Modulation of PAR expression and tryptic enzyme induced IL-4 production in mast cells by IL-29

Interleukin (IL)-29 is a relatively newly discovered cytokine, which has been shown to be actively involved in the pathogenesis of allergic inflammation. However, little is known of the effects of IL-29 on protease activated receptor (PAR) expression and potential mechanisms of cytokine production in mast cells. In the present study, we examined potential influence of IL-29 on PAR expression and cytokine production in P815 and bone marrow derived mast cells (BMMCs) by using flow cytometry analysis, quantitative real time PCR, and ELISA techniques. The results showed that IL-29 downregulated the expression of PAR-1 by up to 56.2%, but had little influence on the expression of PAR-2, PAR-3 and PAR-4. IL-29 also induced downregulation of expression of PAR-1 mRNA. However, when mast cells were pre-incubated with IL-29, thrombin-, trypsin- and tryptase-induced expression of PAR-2, PAR-3 and PAR-4 was upregulated, respectively. IL-29 provoked approximately up to 1.9-fold increase in IL-4 release when mast cells was challenged with IL-29. Administration of IL-29 blocking antibody, AG490 or LY294002 abolished IL-29-induced IL-4 release from P815 cells. It was found that IL-29 diminished trypsin- and tryptase-induced IL-4 release from P815 cells following 16 h incubation. In conclusion, IL-29 can regulate expression of PARs and tryptase- and trypsin-induced IL-4 production in mast cells, through which participates in the mast cell related inflammation.     

IgE enhances Fc epsilon receptor I expression and IgE-dependent release of histamine and lipid mediators from human umbilical cord blood-derived mast cells: synergistic effect of IL-4 and IgE on human mast cell Fc epsilon receptor I expression and mediator release

We investigated the effects of IgE versus IL-4 on Fc epsilon RI surface expression in differentiated human mast cells derived in vitro from umbilical cord blood mononuclear cells. We found that IgE (at 5 micrograms/ml) much more strikingly enhanced surface expression of Fc epsilon RI than did IL-4 (at 0.1-100 ng/ml); similar results were also obtained with differentiated mouse mast cells. However, IL-4 acted synergistically with IgE to enhance Fc epsilon RI expression in these umbilical cord blood-derived human mast cells, as well as in mouse peritoneal mast cells derived from IL-4-/- or IL-4+/+ mice. We also found that: 1) IgE-dependent enhancement of Fc epsilon RI expression was associated with a significantly enhanced ability of these human mast cells to secrete histamine, PGD2, and leukotriene C4 upon subsequent passive sensitization with IgE and challenge with anti-IgE; 2) preincubation with IL-4 enhanced IgE-dependent mediator secretion in these cells even in the absence of significant effects on Fc epsilon RI surface expression; 3) when used together with IgE, IL-4 enhanced IgE-dependent mediator secretion in human mast cells to levels greater than those observed in cells that had been preincubated with IgE alone; and 4) batches of human mast cells generated in vitro from umbilical cord blood cells derived from different donors exhibited differences in the magnitude and pattern of histamine and lipid mediator release in response to anti-IgE challenge, both under baseline conditions and after preincubation with IgE and/or IL-4.     

Modulation of cytokine release from mononuclear cells by prostacyclin, IL-4 and IL-13

In a previous study, we reported that cicaprost, a stable prostacyclin analogue can inhibit the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) from activated human peripheral mononuclear blood cells (PBMCs). Since interleukin (IL-4) and IL-13 have been shown to inhibit the release of cytokines from PBMCs we tested the hypothesis that prostacyclin in combination with IL-4 or IL-13 can act synergistically to modulate the release of IL-10, generally associated with anti-inflammatory properties, and the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-alpha). For this purpose, PBMCs were isolated over Ficoll, stimulated with lipopolysaccharide (LPS) and incubated in the presence of cicaprost, IL-4 or IL-13. There was a significant reduction in TNF-alpha as well as IL-10 secretion from LPS-stimulated PBMCs following incubation with IL-4 or IL-13. In contrast, cicaprost reduced the secretion of TNF-alpha but led to a slight enhancement of IL-10 release from PBMCs. When LPS-activated PBMCs were incubated in the presence of cicaprost and IL-4 or IL-13 there was a selective, synergistic inhibition of the TNF-alpha release which was not observed for IL-10. Thus, our data suggest that prostacyclin can synergize with cytokines to selectively inhibit the release of pro-inflammatory cytokines from PBMCs.     

