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IL-4 signaling promotes IgE class switching through STAT6 activation and the induction of Ig germ-line [epsilon] (GL[epsilon]) transcription. Previously, we and others identified a transcription factor, Nfil3, as a gene induced by IL-4 stimulation in B cells. However, the precise roles of nuclear factor, IL-3-regulated (NFIL3) in IL-4 signaling are unknown. Here, we report that NFIL3 is important for IgE class switching. NFIL3-deficient mice show impaired IgE class switching, and this defect is B-cell intrinsic. The induction of GL[epsilon] transcripts after LPS and IL-4 stimulation is significantly reduced in NFIL3-deficient B cells. Expression of NFIL3 in NFIL3-deficient B cells restores the impairment of IgE production, and overexpression of NFIL3 in the presence of cycloheximide induces GL[epsilon] transcripts. Moreover, NFIL3 binds to I[epsilon] promoter in vivo. Together, these results identify NFIL3 as a key regulator of IL-4-induced GL[epsilon] transcription in response to IL-4 and subsequent IgE class switching. IL-4 signal | immunoglobulin | germ-line transcription www.pnas.org/cgi/doi/10.1073/pnas.0909235107

The CD8 co-receptor can modulate CD[8.sup.+] T cell function through its contributions to T cell receptor (TCR) binding and signaling. Here we show that IFN-[gamma] and IL-4 exert opposing effects on the expression of CD8[alpha] mRNA and surface CD8 protein during CD[8.sup.+] T cell activation. IL-4 caused down-regulation of surface CD8 on ovalbumin [(OVA).sub.257-264]-specific TCR-otransgenic OT-I CD[8.sup.+] T cells activated with [OVA.sub.257-264]-coated antigen presenting cells or polyclonal stimuli, and on wild type CD[8.sup.+] T cells activated with polyclonal stimuli. This effect was enhanced in each case when the cells lacked a functional IFN-[gamma] or IFN-[gamma]R gene. When WT or IFN-[gamma]-deficient OT-I CD[8.sup.+] T cells were analyzed 9 days after co-injection with control or IL-4-expressing [OVA.sup.+] tumor cells into RAG-[2.sup.-/-][gamma][c.sup.-/-] mice, CD8 levels were highest on WT donor cells from mice that received the control tumor and lowest on IFN-[gamma]-deficient donor cells from mice that received the IL-4-expressing tumor. The latter CD[8.sup.low] cells displayed markedly impaired binding of [OVA.sub.257-264]/MHC tetramers and peptide/MHC-dependent degranulation. The data reveal an unexpected role for IFN-[gamma] in tuning the CD8 co-receptor during primary CD[8.sup.+] T cell activation both in vitro and in vivo. CD8low | co-receptor tuning | T cell activation | cytotoxic T lymphocytes | cytokines

Differentiation of T helper (Th) subset 2 effector lymphocytes is thought to foreclose on IFN-[gamma] gene expression. Using an IL-4 locus modified to detect transcriptional induction of this effector cytokine gene in developing Th2 cells, we show here that these cells contributed effectively to a long-term memory population. A memory CD4 subset formed efficiently from an activated population after transcriptional induction of the IL-4 locus and differentiation into an IL-4-producing subset with Th2 characteristics. Memory lymphocytes derived from Th2 cells with IL-4 locus activation remained committed to transcriptional competence of Th2 cytokine genes when reactivated and cultured under strong Th1-polarizing conditions. This commitment to transcriptional competence at Th2 cytokine gene loci upon recall activation indicates that linear differentiation is a substantial component of type 2 memory. Strikingly, however, descendants of the Th2 population could turn on IFN-[gamma] expression when reactivated after a quiescent period, revealing an unexpected flexibility allowing activation of the forbidden IFN-[gamma] gene after reactivation and growth. differentiation | gene expression | immune memory | recall | plasticity

