IL-17 reduces TNF-induced Rantes and VCAM-1 expression

Functional diversity of the memory T-cell-derived cytokine IL-17 was explored at the receptor level. IL-17 inhibited TNF-induced chemokine Rantes expression in human synovial fibroblasts and mouse lung fibroblasts. This inhibitory activity of IL-17 (IC50=0.2 ng/ml) was 6-fold more potent than its stimulatory activity on TNF-alpha-induced IL-6 secretion (ED50=1.2 ng/ml), measured in the same cells. IL-17 also inhibited the TNF-mediated expression of adhesion molecule VCAM-1, and the NF-kappaB binding to the VCAM-1 promoter-specific site, along with the inhibitor of NF-kappaB, IkappaB-beta. Neutralization of the human IL-17 receptor (IL-17R) by M202 antibody competitively reverses the IL-17-induced IL-6 upregulation. However, M202 only partially affected the inhibitions by IL-17. Yet, IL-17R was essential for the Rantes inhibition, as assessed in lung-derived fibroblasts from IL-17R gene deficient mice. Therefore, inhibitory and stimulatory functions of IL-17 involve receptor IL-17R but show distinct dose-responses and in turn different sensitivities to an IL-17R antagonizing antibody.     

Possible involvement of a GTP-binding protein in a late event during endogenous ganglioside-modulated cellular proliferation

The B subunit of cholera toxin, a protein which binds specifically to ganglioside GM1 on the cell surface, stimulates DNA synthesis in quiescent Swiss 3T3 fibroblasts as measured by an increase in [3H]thymidine incorporation. Pertussis toxin pretreatment markedly inhibits B subunit-induced DNA synthesis. The inhibitory effects of pertussis toxin were observed even in the presence of insulin which greatly potentiates the mitogenic response to the B subunit. Treatment with either pertussis toxin or insulin did not alter the binding of the B subunit to the cells. The dose-response for pertussis toxin-induced inhibition of DNA synthesis correlated closely with the dose-response for ADP-ribosylation of a 41-kDa membrane protein, suggesting the involvement of a GTP-binding protein that is a substrate for pertussis toxin (Gi) in mitogenesis induced via cross-linking of endogenous gangliosides. Pertussis toxin, in a similar concentration-dependent manner, also inhibited the mitogenic response to unfractionated fetal calf serum and to bombesin in the absence or presence of insulin. The inhibitory effect of pertussis toxin was clearly unrelated to any effects on known G proteins coupled to adenylate cyclase or phospholipase C. In addition, pertussis toxin did not impair the early increase in cytosolic free Ca2+ induced by the B subunit or bombesin. Pertussis toxin-induced inhibition of DNA synthesis could still be observed even when the toxin was added as late as 6 h after addition of the growth-promoting agents. This suggests the involvement of a GTP-binding protein in a late step of the B subunit- and bombesin-mediated pathways of mitogenesis. The possibility that other growth factors bypass this pathway is shown by their lack of sensitivity to pertussis toxin.     

Complex of soluble human IL-6-receptor/IL-6 up-regulates expression of acute-phase proteins.

IL-6 is a major regulator of acute phase protein synthesis in the liver. It exerts its action via a plasma membrane receptor consisting of two subunits, a ligand binding 80-kDa glycoprotein and a 130-kDa glycoprotein involved in signal transduction. We genetically generated a soluble form of the 80-kDa subunit of the human IL-6R (shIL-6R) in mouse fibroblasts (NIH/3T3 cells). The shIL-6R added to human hepatoma cells (HepG2) amplified the induction of alpha 1-antichymotrypsin and haptoglobin by IL-6 at the mRNA and protein level. Moreover, a model for a liver permanently exposed to high IL-6 concentrations has been developed; HepG2 cells were stably transfected with human IL-6-cDNA; 10(6) of the transfected cells (HepG2-IL-6) synthesized and secreted 2 micrograms of IL-6 within 24 h. Incubation of these cells with endogenous or exogenous IL-6 did not result in acute-phase protein induction. However, these IL-6-desensitized cells responded to other cytokines such as leukemia inhibitory factor, transforming growth factor beta 1, and IFN-gamma, known to modulate acute phase protein synthesis in the liver. Incubation of HepG2-IL-6 cells with shIL-6R reconstituted their responsiveness to IL-6 in a dose- and time-dependent manner. The possible biologic role that might be played by the shIL-6R in disease is discussed.     