IL-10 suppresses mast cell IgE receptor expression and signaling in vitro and in vivo

Mast cells are known for their roles in allergy, asthma, systemic anaphylaxis, and inflammatory disease. IL-10 can regulate inflammatory responses and may serve as a natural regulator of mast cell function. We examined the effects of IL-10 on in vitro-cultured mouse and human mast cells, and evaluated the effects of IL-10 on FcepsilonRI in vivo using mouse models. IgE receptor signaling events were also assessed in the presence or absence of IL-10. IL-10 inhibited mouse mast cell FcepsilonRI expression in vitro through a Stat3-dependent process. This down-regulation was consistent in mice tested in vivo, and also on cultured human mast cells. IL-10 diminished expression of the signaling molecules Syk, Fyn, Akt, and Stat5, which could explain its ability to inhibit IgE-mediated activation. Studies of passive systemic anaphylaxis in IL-10-transgenic mice showed that IL-10 overexpression reduced the IgE-mediated anaphylactic response. These data suggest an important regulatory role for IL-10 in dampening mast cell FcepsilonRI expression and function. IL-10 may hence serve as a mediator of mast cell homeostasis, preventing excessive activation and the development of chronic inflammation.     

Functional expression of neurokinin 1 receptors on mast cells induced by IL-4 and stem cell factor

It is widely accepted that neurokinin 1 (NK(1)) receptors are not generally expressed on mast cells but little is known about their expression in inflammation. The present study shows expression of NK(1) receptors on bone marrow-derived mast cells (BMMC) under the influence of IL-4 or stem cell factor (SCF). Highest expression was found when both cytokines are present. Six days of coculture with the cytokines IL-4 and SCF showed significant expression of NK(1) receptors (NK(1) receptor(+)/c-kit(+) BMMC; control: 7%, IL-4/SCF: 16%), while 12 days of cytokine coculture increased this expression to 37% positive cells. A longer coculture with IL-4 and SCF did not give an additional effect. Increased expression in IL-4/SCF-treated BMMC was further confirmed using Western blot analysis. Next, we demonstrated the functional relevance of NK(1) receptor expression for mast cell activation, resulting in an enhanced degranulation upon stimulation by substance P. BMMC activation was significantly diminished by the NK(1) receptor antagonist RP67580 (10 micro M) when stimulated with low concentrations of substance P. The inactive enantiomer RP65681 had no effect. In addition, BMMC cultured from bone marrow of NK(1) receptor knockout mice showed significantly decreased exocytosis to low concentrations of substance P. The present study clearly shows that NK(1) receptor-induced activation contributes significantly at low physiological substance P concentrations (<100 micro M). In conclusion, BMMC were shown to express NK(1) receptors upon IL-4/SCF coculture. This expression of NK(1) receptors has been demonstrated to be of functional relevance and leads to an increase in the sensitivity of BMMC to substance P.     