Little is known concerning the possible contribution of T helper 2 (Th2)-type cytokines to the recruitment of neutrophils into the lung tissue. In the present study, endothelial cells from equine pulmonary arteries were cultured in the presence of recombinant equine (re) IL-4 and reIL-5, and the cytokine mRNA expression of molecules implicated in the chemotaxis and migration of neutrophils was studied using real-time RT-PCR. The functional response of reIL-4-induced endothelial cell stimulation on neutrophil migration was also studied using a chemotaxis chamber. ReIL-4 either increased the expression of CXCL-8, E-selectin, vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS), or potentiated the coeffects of lipopolysaccharide (LPS) and tumor necrosis factor-[alpha] (TNF-[alpha] on CXCL-8. Supernatants collected from cultured endothelial cells stimulated with reIL-4 significantly promoted neutrophil migration in a dose-dependent manner. Dexamethasone (DXM) decreased the expression of CXCL-8, VEGF, and iNOS induced by reIL-4, while 1400W dihydrochloride (1400W), a selective inhibitor of iNOS, decreased the expression of E-selectin, VEGF, and iNOS. DXM and 1400W attenuated the mRNA expression of E-selectin and iNOS induced by the costimulation of reIL-4, reTNF-[alpha], and LPS. Neither equine nor human recombinant IL-5 influenced the mRNA expression of CXCL-8, E-selectin, or VEGF. These findings suggest that Th2-type cytokines may contribute to pulmonary neutrophilia during allergic inflammation by the increased expression of neutrophil chemokines and adhesion molecules by endothelial cells. DXM and the iNOS inhibitors may decrease pulmonary neutrophilia due, in part, to a direct inhibition of some of these factors. interleukin-4; neutrophil chemokines doi:10.1152/ajplung.00294.2006

The airway surface is covered by a fluid, the airway surface liquid, interposed between the mucous layer and the epithelium. The airway surface liquid contains proteins, secreted by different cell types, that may have pro-/anti-inflammatory or bactericidal functions or have a role in the mucociliary clearance. We have used a proteomics approach to identify the proteins secreted by an isolated in vitro model of human airway epithelium, at resting and under proinflammatory conditions, as a strategy to define the factors involved in epithelial barrier function. To this aim, we have analyzed the airway surface liquid from human bronchial epithelial cells grown as polarized monolayers in the presence and absence of inflammatory stimuli such as IL-4, IL-1[beta], TNF-[alpha], and IFN-[gamma]. Two-dimensional electrophoresis followed by mass spectrometry analysis has allowed the identification of ~175 secreted protein spots, among which are immune-related proteins, structural proteins, an actin severer, some protease inhibitors, and a metalloproteinase. Comparisons between treated and untreated conditions have shown that the expression of several proteins was significantly modified by the different cytokines. Our results indicate that the surface epithelium is an active player in the epithelial barrier function and that inflammatory conditions may modulate protein secretion. human bronchial epithelial cells; interleukin-4; interleukin-1 [beta]; tumor necrosis factor-[alpha]; interferon-[gamma]

Intestinal mucosal inflammation can lead to altered function of the underlying smooth muscle, which becomes hyperreactive to most contractile stimuli. Through nematode parasite infection models, T helper type 2 (Th2) cytokines have been implicated in intestinal muscle dysfunction; however, the mechanisms involved and the relevance of these findings to other forms of intestinal inflammation are unclear. Through gene transfer, we explored whether the Th2 cytokine IL-4 can mediate changes in longitudinal muscle function in the context of an adenoviral infection. Following abdominal surgery on mice, control [beta]-galactosidase-encoding recombinant adenoviruses and IL-4-encoding adenoviruses were applied to the serosal surface of the jejunum, leading to infection of cells in the serosa and in the mesentery. Marker transgene expression lasted for 3 wk and was accompanied by the recruitment of macrophages, lymphocytes, and neutrophils into the peritoneal cavity and mild inflammation at the site of infection. IL-4 transgene expression led to a stronger inflammatory response characterized by tissue eosinophilia and increased numbers of peritoneal mast cells and plasma cells. Whereas control virus infection had no effect on intestinal muscle function, infection with the IL-4 virus led to significant jejunal muscle hypercontractility, evident by day 7 postinfection. This modulation of smooth muscle function was shown to be IL-4 specific, since the application of an IL-5-encoding adenovirus induced tissue eosinophilia but did not alter muscle function. These results highlight an important causal role for IL-4 in the pathological regulation of enteric smooth muscle function and identify a novel strategy for gene transfer to the intestine. adenovirus; immunomodulation; motility; enteric infections; pathophysiology; interleukin-4