Signal transduction pathway for IL-1. Involvement of a pertussis toxin-sensitive GTP-binding protein in the activation of adenylate cyclase

Human Il-1 alpha induces the synthesis of kappa Ig L chains by the pre-B cell line 7OZ/3, IL-2R alpha by the human NK cell line YT, and PGE2 by human rheumatoid synovial cells. Pertussis toxin (PT) markedly inhibited all three IL-1-induced activation events. The inhibition by PT was associated with a decrease in IL-1-mediated cAMP production. PT also inhibited IL-1-stimulated cAMP production in crude membrane fractions from 7OZ/3, YT, and 3T3 fibroblasts. In addition, IL-1 stimulated GTPase activity present in the membranes IL-1-responsive cells. Furthermore, the IL-1-induced GTPase activity was sensitive to PT. PT induced the ADP-ribosylation of a 46-kDa substrate in membrane preparations from IL-1-responsive cells. Cholera toxin also induced the ADP-ribosylation of a 46-kDa substrate in the same membrane preparations. The present findings indicate that the IL-1R is linked to a PT-sensitive G protein that stimulates the activity of adenylate cyclase.     

Binding of ATP by pertussis toxin and isolated toxin subunits.

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [3H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP greater than ATP greater than GTP greater than CTP greater than TTP for pertussis toxin and ATP greater than GTP greater than TTP greater than CTP for the B oligomer. Phosphate ions inhibited the binding of [3H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [3H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.     

IL-27 suppresses CD28-mediated [correction of medicated] IL-2 production through suppressor of cytokine signaling 3

IL-27 is a novel IL-6/IL-12 family cytokine that not only plays a role in the early regulation of Th1 differentiation, but also exerts an inhibitory effect on immune responses, including the suppression of proinflammatory cytokine production. However, the molecular mechanism by which IL-27 exerts the inhibitory effect remains unclear. In this study we demonstrate that IL-27 inhibits CD28-mediated IL-2 production and that suppressor of cytokine signaling 3 (SOCS3) plays a critical role in the inhibitory effect. Although IL-27 enhanced IFN-gamma production from naive CD4+ T cells stimulated with plate-coated anti-CD3 and anti-CD28 in the presence of IL-12, IL-27 simultaneously inhibited CD28-mediated IL-2 production. Correlated with the inhibition, IL-27 was shown to augment SOCS3 expression. Analyses using various mice lacking a signaling molecule revealed that the inhibition of IL-2 production was dependent on STAT1, but not on STAT3, STAT4, and T-bet, and was highly correlated with the induction of SOCS3 expression. Similar inhibition of CD28-mediated IL-2 production and augmentation of SOCS3 expression by IL-27 were observed in a T cell hybridoma cell line, 2B4. Forced expression of antisense SOCS3 or dominant negative SOCS3 in the T cell line blocked the IL-27-inudced inhibition of CD28-mediated IL-2 production. Furthermore, pretreatment with IL-27 inhibited IL-2-mediated cell proliferation and STAT5 activation, although IL-27 hardly affected the induction level of CD25 expression. These results suggest that IL-27 inhibits CD28-mediated IL-2 production and also IL-2 responses, and that SOCS3, whose expression is induced by IL-27, plays a critical role in the inhibitory effect in a negative feedback mechanism.     

IL-12: monoclonal antibodies specific for the 40-kDa subunit block receptor binding and biologic activity on activated human lymphoblasts.