IL-1 and IL-4 modulate IL-1 receptor expression in a murine T cell line

The combination of IL-1 and IL-4 stimulates the proliferation of certain murine T cell populations. Although this effect has been best characterized for a number of murine type 2 Th cell (Th2) clones, the mechanism(s) by which these cytokines effect this response is unclear. We have examined the effects of IL-1 and IL-4 on IL-1R expression by MD10 cells, and IL-1-responsive murine T cell line. These cells bear specific IL-1R, which bind human and murine IL-1 alpha and -beta. The measured apparent IL-1R dissociation constant ranged from 41 to 255 pM using 125I-HrIL-1 alpha. Cross-linking studies demonstrated two different 125I-HrIL-1 alpha binding complexes having Mr of 70,000 and 130,000 to 156,000. When removed from passage conditions and placed in non-growth factor-supplemented media, MD10 IL-1R expression spontaneously increased two- to fourfold over the first 11 to 12 h of culture followed by a decline. This phenomenon is partially inhibitable by cycloheximide suggesting that protein synthesis is involved. In agreement with other reports, HrIL-1 alpha down-regulated the expression of its own receptor with an ED50 of between 1 and 10 pM HrIL-1 alpha for this effect. In most experiments, low amounts of HrIL-1 alpha (1.0, 0.1 pM) significantly augmented IL-1R expression. Scatchard analysis of data obtained with all HrIL-1 alpha treatment conditions showed that the effects were due to a change in receptor number, not affinity. Significantly, purified murine IL-4 (MpIL-4) augmented MD10 IL-1R expression in both a time- and dose-dependent fashion. In the presence of 50 U/ml MpIL-4, MD10 IL-1R expression increased two- to threefold after 24 h without a change in receptor affinity. When MpIL-4 (50 U/ml) and various amounts of HrIL-1 alpha (.01-1000 pM) were co-added, the down-regulatory effect of high levels of HrIL-1 alpha was significantly antagonized. When added to cultures after 24 h of HrIL-1 alpha (100 pM) treatment, MpIL-4 reversed the IL-1R down-regulatory effect induced by high levels of HrIL-1 alpha. Finally, when combined in MD10 proliferation assays, MpIL-4 synergistically enhanced the proliferation of MD10 cells treated with suboptimal levels of HrIL-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of cardiac contractility through endothelin-1 release and myocardial mast cell degranulation

The aim of this study was to outline the consequences of a hypertonic saline-dextran-40 (HSD) infusion-induced peripheral flow stimulus on the ventricular function in closed-chest, pentobarbital-anesthetized dogs. We hypothesized that HSD-induced elevation in endothelin-1 (ET-1) and nitric oxide (NO) release can have a role in myocardial contractile responses; and that cardiac mast cells (MC) degranulation may be involved in this process. The consequences of disodium cromoglycate (a MC stabilizer) or ETR-p1/fl peptide (an endothelin-A receptor antagonist) treatment were evaluated. A 4 ml/kg iv HSD40 infusion significantly increased cardiac index and myocardial contractility, and resulted in a decreased peripheral resistance. The postinfusion period was characterized by significant plasma NO and ET-1 elevations, these hemodynamic and biochemical changes being accompanied by a decreased myocardial ET-1 content, NO synthase activity and enhanced myocardial MC degranulation. Disodium cromoglycate treatment inhibited the HSD40-induced elevations in myocardial contractility and MC degranulation, and similar hemodynamic changes were noted after treatment with ETR-p1/fl peptide, together with a normalized myocardial myocardial ET-1 content, NO synthesis and a significant reduction in MC degranulation. These results indicate that peripheral NO and ET-1 release modulates the cardiac contractility through myocardial ET-A receptor activation and MC degranulation.     

Modulation of IL-2- and IL-4-dependent human B cell proliferation by cyclic AMP.

The second messenger cAMP is a modulator of cellular growth possessing both inhibitory and stimulatory properties. In this report, we show that IL-2- and IL-4-dependent DNA synthesis of anti-mu-activated human B cells is modulated in opposite ways by agents increasing intracellular levels of cAMP. Forskolin and 2'-O-dibutyriladenosine-3',5'-cyclic monophosphate had no proliferative effect by themselves. Nevertheless they decreased IL-2-driven proliferation and increased IL-4-mediated DNA synthesis. IL-4 and cAMP each inhibited the IL-2-dependent proliferation with similar patterns of reactivity. Both IL-4 and forskolin needed to be present during the first 48 h of culture to display inhibitory activity, and preactivation of B cells for 16 h with forskolin and IL-4 did not prevent further B cell response to IL-2. This suggests that cAMP and IL-4 directly interact with IL-2 signaling. In addition, we show that the cAMP-dependent protein kinase inhibitor N-(2-methylamino-ethyl)-5-iso-quinoline-sulfamide reversed the IL-4-inhibitory effect on IL-2-driven proliferation. Our data suggest that the IL-4-inhibitory signal to IL-2-driven human B cell proliferation involves cAMP-dependent protein kinase activation.     

IL-12 receptor. II. Distribution and regulation of receptor expression.