T cell helper type 2 (Th2) differentiation is driven by a source of IL-4 receptor (IL-4R) that mobilizes IL-4R signaling pathways and the transcription factor GATA-3. Naive CD4 cells can secrete IL-4 independently of IL-4R signals, but how this secretion is regulated is not understood. Here we demonstrate that costimulation through the tumor necrosis factor receptor family molecule OX40, in synergy with CD28, is essential for high levels of nuclear factor of activated T cells c1 to accumulate in the nucleus of a recently activated naive T cell. This action is not dependent on either IL-4R or IL-2R signals and results in OX40 controlling initial naive T cell IL-4 transcription. OX40 signals subsequently enhance nuclear GATA-3 accumulation through an IL-4R-dependent action, leading to Th2 differentiation. These data show that, in the absence of an exogenous source of IL-4, OX40 provides a critical synergistic and temporal signal with other noncytokine receptors to modulate nuclear factor of activated T cells c1 and to promote optimal Th2 generation. IL-4 | GATA-3

Crohn's disease is an idiopathic inflammatory condition. However, little is known about the changes that occur in the muscularis externa, despite the fact that this tissue contributes to motility changes and stricture formation. We characterized immune activity in the muscularis externa from intestinal segments of Crohn's disease patients and evaluated the role of IL-4 and -13 as well as signal transducer and activator of transcription (STAT)6 in the contractility of the cultured human intestinal smooth muscle cells. CD3+ve cells (P < 0.01) and IL-4 protein (P < 0.01) were significantly increased in the muscularis externa of Crohn's disease patients compared with noninflamed controis. Preincubation of human cultured smooth muscle cells with IL-4 (P < 0.001) or IL-13 (P < 0.05) significantly enhanced carbachol-induced contraction, and this was significantly inhibited by the STAT6 inhibitor leflunomide (P < 0.0001). A similar profile was observed in muscle cells isolated from Crohn's disease patients. Both IL-4 and IL-13 increased specific STAT6-DNA binding in control cells, and this was inhibited by anti-STAT6 Ab (P < 0.05) or leflunomide (P < 0.05). IL-4 and IL-13 mediate the hypercontractility of intestinal muscle via a STAT6 pathway at the level of the smooth muscle cell. The STAT6 pathway may contribute to the hypercontractility of intestinal muscle in Crohn's disease. intestinal smooth muscle cell; interleukin-4; interleukin-13

The role of tobacco smoking in the development and outcome of pulmonary fibrosis is uncertain. To approach the effects of cigarette smoke on bleomycin-induced lung fibrosis, we studied five groups of guinea pigs: 1) controls, 2) instilled with bleomycin (B), 3) exposed to tobacco smoke for 6 wk (TS), 4) bleomycin instillation plus tobacco smoke exposure for 6 wk (B+TS), and 5) tobacco smoke exposure for 6 wk and bleomycin after smoking (TS/B). Guinea pigs receiving bleomycin and tobacco smoke exposure exhibited higher fibrotic lesions including a significant increase in the number of positive [alpha]-smooth muscle actin cells compared with bleomycin alone (B+TS, 3.4 [+ or -] 1.2%; TS/B, 3.7 [+ or -] 1.5%; B, 2.3 [+ or -] 1.5%; P < 0.01). However, only the TS/B group reached a significant increase in lung collagen compared with the bleomycin group (TS/B, 3.5 [+ or -] 0.7; B [+ or -] TS, 2.9 [+ or -] 0.4; B, 2.4 [+ or -] 0.2 mg hydroxyproline/lung; P < 0.01). Bronchoalveolar lavage (BAL) from TS/B showed an increased number of eosinophils and higher levels of IL-4 and tissue inhibitor of metalloproteinase-2 (P < 0.01 for all comparisons) and induced a significant increase in fibroblast proliferation (P < 0.05). Importantly, smoke exposure alone induced an increase in BAL neutrophils, matrix metalloproteinase-9, and fibroblast proliferation compared with controls, suggesting that tobacco smoke creates a profibrotic milieu that may contribute to the increased bleomycin-induced fibrosis. matrix metalloproteinase-9; fibroblast proliferation; interleukin-4; tissue inhibitor of metalloproteinase-2; pulmonary fibrosis