IL-12, formerly known as cytotoxic lymphocyte maturation factor, is a cytokine that stimulates proliferation of PHA-activated human peripheral blood lymphoblasts and synergizes with low concentrations of IL-2 in the induction of lymphokine-activated killer cells. IL-12 is a 75-kDa heterodimer composed of disulfide-bonded 40-kDa and 35-kDa subunits. mAb prepared against a partially purified preparation of natural IL-12 have been characterized by 1) immunoprecipitation of 125I-labeled IL-12, 2) immunodepletion of IL-12 bioactivity, 3) Western blotting of IL-12, 4) inhibition of [125I]IL-12 binding to its cellular receptor, and 5) neutralization of IL-12 bioactivity. Twenty antibodies immunoprecipitate 125I-labeled IL-12 and immunodeplete IL-12 bioactivity as assessed in the T cell proliferation and lymphokine-activated killer cell induction assays. Western blot analysis demonstrated that each antibody binds to the 75-kDa heterodimer and to the 40-kDa subunit. An IL-12R-binding assay identified 12 individual antibodies that inhibited the binding of [125I]IL-12 to its cellular receptor. Two inhibitory antibodies, 4A1 and 7B2, were tested in the neutralization assay and found to block IL-12 bioactivity whereas one noninhibitory antibody, 8E3, was shown not to neutralize IL-12 bioactivity. Antibodies 4A1 and 8E3 can simultaneously bind to the 75-kDa heterodimer demonstrating that inhibitory and noninhibitory epitopes are spatially distinct on the 40-kDa protein. The ability of antibodies specific for the 40-kDa subunit of IL-12 to block receptor binding of [125I]IL-12 and to neutralize IL-12 bioactivity suggests that localized determinants on the 40-kDa subunit may be necessary for binding to the IL-12 cellular receptor.     

Membrane gangliosides modulate interleukin-2-stimulated T-lymphocyte proliferation.

Membrane gangliosides appear to modulate signal transduction by several growth factor receptors. We have investigated the possible regulation of IL-2-induced proliferation signals by gangliosides. Low concentrations of cholera toxin B subunit (CT-B), which binds specifically to GM1 ganglioside, greatly inhibited IL-2-stimulated DNA synthesis in the IL-2-dependent cell line CTLL-2, but had no effect on proliferation of HT-2. GM1 levels proved to be very low in HT-2 compared to CTLL-2. Large increases in membrane-associated GM1 could be achieved in both cell lines by incubation with exogenous GM1, resulting in a high degree of inhibition of proliferation by CT-B for both CTLL-2 and HT-2. Inhibition was blocked by large unilamellar vesicles containing GM1, but not by vesicles of lipid alone. The time course of CT-B inhibition for CTLL-2 synchronized in G0-G1, indicated that the negative growth signal acts relatively early in the IL-2 activation pathway. CT-B did not affect binding of IL-2 to high-affinity IL-2r. The inhibitory effects of CT-B could not be reversed by pertussis toxin, suggesting that a G protein is probably not involved. These results show that CT-B binding to either endogenous or inserted GM1 can modulate IL-2-induced lymphocyte proliferation.     

Induction of IgG2a class switching in B cells by IL-27

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. However, its role in B cells remains unexplored. We here show a role for IL-27 in the induction of T-bet expression and regulation of Ig class switching in B cells. Expression of WSX-1, one subunit of IL-27R, was detected at the mRNA level in primary mouse spleen B cells, and stimulation of these B cells by IL-27 rapidly activated STAT1. IL-27 then induced T-bet expression and IgG2a, but not IgG1, class switching in B cells activated with anti-CD40 or LPS. In contrast, IL-27 inhibited IgG1 class switching induced by IL-4 in activated B cells. Similar induction of STAT1 activation, T-bet expression and IgG2a class switching was observed in IFN-gamma-deficient B cells, but not in STAT1-deficient ones. The induction of IgG2a class switching was abolished in T-bet-deficient B cells activated with LPS. These results suggest that primary spleen B cells express functional IL-27R and that the stimulation of these B cells by IL-27 induces T-bet expression and IgG2a, but not IgG1, class switching in a STAT1-dependent but IFN-gamma-independent manner. The IL-27-induced IgG2a class switching is highly dependent on T-bet in response to T-independent stimuli such as LPS. Thus, IL-27 may be a novel attractive candidate as a therapeutic agent against diseases such as allergic disorders by not only regulating Th1 differentiation but also directly acting on B cells and inducing IgG2a class switching.     