IL-12 is a heterodimeric lymphokine that induces IFN-gamma production by resting PBMC, enhances the lytic activity of NK/lymphokine activated killer cells, and causes the proliferation of activated T cells and NK cells. In this report, we have investigated the expression of IL-12R on mitogen- and IL-2-activated PBMC or tonsillar lymphocytes as well as on a variety of cell lines. The results of radiolabeled IL-12-binding assays indicated that high affinity IL-12R are present on PBMC activated by various T cell mitogens or by IL-2. High affinity IL-12R were also found to be expressed constitutively on a transformed marmoset NK-like cell line HVS.SILVA 40. At the time of peak IL-12R expression, mitogen- or IL-2-activated cells displayed approximately 1000 to 9000 IL-12 binding sites/cell with an apparent Kd of 100 to 900 pM. Kinetic studies revealed that maximum expression of IL-12R occurred earlier on PHA-activated PBMC as compared with PBMC activated by IL-2, and that expression of IL-12R on these cells correlated with their ability to proliferate in response to IL-12. Although IL-2 could up-regulate IL-12R expression on resting PBMC, the ability of mitogen-activated PBMC to up-regulate IL-12R was found to be independent of IL-2. Analysis of IL-12R expression by flow cytometry revealed that receptors for IL-12 are present on activated T cells of both the CD4+ and CD8+ subsets and on activated CD56+ NK cells. In contrast, neither resting PBMC or tonsillar B cells nor tonsillar B cells activated by anti-IgM/Dx, anti-IgM/Dx + IL-2, or SAC + IL-2 displayed IL-12R detectable by flow cytometry or by the radiolabeled IL-12-binding assay. In summary, these results indicate that activation of T cells or NK cells results in up-regulation of IL-12R expression; on the other hand, B cell activation, at least under some circumstances, appears not to be associated with enhanced expression of IL-12R.     

Regulation of mast cell histamine release by neurotensin

Neurotensin (NT), a neuropeptide found both centrally and peripherally, stimulated release of histamine from rat peritoneal mast cells in a dose-dependent manner. Release was evident by 10 nM and reached a plateau of 15-20% total cellular histamine by 10(-7)-10(-6) M NT. Optimal conditions for stimulation occurred at pH 6.5-7.5, 37 degrees C and at calcium concentrations of less than 1 mM. Release was complete within 2 minutes of peptide addition. Studies of histamine release by NT analogues indicted that the C-terminus is the biologically active portion of the molecule in this system, as is true of all other systems responsive to NT (1). D-Trp11-NT, which acts as a NT antagonist in several peripheral NT-sensitive tissues (2,3), also inhibited NT action on mast cells. Manipulations involving Ca2+ availability suggest that the mechanism of NT stimulation may involve use of intracellular Ca2+ to a greater extent than extracellular Ca2+. Lowering the extracellular Ca2+ concentration or blocking influx of extracellular Ca2+ with lanthanum (La3+), had little effect on NT-induced release, whereas Ca2+ depletion by treatment with ethylenediaminetetracetic acid (EDTA) or blockade of intracellular Ca2+ mobilization by N,N-(diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), inhibited the response to NT. Increasing cellular levels of adenosine 3',5'-cyclic monophosphate (cAMP), by treatment with 8-bromo-cAMP or stimulation with prostaglandin E2 (PGE2) in the presence of isobutylmethylxanthine (IBMX), served to reduce histamine release by NT, indicating that cAMP may play a role in NT stimulation.     

IL-4 induces the proteolytic processing of mast cell STAT6

IL-4 is a potent, pleiotropic cytokine that, in general, directs cellular activation, differentiation, and rescue from apoptosis. However, in mast cells, IL-4 induces the down-regulation of activation receptors and promotes cell death. Mast cells have been shown to transduce IL-4 signals through a unique C-terminally truncated isoform of STAT6. In this study, we examine the mechanism through which STAT6 is processed to generate this isoform. We demonstrate that STAT6 processing in mast cells is initiated by IL-4-induced phosphorylation and nuclear translocation of full-length STAT6 and subsequent cleavage by a nuclear serine-family protease. The location of the protease in the nucleus ensures that the truncated STAT6 has preferential access to bind DNA. IL-4-responsive target genes in mast cells are identified by chromatin immunoprecipitation of STAT6, including the IL-4 gene itself. These results suggest a molecular explanation for the suppressive effects of IL-4 on STAT6-regulated genes in mast cells.     