Recent data support a modulatory role for CD4 T cells in experimental renal ischemia-reperfusion injury (IRI). CD4 T cells can functionally differentiate to either a Th1 (IFN-[gamma] producing) or the counterbalancing Th2 (IL-4) phenotype. The enzymes signal transducers and activators of transcription (STAT) 4 and STAT6 regulate Th1 or Th2 differentiation and cytokine production, respectively. We therefore hypothesized that mice that were STAT4 deficient would be protected from renal IRI and that STAT6-deficient mice would have a more severe course. Intracellular cytokine staining of splenocytes from STAT4-/- or STAT6-/- exhibited distinct IFN-[gamma] and IL-4 cytokine expression profiles. STAT6-/- had markedly worse renal function and tubular injury postischemia compared with wild type. STAT4-/had only mildly improved function. Renal phagocyte infiltration and ICAM-1 upregulation were similar in STAT4-/-, STAT6-/-, and wild type. To evaluate if the mechanism of the marked worsening in the STAT6-/- mice could be due to IL-4 deficiency, IL-4-deficient mice were studied and had similar postischemic phenotype to STAT6-/- mice. These data demonstrate that the STAT6 pathway has a major protective role in renal IRI. IL-4 deficiency is a likely mechanism underlying the STAT6 effect. A 'yin-yang' role for inflammation is emerging in renal IRI, similar to recent observations in atherosclerosis. Th1/Th2 cells; inflammation; interleukin-4

IL-4 inhibits vasoactive intestinal peptide production by macrophages. Am J Physiol Gastroin-test Liver Physiol 283: G115-G121, 2002. First published March 6, 2002; 10.1152/ajpgi.00491.2001.--In schistosomiasis, eggs induce granulomas that have a vasoactive intestinal peptide (VIP) immunoregulatory circuit. This study explored the regulation of VIP production at sites of inflammation. Splenocytes from uninfected C57BL/6 mice expressed VIP mRNA and protein, which stopped following egg deposition. Eggs induce a Th2 response, suggesting that Th2 cytokines like interleukin (IL)-4 can regulate VIP. To address this issue, splenocytes from uninfected mice were incubated for 4 h with or without recombinant IL-4. IL-4 inhibited VIP mRNA expression. F4/[80.sup.+] macrophages were the source of constitutively expressed VIP, subject to IL-4 regulation. In IL-4 knockout mice, splenic VIP production did not down-modulate during schistosome infection, suggesting that IL-4 is a critical cytokine regulating VIP production in wild-type mouse spleen. IL-4-producing granulomas in schistosomiasis made VIP. Experiments showed that granuloma VIP derived from F4/80-(nonmacrophage) cell populations, explaining this paradox. Granuloma F4/[80.sup.+] cells from IL-4 knockout mice expressed VIP. Thus macrophages can make VIP, which is subject to IL-4 regulation. However, in the Th2 granulomas, other cell types produce VIP, which compensates for loss of macrophages as a source of this molecule. granulomas;interleukin-4; Th1; Th2