IL-10 inhibits mitogen-induced T cell proliferation by selectively inhibiting macrophage costimulatory function.

IL-10, a newly designated cytokine primarily produced by the Th2 subset of CD4+ T lymphocytes and Ly-1+ B lymphocytes, has recently been hypothesized to inhibit cytokine production by Th1 T cell clones by blocking accessory cell- (AC) dependent costimulatory function. To evaluate the effect of IL-10 on Con A-induced proliferative responses of resting murine T cells, purified T cells were cultured with different types of AC. The addition of IL-10 produced a 70 to 90% inhibition of resting T lymphocyte proliferation when purified populations of macrophages were used as AC, but had no effect on the AC function of T-depleted spleen cells, activated B cells, dendritic cells, or L cells. The inhibitory effects of IL-10 were inversely related to the concentration of mitogen and could be reversed by the addition of the neutralizing anti-IL-10 mAb, SXC1. The inhibition of macrophage AC function was not related to the induction of a suppressor cytokine as stimulation by mixtures of macrophages and limiting numbers of dendritic cells was not inhibited. The decrease in proliferative responses was primarily secondary to inhibition of IL-2 production although the failure of exogenous IL-2 to completely reconstitute the response suggested that IL-10 may also exert inhibitory effects on the induction of expression of a functional IL-2R. These results are most consistent with a model in which IL-10 inhibits the induction of expression on macrophages of a critical costimulatory molecule that may be constitutively expressed on other types of AC.     

Ovarian carcinoma cells inhibit T cell proliferation: suppression of IL-2 receptor beta and gamma expression and their JAK-STAT signaling pathway

Deficient T cell immune function and intracellular signaling in cancer patients may result from effects of tumors or their products on lymphocytes. Recently, it was demonstrated that several ovarian carcinoma cell lines could produce soluble factors that inhibited T cell proliferation. The aim of this study is to assess the effect of supernatants from 3 ovarian carcinoma cell lines (OVCAR3, CAOV3, SKOV3) on signal transduction elements that are linked to the IL-2R and its JAK-STAT pathway. A profound inhibition of proliferation, lower level of IFN-gamma and higher level of IL-10 gene expression were observed when CD8+ T cells were co-cultured with supernatants from 3 ovarian carcinoma cell lines. Cell cycle studies on inhibited CD8+ T cells showed most of them were growth arrested in G0/G1 phase. Western blot analysis showed that tumor supernatants suppressed expression of JAK3 and tyrosine phosphorylation of STAT5. JAK1 was not altered and the inhibition of STAT3 only appeared in OVCAR3 cells. Tumor supernatants also partially blocked induction of IL-2R beta and gamma chains expression. These findings suggest that ovarian carcinoma cells may suppress T cell proliferation through inhibition IL-2 dependent signaling pathways, which may be a mechanism of ovarian carcinoma induced immunosuppression.     