Glucocorticoid suppresses autocrine survival of mast cells by inhibiting IL-4 production and ICAM-1 expression

When mast cells are activated through their high affinity IgE receptors (FcepsilonRI), release of chemical mediators is followed by secretion of multiple cytokines. In this work, we report that IL-3-dependent mast cell line MC9 undergoes apoptosis when IL-3 is withdrawn. However, cross-linking of FcepsilonRI prevents apoptosis of MC9 by an autocrine mechanism, producing IL-3, IL-4, and GM-CSF. Although stimulated MC9 synthesizes mRNAs and proteins of these cytokines, secretion of endogenous IL-3 and GM-CSF is not enough for cell survival, whereas IL-4 itself does not have survival effect on MC9, but it induces cell aggregation by expressing LFA-1 and makes it reactive to endogenous growth factors. Addition of dexamethazone (DXM) to MC9 results in significant down-regulation of IL-4 mRNA in activated MC9. However, mRNA levels of IL-3 and GM-CSF are not changed by DXM. DXM also directly down-regulates the expression of ICAM-1 that is the high affinity ligand of LFA-1, by which the self-aggregation of MC9 is inhibited. Thus, glucocorticoids suppress autocrine survival of mast cells by inhibiting IL-4 production and ICAM-1 expression.     

Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4)

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.     

Modulation of CLA, IL-12R, CD40L, and IL-2Ralpha expression and inhibition of IL-12- and IL-23-induced cytokine secretion by CNTO 1275

Cytokines interleukin (IL)-12 and IL-23 are implicated in the pathogenesis of psoriasis. IL-12 causes differentiation of CD4+ T cells to interferon-gamma (IFN-gamma)-producing T helper 1 (Th1) cells, while IL-23 induces differentiation to IL-17-producing pathogenic Th17 cells. The effects of the monoclonal antibody to IL-12/23 p40 subunit (CNTO 1275) on IL-12 receptor (IL-12R) expression, markers associated with skin homing, activation, and cytokine secretion were investigated in vitro using human peripheral blood mononuclear cells (PBMCs) from healthy donors. PBMCs were activated in the presence or absence of recombinant human (rh) IL-12 or rhIL-23, with or without CNTO 1275. CNTO 1275 inhibited upregulation of CLA, IL-12R, IL-2Ralpha and CD40L expression and also inhibited IL-12- and IL-23-induced IFN-gamma, IL-17A, tumor necrosis factor (TNF)-alpha, IL-2, and IL-10 secretion. Thus, the therapeutic effect of CNTO 1275 may be attributed to the IL-12/23 neutralization, resulting in decreased expression of skin homing and activation markers, and IL-12- and IL-23-induced cytokine secretion.     

IL-4-dependent up-regulation of IL-4 receptor expression in murine T and B cells

This study examined mRNA levels and cell surface expression of IL-4 receptor (IL-4R) in murine T and B cells after incubation with IL-4. Northern blot analysis of mRNA levels of T cells isolated from mesenteric lymph nodes and spleen revealed that IL-4 induced a transient augmentation of IL-4R mRNA in a dose-dependent manner. Maximal levels of mRNA were detected as early as 5 h after initiation of culture. These data were complemented by studies examining the cell surface expression of IL-4R using an anti-IL-4R mAb. Resting T and B lymphocytes express IL-4R (T greater than B) and incubation of these cells with exogenous IL-4 increased IL-4R expression to a maximum after 24 h. This effect was abolished after addition of anti-IL-4 antibody. Continuous incubation of T cells in the presence of high concentrations of IL-4 resulted in a down-regulation of IL-4R expression. Addition of the protein synthesis inhibitor cycloheximide blocked the induced increases in IL-4R expression, indicating the requirement for de novo protein synthesis. Both the levels of mRNA and cell surface expression of IL-4R were not affected by addition of exogenous IL-2, and IL-4 regulation of IL-4R expression was not influenced by the immunosuppressive drug cyclosporin A. These data demonstrate that in T and B cells, IL-4 induces a transient up-regulation of IL-4 mRNA levels that is subsequently reflected in increased numbers of IL-4R displayed on the cell surface. This regulation of IL-4R expression by IL-4 provides an important mechanism for amplification of IL-4-dependent activation pathways.