Interferons (IFNs) inhibit induction by IL-4 of multiple genes in human monocytes. However, the mechanism by which IFNs mediate this inhibition has not been defined. IL-4 activates gene expression by inducing tyrosine phosphorylation, homodimerization, and nuclear translocation of the latent transcription factor, STAT6 (signal transducer and activator of transcription-6). STAT6-responsive elements are characteristically present in the promoters of IL-4-inducible genes. Because STAT6 activation is essential for IL-4-induced gene expression, we examined the ability of type I and type II IFNs to regulate activation of STAT6 by IL-4 in primary human monocytes. Pretreatment of monocytes with IFN-[Beta] or IFN-[Gamma], but not IL-1, IL-2, macrophage colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, IL-6, or transforming growth factor [Beta] suppressed activation of STAT6 by IL-4. This inhibition was associated with decreased tyrosine phosphorylation and nuclear translocation of STAT6 and was not evident unless the cells were preincubated with IFN for at least 1 hr before IL-4 stimulation. Furthermore, inhibition by IFN could be blocked by cotreatment with actinomycin D and correlated temporally with induction of the JAK/STAT inhibitory gene, SOCS-1. Forced expression of SOCS-1 in a macrophage cell line, RAW264, markedly suppressed trans-activation of an IL-4-inducible reporter as well as IL-6- and IFN-[Gamma]-induced reporter gene activity. These findings demonstrate that IFNs inhibit IL-4-induced activation of STAT6 and STAT6-dependent gene expression, at least in part, by inducing expression of SOCS-1.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is highly expressed in macrophages from thioglycollate-elicited peritonitis. Cytokines and other factors known to control macrophage differentiation and function were evaluated to determine their potential effects on PPAR-gamma expression. Interleukin-4 is shown to induce the expression of PPAR-gamma and 12-15-lipoxygenase mediates interleukin-4-dependent transcription of the CD36 gene in macrophages.

Interleukin-4 (IL-4) was evaluated either alone or together with tumor necrosis factor (TNF)-alpha costimulation if it would modulate CD54 expression by human bronchial epithelial cells (HBECs). IL-4 elicited increased positivity from CD54 while greater induction of CD54 resulted from TNF-alpha. The results showed that IL-4 augmented epithelial cell intercellular adhesion molecule-1 expression and it potentiated the adhesion of THP-1 monocyte/macrophage cells to epithelial cells.

There has been increased interest in the study of nutrition and immunity. This is especially true with respect to the hypothesis that consumption of specific foods may reduce an individual's susceptibility to the establishment and/or progression of immunologic disease. Although an increased intake of a specific food may improve health status in select cases, chronic consumption of large amounts of one specific food may in fact be detrimental. The studies described here examined the long-term effect of yogurt consumption on two different age populations, young adults (20-40 y) and senior adults (55-70 y). There were three study groups per age group, live-culture yogurt, pasteurized yogurt and control (no yogurt), given 200 g/d of yogurt for 1 y. The subjects completed a questionnaire detailing health parameters on a weekly basis and a 4-d food record was taken monthly. Blood was taken every 3 mo and complete blood chemistry, blood count, total and specific immunoglobulin (Ig)E, and interferon-[Gamma] (IFN-[Gamma]) production measured. Yogurt consumption, especially for the live-culture groups, was associated with a decrease in allergic symptoms in both age groups. Seniors in the control group experienced an increase in both total and LDL cholesterol, whereas those in the yogurt groups remained stable during the course of the study. There was little effect on IFN-[Gamma] and IgE production, although seniors in the yogurt group had lower levels of total IgE throughout the year. KEY WORDS: * immunity * yogurt * immunoglobulin E * interleukin-2 * interleukin-4

A new pathway for mechanotransduction in human articular chondrocytes (HAC) is discussed. Chondrocytes are found in collagen-rich extracellular matrices in human cartilage. With adequate mechanical stimulation, chondrocyte function in HAC is enhanced. Scientific explanation for this remains elusive. However, integrins and integrin-associated signalling pathways have been found to affect the activation of plasma membrane apamin-sensitive CA2+-activated K+ channels that results in the hyperpolarization and enhancement of HAC.

