, several clones highly reactive to rIL‐12 alone were obtained. One of these clones, designated 2D6, was found to proliferate strongly in response to less than 1 pg/mL of rIL‐12. This clone exhibited the following surface phenotypes: CD3+, T cell receptor (TCR) αβ+, Vβ11+, NK‐1.1‐; CD4‐ CD8‐; LFA‐1+, ICAM‐1+; and CD28+, CD80+, CD86+, CTLA‐4‐. In accordance with high responsiveness to IL‐12, 2D6 cells were also found to express IL‐12 receptor (IL‐12R) as detected by incubation with rIL‐12 and then staining with anti‐IL‐12 monoclonal antibody (mAb). Stimulation of 2D6 with rIL‐12 resulted in the expression of interferon‐γ (IFN‐γ) and IL‐10 mRNAs and production of these cytokines. The 2D6 clone responded to IL‐2 (vigorously), IL‐7 (moderately), and IL‐4 (mildly) in addition to IL‐12. However, the Ab capture assay using anti‐IL‐12 mAb enabled us to quantify IL‐12‐specific activity contained in a given sample. Thus, this study describes the unique features of the IL‐12‐responsive T cell clone and demonstrates the utilization of this clone in the quantitation of a specific IL‐12 activity. J. Leukoc. Biol. 61: 346–352; 1997.

A Mandatory Role for STAT4 in IL-12 Induction of Mouse T Cell CCR5

IL-12 was recently shown to induce CCR5 on TCR-triggered mouse T cells. Considering that STAT4 is the most critical of IL-12 signaling molecules, this study investigated the role for STAT4 in the induction of CCR5 expression. IL-12R was induced by stimulation with anti-CD3 plus anti-CD28 mAb similarly on T cells from wild-type (WT) and STAT4-deficient (STAT4−/−) mice, but the levels of IL-12R induced on IFN-γ-deficient (IFN-γ−/−) T cells were lower compared with WT T cells. Exposure of TCR-triggered WT T cells to IL-12 induced CCR5 expression. In contrast, TCR-triggered STAT4−/− T cells failed to express CCR5 in response to IL-12. IL-12 stimulation induced detectable albeit reduced levels of CCR5 expression on IFN-γ−/− T cells. Addition of rIFN-γ to cultures of IFN-γ−/− T cells, particularly to cultures during TCR triggering resulted in restoration of CCR5 expression. However, CCR5 expression was not induced in STAT4−/− T cells by supplementation of rIFN-γ. These results indicate that for the induction of CCR5 on T cells, 1) STAT4 plays an indispensable role; 2) such a role is not substituted by simply supplementing rIFN-γ; and 3) IFN-γ amplifies CCR5 induction depending on the presence of STAT4.

IFN-α Acts on T-Cell Receptor-Triggered Human Peripheral Leukocytes to Up-Regulate CCR5 Expression on CD4+ and CD8+ T Cells

Interleukin-12 (IL-12) as well as IL-2 was recently shown to up-regulate CCR5 expression on T-cell receptor (TCR)-triggered human T cells. Because of the functional similarity between interferon-alpha (IFN-α) and IL-12, the present study investigated whether IFN-α also up-regulates T cell CCR5 expression. CCR5 was marginally detected on T cells from unstimulated human peripheral blood leukocytes (PBLs) and only slightly induced on PBL T cells following stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies (mAbs). When anti-CD3/anti-CD28-triggered PBLs were exposed to IFN-α, T cells expressed high levels of CCR5. The levels of CCR5 expression were comparable to those induced by IL-12. However, when purified T cells instead of unfractionated PBL were stimulated with anti-CD3/CD28 and then exposed to IL-12 or IFN-α, CCR5 expression was induced by IL-12 but not by IFN-α. IFN-α was found to act on anti-CD3/anti-CD28-stimulated PBL to promote their IL-12 production. Moreover, addition of anti-IL-12 mAb to IFN-α-stimulated cultures of anti-CD3/CD28-pretreated PBL resulted in considerable inhibition of CCR5 expression. Together, these results indicate that IFN-α as well as IL-12 up-regulates CCR5 expression on TCR-triggered T cells and that IFN-α functions not by acting directly on T cells but via enhancing IL-12 production by PBL.

IL-12 plays a pivotal role in LFA-1-mediated T cell adhesiveness by up-regulation of CCR5 expression

The chemokine receptor CCR5 has been implicated in the recruitment of T cells to inflammatory sites. However, the regulation of CCR5 induction on T cells and its contribution to T cell adhesiveness are poorly understood. Using a Th1 clone, 2D6, that can be maintained with interleukin (IL)‐12 or IL‐2 alone (designated 2D6IL‐12 or 2D6IL‐2, respectively), we investigated how CCR5 is induced on T cells and whether CCR5 is responsible for up‐regulating the function of adhesion molecules. 2D6IL‐12 grew, forming cell aggregates, in culture containing IL‐12. This was due to lymphocyte function‐associated antigen (LFA)‐1–intercellular adhesion molecule (ICAM)‐1 interaction, because 2D6IL‐12 expressed both LFA‐1 and ICAM‐1 and cell aggregation was inhibited by anti‐ICAM‐1 monoclonal antibody. Despite comparable levels of LFA‐1 and ICAM‐1 expression, 2D6IL‐2 cells did not aggregate in culture with IL‐2. It is important that there was a critical difference in CCR5 expression between 2D6IL‐12 and 2D6IL‐2; the former expressed high levels of CCR5, and the latter expressed only marginal levels. Both types of cells expressed detectable albeit low levels of RANTES (regulated on activation, normal T expressed and secreted) mRNA. Unlike IL‐12 or IL‐2, IL‐18 induced high levels of RANTES mRNA expression without modulating CCR5 expression. Therefore, combined stimulation with IL‐12 and IL‐18 strikingly up‐regulated 2D6 cell aggregation. Notably, LFA‐1‐mediated aggregation of 2D6IL‐12 cells was suppressed by anti‐CCR5 antibody. These results indicate that IL‐12 plays a critical role in CCR5 expression on Th1 cells and consequently contributes to CCR5‐mediated activation of LFA‐1 molecules.

CD28 costimulation is required not only to induce IL-12 receptor but also to render Janus kinases / STAT4 responsive to IL-12 stimulation in TCR-triggered T cells

The activation of resting T cells for the acquisition of various functions depends on whether CD28 costimulatory signals are provided upon T cell receptor stimulation. Here, we investigated how CD28 costimulation functions to allow TCR‐triggered resting T cells to acquire IL‐12 responsiveness. When T cells are stimulated with low doses of anti‐CD3 mAb, CD28 costimulation was required for the optimal levels of IL‐12 receptor (IL‐12R) expression. However, stimulation of T cells with high doses of anti‐CD3 alone induced comparable levels of IL‐12R expression to those induced upon CD28 costimulation. Nevertheless, there was a substantial difference in IL‐12 responsiveness between these two groups of T cells: compared to anti‐CD28‐costimulated T cells, T cells that were not costimulated with anti‐CD28 exhibited decreased levels of Janus kinases (JAK) JAK2u2009/u2009TYK2 and STAT4 phosphorylation and IFN‐γ production following IL‐12 stimulation. Importantly, STAT6 phosphorylation following IL‐4 stimulation was not decreased in anti‐CD28‐uncostimulated T cells. These resutls indicate that CD28 costimulation not only contributes to up‐regulating IL‐12R expression but is also required to render JAKsu2009/u2009STAT4 responsive to IL‐12 stimulation.