Mechanism of action of interleukin-13 antagonist (IL-13E13K) in cells expressing various types of IL-4R

As interleukin (IL)-13 and IL-4 play a major role in various diseases including asthma, allergy, and malignancies, it is desirable to generate a molecule that blocks the effects of both cytokines. We previously generated a human IL-13 mutant (IL-13E13K), which is a powerful antagonist of IL-13, blocking the biological activities of IL-13. We now show that IL-13E13K also competitively inhibits signaling and biological activities of IL-4 through type II and partially through type III IL-4 receptor (R) system. IL-13E13K completely blocked the IL-4-induced phosphorylation of STAT6 and IL-4-dependent protein synthesis in cells expressing type II and partially type III IL-4R but not type I IL- 4R. Consistent with the inhibition of biological activities, IL-13E13K inhibited IL-4 binding to type II IL-4R-expressing cells but not to type I IL-4R-expressing cells. The inhibition efficiency of IL-4 binding by IL-13E13K was relatively lower compared to wtIL-13 even though IL-13E13K bound to IL-13Ralpha1 positive cells with a similar affinity to wtIL-13. These results indicate that Glu13 in IL-13 associates with IL-4Ralpha, and mutation to lysine decreases its binding ability to IL-4Ralpha chain. IL-13E13K binds to IL- 13Ralpha1, which is shared by both IL-13R and IL-4R systems. Consequently, IL-13E13K inhibits IL-4 binding to these cells and prevents heterodimer formation between IL-13Ralpha1 and IL-4Ralpha chains. This interference by IL-13E13K blocks the biological activities of not only IL-13 but also partially of IL-4. Thus, IL-13E13K may be a useful agent for the treatment of diseases such as asthma, allergic rhinitis, and cancer, which are dependent on signaling through both IL-4 and IL-13 receptors.     

Identification of a novel protein capable of interacting with the IL-3 receptor

IL-3 has numerous functions in hematopoiesis yet its receptor has not been fully characterized. We have developed two mAb, 4G8 and 2F2, that markedly inhibited IL-3-dependent proliferation whereas only marginally affecting IL-2 or IL-4-induced proliferation. On Western blots, both antibodies identified the same protein, which varied in size from 115 to 145 kDa in six cell lines tested. The 4G8/2F2 Ag was detected at moderate density, on a wide variety of cells including IL-3-dependent cell lines and T lymphocytes. Radioligand binding studies revealed that 4G8, but not 2F2, could inhibit the binding of 125I-IL-3 to the high affinity IL-3R. These data suggest that the mAb 4G8 and 2F2 recognize different epitopes on the same Ag, and suggest furthermore that the inhibition of IL-3-dependent proliferation mediated by 2F2, in particular, does not occur via inhibition of ligand binding. Neither antibody showed an enhanced level of fluorescent staining of Cos 7 cells transfected with the low affinity IL-3R cDNA. In addition, 4G8 did not inhibit IL-3 binding to L cells transfected with the cloned IL-3R or IL-4R despite the fact that 4G8 was expressed on these cells. These data suggest that the 4G8/2F2 Ag is a unique cell surface protein that can interact with the endogenous functional IL-3R.     

Suppressor of cytokine signaling 1 inhibits IL-10-mediated immune responses

IL-10 has proved to be a key cytokine in regulating inflammatory responses by controlling the production and function of various other cytokines. The suppressor of cytokine signaling (SOCS) gene products are a family of cytoplasmic molecules that are essential mediators for negatively regulating cytokine signaling. It has been previously shown that IL-10 induced SOCS3 expression and that forced constitutive expression of SOCS3 inhibits IL-10/STAT3 activation and LPS-induced macrophage activation. In this report, we show that, in addition to SOCS3 expression, IL-10 induces SOCS1 up-regulation in all cell lines tested, including Ba/F3 pro-B cells, MC/9 mast cells, M1 leukemia cells, U3A human fibroblasts, and primary mouse CD4(+) T cells. Induction of SOCS molecules is dependent on STAT3 activation by IL-10R1. Cell lines constitutively overexpressing SOCS proteins demonstrated that SOCS1 and SOCS3, but not SOCS2, are able to partially inhibit IL-10-mediated STAT3 activation and proliferative responses. Pretreatment of M1 cells with IFN-gamma resulted in SOCS1 induction and a reduction of IL-10-mediated STAT3 activation and cell growth inhibition. IL-10-induced SOCS is associated with the inhibition of IFN-gamma signaling in various cell types, and this inhibition is independent of C-terminal serine residues of the IL-10R, previously shown to be required for other anti-inflammatory responses. Thus, the present results show that both SOCS1 and SOCS3 are induced by IL-10 and may be important inhibitors of both IL-10 and IFN-gamma signaling. IL-10-induced SOCS1 may directly inhibit IL-10 IFN-gamma signaling, while inhibition of other proinflammatory cytokine responses may use additional IL-10R1-mediated mechanisms.     