Byline: Byoung-Soo Cho (1), Suk-Ran Yoon (2), Ji-Young Jang (3), Kwan-Ho Pyun (2), C.-E. Lee (3) Keywords: Key wordsaMinimal change nephrotic syndrome; Type II IgE receptor; Interleukin-4; Interleukin-4 mRNA; Upregulation Abstract: aAlthough the pathogenesis of childhood minimal change nephrotic syndrome (MCNS) has not been clearly defined, the current hypothesis favors an involvement of T cell dysfunction. The symptom onset and the relapse of MCNS are frequently associated with allergy and increased IgE levels in sera. Since a T cell-derived cytokine interleukin-4 (IL-4) plays a key role in the regulation of IgE production and allergic response, we investigated the role of IL-4 in the pathophysiology of MCNS. Using fluorescence-activated cell scanning we observed a significantly higher expression of CD23, the type II IgE receptor (Fc E RII), on fresh B cells from active MCNS patients (n=22) compared with age-matched healthy normal controls (n=12). The upregulation of CD23 correlates with greater IL-4 activity in the culture supernatant of MCNS peripheral blood lymphocytes (PBLs) than normal PBLs stimulated by mitogens, as assessed by the CD23-inducing effect of the PBL supernatant on tonsillar B cells. Furthermore, Northern blot and reverse transcription-based polymerase chain reaction analysis have revealed significantly elevated levels of IL-4 mRNAs both in mitogen-stimulated and unstimulated MCNS PBLs, compared with healthy normals or disease controls with other renal disorders. Together these results strongly suggest that the upregulation of IL-4 in T cells may be part of the T cell dysfunction involved in MCNS. Author Affiliation: (1) Department of Pediatrics, College of Medicine, Kyung Hee University, Seoul, 130--702, Korea, KR (2) Korea Research Institute of Bioscience and Biotechnology, KIST, Taejon, 305--600, Korea, KR (3) Department of Biology and Institute of Basic Science, Sung Kyun Kwan University, Suwon, 440--746, Korea e-mail: celee@yurim.skku.ac.kr Tel.: +82-331-290-7006, Fax: +82-331-290-7006/7015, KR Article note: Received: 10 March 1997 / Revised: 25 June 1998 / Accepted: 6 July 1998

An important signaling pathway for the differentiation of T helper type 2 (TH2) cells from uncommitted CD4 T cell precursors is activation of the STAT6 transcription factor by interleukin 4 (IL-4). The protooncogene BCL-6 is also involved in TH2 differentiation, as BCL-6 -/- mice develop an inflammation of the heart and lungs associated with an overproduction of TH2 cells. Surprisingly, IL-4 -/BCL-6 -/- and STAT6 -/- BCL-6 -/- double-mutant mice developed the same TH2-type inflammation of the heart and lungs as is characteristic of BCL-6 -/- mice. Furthermore, a TH2 cytokine response developed in STAT6 -/BCL-6 -/- and IL-4 -,/- BCL-6 -/- mice after immunization with a conventional antigen in adjuvant. In contrast to these in vivo findings, STAT6 was required for the in vitro differentiation of BCL-6 -/- T cells into TH2 cells. BCL-6, a transcriptional repressor that can bind to the same DNA binding motifs as STAT transcription factors, seems to regulate TH2 responses in vivo by a pathway independent of IL-4 and STAT6.

The anti-common gamma chain ([[Gamma].sub.c]) mAb CP.B8 is shown to inhibit interleukin 4 (IL-4)-dependent proliferation of phytohemagglutinin (PHA) activated T cells noncompetitively with respect to cytokine by blocking the IL-4-induced heterodimerization of IL-4R[Alpha] and [[Gamma].sub.c] receptor chains. Affinities for the binding of IL-4 to Cos-7 cells transfected with huIL-4R[Alpha], and to PHA blasts expressing both IL-4R[Alpha] and [[Gamma].sub.c], were used to estimate the affinity of the key interaction between [[Gamma].sub.c] and the binary IL-4R[Alpha][multiplied by]IL-4 complex on the cell surface. This affinity was defined in terms of the dimensionless ratio [IL-4R[Alpha][multiplied by]IL-4[multiplied by][[Gamma].sub.c]]/[IL-4R[Alpha][multiplied by]IL-4], which we designate [K.sub.R]. The results show that on PHA blasts this interaction is relatively weak; [K.sub.R] [approximately equal to] 9, implying that [approximately equal to] 10% of the limiting IL-4R[Alpha] chain remains free of [[Gamma].sub.c] even at saturating concentrations of IL-4. This quantitative treatment establishes [K.sub.R] as a key measure of the coupling between ligand binding and receptor activation, providing a basis for functional distinctions between different receptors that are activated by ligand-induced receptor dimerization.

