Hyun-Jong Ahn

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.

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.

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.

Differential requirements for JAK2 and TYK2 in T cell proliferation and IFN‐γ production induced by IL‐12 alone or together with IL‐18

IL‐12 activates TYK2 and Janus kinase (JAK)‐2 to induce the phosphorylation of various signal transducers and activators of transcription (STAT) proteins. However, little is known regarding how these JAK exhibit the distinct biological effects of IL‐12. Using two JAK inhibitors, tyrphostin A1 (A1) for TYK2 and tyrphostin B42 (B42) for JAK2, we investigated the involvement of JAK2 and TYK2 in IL‐12‐induced T cell proliferation and IFN‐γ production. B42, but not A1, inhibited T cell proliferation along with down‐regulation of IL‐12‐induced c‐Myc expression and STAT5 phosphorylation. In contrast, A1 but not B42 inhibited STAT4/STAT3 phosphorylation and IFN‐γ production. IL‐18, but not IL‐12, induced activator protein‐1 (AP‐1) responsible for high levels of IFN‐γ promoter activation. However, this IL‐18 effect depended on the interaction of AP‐1 with STAT4. A1 prevented AP‐1 binding by inhibiting STAT4 involvement and down‐regulated synergistic IFN‐γ promoter activation. These results indicate that JAK2 activation is required for IL‐12‐mediated T cell growth, whereas the TYK2‐STAT4 signaling pathway is critical for IFN‐γ expression that is mediated by IL‐12 alone and enhanced synergistically by combination with IL‐18.

Requirement for Distinct Janus Kinases and STAT Proteins in T Cell Proliferation Versus IFN-γ Production Following IL-12 Stimulation

While IL-12 is known to activate JAK2 and TYK2 and induce the phosphorylation of STAT4 and STAT3, little is known regarding how the activation of these signaling molecules is related to the biologic effects of IL-12. Using an IL-12-responsive T cell clone (2D6), we investigated their requirements for proliferation and IFN-gamma production of 2D6 cells. 2D6 cells could be maintained with either IL-12 or IL-2. 2D6 lines maintained with IL-12 (2D6(IL-12)) or IL-2 (2D6(IL-2)) exhibited comparable levels of proliferation, but produced large or only small amounts of IFN-gamma, respectively, when restimulated with IL-12 after starvation of either cytokine. 2D6(IL-12) induced TYK2 and STAT4 phosphorylation. In contrast, their phosphorylation was marginally induced in 2D6(IL-2). The reduced STAT4 phosphorylation was due to a progressive decrease in the amount of STAT4 protein along with the passages in IL-2-containing medium. 2D6(IL-12) and 2D6(IL-2) similarly proliferating in response to IL-12 induced comparable levels of JAK2 activation and STAT5 phosphorylation. JAK2 was associated with STAT5, and IL-12-induced STAT5 phosphorylation was elicited in the absence of JAK3 activation. These results indicate that IL-12 has the capacity to induce/maintain STAT4 and STAT5 proteins, and that TYK2 and JAK2 activation correlate with STAT4 phosphorylation/IFN-gamma induction and STAT5 phosphorylation/cellular proliferation, respectively.

Melatonin inhibits the development of 2,4‐dinitrofluorobenzene‐induced atopic dermatitis‐like skin lesions in NC/Nga mice

Abstract:  Atopic dermatitis (AD) is a common disease in children, and epicutaneous treatment with a chemical hapten such as 2,4‐dinitrofluorobenzene (DNFB) evokes an AD‐like reaction in NC/Nga mice under specific pathogen‐free conditions. Melatonin (N‐acetyl‐5‐methoxytryptamine) is synthesized by the pineal gland, has several different physiologic functions, which include seasonal reproduction control, immune system modulation, free radical scavenging, and inflammatory suppression. In the present study, we investigated whether melatonin suppresses DNFB‐induced AD‐like skin lesions in NC/Nga mice. The topical administration of melatonin to DNFB‐treated NC/Nga mice was found to inhibit ear thickness increases and the skin lesions induced by DNFB. Furthermore, interleukin (IL)‐4 and interferon (IFN)‐γ secretion by activated CD4+ T cells from the draining lymph nodes of DNFB‐treated NC/Nga mice were significantly inhibited by melatonin, and total IgE levels in serum were reduced. Our findings suggest that melatonin suppresses the development of AD‐like dermatitis in DNFB‐treated NC/Nga mice by reducing total IgE in serum, and IL‐4 and IFN‐γ production by activated CD4+ T cells.