Inhibition of protein kinase C-dependent cellular proliferation by interaction of endogenous ganglioside GM1 with the B subunit of cholera toxin

In quiescent Swiss 3T3 fibroblasts, the B subunit of cholera toxin, a protein which binds specifically to ganglioside GM1 on the cell surface, stimulates DNA synthesis and potentiates the effects of several other growth factors such as insulin, epidermal growth factor, bombesin, and even unfractionated serum. In contrast to its synergistic effect with other known growth factors, the B subunit markedly inhibited DNA synthesis induced by the phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA). The inhibitory effect of the B subunit was observed even in the presence of insulin, which greatly potentiates the mitogenic response to TPA or the B subunit. In contrast to the effect of the B subunit, calcium ionophores and cholera toxin stimulated DNA synthesis induced by TPA. The antagonism between the B subunit and TPA is not simply due to their abilities to modify their mutual binding sites or known effector systems. TPA did not block the early rise in cytosolic free calcium in response to the B subunit, and conversely, the B subunit did not modify the ability of TPA to activate protein kinase C. However, in protein kinase C-deficient cells, the antagonistic effect between TPA and the B subunit was abolished. In addition, there was no indication for the involvement of a pertussis toxin-sensitive G protein in the antagonism. Maximum inhibition was found when the B subunit was added 2 h after the addition of TPA. Significant inhibition was still evident when the time of addition of the B subunit was delayed until 6 h after the addition of TPA. This suggests that the cross-talk between signal transduction induced through endogenous gangliosides and protein kinase C is a late step in mitogenesis.     

Interleukin-18 enhances IL-18R/Nox1 binding,and mediates TRAF3IP2-dependent smooth muscle cell migration. Inhibition by simvastatin

We investigated the role of TRAF3 interacting protein 2 (TRAF3IP2), a redox-sensitive adapter protein and an upstream regulator of IKK and JNK in interleukin (IL)-18 induced smooth muscle cell migration, and the mechanism of its inhibition by simvastatin. The pleiotropic cytokine IL-18 induced human coronary artery SMC migration through the induction of TRAF3IP2. IL-18 induced Nox1-dependent ROS generation, TRAF3IP2 expression, and IKK/NF-κB and JNK/AP-1 activation. IL-18 induced its own expression and that of its receptor subunit IL-18Rα. Using co-IP/IB and GST pull-down assays, we show for the first time that the subunits of the IL-18R heterodimer physically associate with Nox1 under basal conditions, and IL-18 appears to enhance their binding. Importantly, the HMG-coA reductase inhibitor simvastatin attenuated IL-18-induced TRAF3IP2 induction. These inhibitory effects were reversed by mevalonate and geranylgeranylpyrophosphate (GGPP), but not by farnesylpyrophosphate (FPP). Interestingly, simvastatin, GGPP, FPP, or Rac1 inhibition did not modulate ectopically expressed TRAF3IP2. These results demonstrate that the promigratory effects of IL-18 are mediated through TRAF3IP2 in a redox-sensitive manner, and this may involve IL-18R/Nox1 physical association. Further, Simvastatin inhibits inducible, but not ectopically-xpressed TRAF3IP2. Targeting TRAF3IP2 may blunt progression of hyperplastic vascular diseases in vivo.