Interleukin 4 (IL-4) is a pleiotropic cytokine. Of the cell types responsive to IL-4, T cells express one IL-4 receptor (IL-4R) type, IL-4R[Alpha]/IL-2R[Gamma] (class I IL-4R), whereas endothelial cells express another type, IL-4R[Alpha]/IL-13R[Alpha](class II IL-4R). It was hypothesized that IL-4 variants could be generated that would be selective for cell types expressing the different IL-4Rs. A series of IL-4 muteins were generated that were substituted in the region of IL-4 implicated in interactions with IL-2R[Gamma]. These muteins were evaluated in T cell and endothelial cell assays. One of these muteins, containing the mutation Arg-121 to Glu (IL-4/R121E), exhibited complete biological selectivity for T cells, B cells, and monocytes, but showed no activity on endothelial cells. Receptor binding studies indicated that IL-4/RI21E retained physical interaction with IL-2R[Gamma] but not IL-13R[Alpha]; consistent with this observation, IL-4/R121E was an antagonist of IL-4-induced activity on endothelial cells. IL-4/R121E exhibits a spectrum of activities in vitro that suggest utility in the treatment of certain autoimmune diseases.

We report that interleukin 4 (IL-4) inhibits the propagation of non-syncytia-inducing and increases the propagation of syncytia-inducing HIV-1 isolates by two mechanisms. It differentially regulates the two major HIV-1 coreceptors, CCR5 and CXCR4, in human peripheral blood mononuclear cells, increasing CXCR4 and decreasing CCR5 expression in primary CD4(+) T-lymphocytes. In addition, IL-4 stimulates the expression of all HIV-1 isolates via a transcriptional activation mechanism. The combination of these effects results in increased propagation of CXCR4-using and inhibition of CCR5-using HIV-1 strains. IL-4 also activates HIV-1 expression in primary monocytes/macrophages but does not affect CCR5 expression. These results identify IL-4 as an important regulator of HIV-1 and suggest a critical role for this cytokine in the control of viral evolution and in the phenotypic switch from non-syncytia-inducing to syncytia-inducing, which leads to accelerated disease progression.

Mice lacking CD81 (TAPA-1), a widely expressed tetraspanin molecule, have impaired antibody responses to protein antigens. This defect is specific to antigens that preferentially stimulate a T helper 2 response (ovalbumin or keyhole limpet hemocyanin in alum) and is only seen with T cell-dependent antigens. Absence of CD81 on B cells is sufficient to cause the defect. Also, antigen-specific interleukin (IL) 4 production is greatly reduced in the spleen and lymph nodes of CD81-null mice compared with heterozygous littermates. Thus, expression of CD81 on B cells is critical for inducing optimal IL-4 and antibody production during T helper 2 responses. These findings suggest that CD81 may interact with a ligand on T cells to signal IL-4 production. By using a soluble form of CD81 as a probe, a putative ligand for CD81 was identified on a subset of B and T cells. Two possible models for the interaction of CD81 on B cells with a potential ligand on either B or T cells are proposed.