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) JAK2 / TYK2 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 JAKs / STAT4 responsive to IL‐12 stimulation.

SYNERGY BETWEEN CD28 AND CD9 COSTIMULATION FOR NAIVE T-CELL ACTIVATION

Our previous study demonstrated that CD9 is expressed on most mature naive T-cells and delivers a potent costimulatory signal that functions independently of CD28. Here, we investigated whether this CD9-mediated signal is different from the CD28-mediated signal in the mode of costimulation and whether both signals function synergistically for T-cell activation. Anti-CD9 or anti-CD28 monoclonal antibody (mAb) increased [3H]TdR incorporation of naive T-cells in the absence of antigen-presenting cells (APC) when coimmobilized with submitogenic doses of anti-CD3 mAb. The levels of costimulation induced by ligation of CD9 and CD28 were comparable. However, the costimulatory effect differed between soluble anti-CD9 and CD28 mAb. A soluble form of anti-CD28 mAb could costimulate anti-CD3-triggered T-cells, whereas soluble anti-CD9 mAb failed to costimulate. Although anti-CD28 costimulated naive T-cells treated with phorbol myristate acetate (PMA) instead of anti-CD3 mAb, a combination of PMA plus anti-CD9 mAb could not induce T-cell activation. The combined costimulation of anti-CD3-triggered T-cells with anti-CD9 and anti-CD28 mAbs resulted in strikingly enhanced [3H]TdR uptake and lymphokine (IL-2 and IFN-gamma) production when compared to those induced by each costimulation. These results suggest that CD9 and CD28 induce T-cell costimulation using different signaling pathways, thereby inducing synergy in T-cell activation.

Original ArticleAspartame Attenuates 2, 4-Dinitrofluorobenzene-Induced Atopic Dermatitis–Like Clinical Symptoms in NC/Nga Mice

Atopic dermatitis (AD) is a common multifactorial chronic skin disease that has a multiple and complex pathogenesis. AD is gradually increasing in prevalence globally. In NC/Nga mice, repetitive applications of 2, 4-dinitrofluorobenzene (DNFB) evoke AD-like clinical symptoms similar to human AD. Aspartame (N-L-α-aspartyl-L-phenylalanine 1-methyl ester) is a methyl ester of a dipeptide, which is used as an artificial non-nutritive sweetener. Aspartame has analgesic and anti-inflammatory functions that are similar to the function of nonsteroidal anti-inflammatory drugs such as aspirin. We investigated whether aspartame can relieve AD-like clinical symptoms induced by DNFB treatment in NC/Nga mice. Sucrose did not relieve AD-like symptoms, whereas aspartame at doses of 0.5 μg kg(-1) and 0.5 mg kg(-1) inhibited ear swelling and relieved AD-like clinical symptoms. Aspartame inhibited infiltration of inflammatory cells including eosinophils, mast cells, and CD4(+) T cells, and suppressed the expression of cytokines including IL-4 and IFN-γ, and total serum IgE levels. Aspartame may have therapeutic value in the treatment of AD.

Aspartame Attenuates 2, 4-Dinitrofluorobenzene-Induced Atopic Dermatitis|[ndash]|Like Clinical Symptoms in NC|[sol]|Nga Mice

Atopic dermatitis (AD) is a common multifactorial chronic skin disease that has a multiple and complex pathogenesis. AD is gradually increasing in prevalence globally. In NC/Nga mice, repetitive applications of 2, 4-dinitrofluorobenzene (DNFB) evoke AD-like clinical symptoms similar to human AD. Aspartame (N-L-α-aspartyl-L-phenylalanine 1-methyl ester) is a methyl ester of a dipeptide, which is used as an artificial non-nutritive sweetener. Aspartame has analgesic and anti-inflammatory functions that are similar to the function of nonsteroidal anti-inflammatory drugs such as aspirin. We investigated whether aspartame can relieve AD-like clinical symptoms induced by DNFB treatment in NC/Nga mice. Sucrose did not relieve AD-like symptoms, whereas aspartame at doses of 0.5 μg kg(-1) and 0.5 mg kg(-1) inhibited ear swelling and relieved AD-like clinical symptoms. Aspartame inhibited infiltration of inflammatory cells including eosinophils, mast cells, and CD4(+) T cells, and suppressed the expression of cytokines including IL-4 and IFN-γ, and total serum IgE levels. Aspartame may have therapeutic value in the treatment of AD.