Byline: Jie Cai (1), Tong Zheng (1), John Murphy (2), Cory A. Waters (3), George Y. Lin (1), Parkash S. Gill (1) Keywords: AIDS-KS; IL-4R; IL-4 toxin Abstract: Interleukin-4 (IL-4) is a pleiotropic cytokine affecting growth and differentiation of various cell types as well as regulating other cytokines. To study the effect of IL-4 on AIDS-related Kaposi's sarcoma (AIDS-KS) cells, we first examined the tumor cells for IL-4 receptor (IL-4R) expression. KS cells express a single 4 kB IL-4R-specific mRNA and 1828 +- 408 high affinity IL-4 binding sites per cell with a dissociation constant (Kd) of 154 +- 37 pM. Addition of recombinant human IL-4 (rIL-4) minimally inhibited AIDS-KS cell growth and expression of IL-6. We then studied the effects of a chimeric fusion toxin DAB.sub.389-IL-4 which exerts cellular toxicity only on cells expressing IL-4R. DAB.sub.389-IL-4 inhibited protein synthesis in AIDS-KS cells at low concentrations (IC.sub.50 of 5 x 10.sup.-11 M). This effect was abrogated by neutralizing antibody to IL-4 (25D2). We conclude that KS cells express a functional IL-4R and this receptor could serve as a target for novel therapy with agents such as DAB.sub.389-IL-4. Author Affiliation: (1) Department of Medicine, University of Southern California, Los Angeles, California, USA (2) Evans Department of Clinical Research and the Department of Medicine and Microbiology, Boston University, Boston, Massachusetts, USA (3) Seragen Inc., Hopkinton, Massachusetts, USA Article History: Registration Date: 29/09/2004

Extravascular procoagulant activity often accompanies cell-mediated immune responses and systemic administration of pharmacologic anticoagulants prevents cell-mediated delayed-type hypersensitivity reactions. These observations suggest a direct association between coagulation and cell-mediated immunity. The cytokine interleukin (IL)-4 potently suppresses cell-mediated immune responses, but its mechanism of action remains to be determined. Herein we demonstrate that the physiologic anticoagulant protein S is IL-4-inducible in primary T cells. Although protein S was known to inhibit the classic factor Va-dependent prothrombinase assembled by endothelial cells and platelets, we found that protein S also inhibits the factor Va-independent prothrombinase assembled by lymphoid cells. Thus, protein S-mediated down-regulation of lymphoid cell procoagulant activity may be one mechanism by which IL-4 antagonizes cell-mediated immunity.

IL-4 receptor [Alpha] chain (IL-4R[Alpha])-deficient mice were generated by gene-targeting in BALB/c embryonic stem cells. Mutant mice showed a loss of IL-4 signal transduction and functional activity. The lack of IL-4R[Alpha] resulted in markedly diminished, but not absent, TH2 responses after infection with the helminthic parasite Nippostrongylus brasiliensis. CD4+, CD62L-high, and CD62L-low T cell populations from uninfected IL-4R[[Alpha].sup.-/-] mice were isolated by cell sorting. Upon primary stimulation by T cell receptor cross-linkage, the CD62L-low, but not the CD62L-high, cells secreted considerable amounts of IL-4, which was strikingly enhanced upon 4-day culture with anti-CD3 in the presence or absence of IL-4. CD62L-low cells isolated from IL-4R[a[Alpha].sup.-/-], [[Beta].sub.2]-microglobu[lin.sup.-/-] double homozygous mice produced less IL-4 than did either IL-4R[[Alpha].sup.-/-] or wild-type mice. These results indicate that an IL-4-independent, [[Beta].sub.2]-microglobulin-dependent pathway exists through which the CD62L-low CD[4.sup.+] population has acquired IL-4-producing capacity in vivo, strongly suggesting that these cells are NK T cells.

To investigate the contribution of interleukin-4 (IL-4) to airway inflammation in vivo and to explore directly its relationship to airway reactivity, we created transgenic mice in which the murine cDNA for IL-4 was regulated by the rat Clara cell 10 protein promoter. Expression was detected only in the lung and not in thymus, heart, liver, spleen, kidney, or uterus. The expression of IL-4 elicited hypertrophy of epithelial cells of the trachea, bronchi, and bronchioles. Hypertrophy is due, at least in part, to the accumulation of mucus glycoprotein. Histologic examination of parenchyma revealed multinucleated macrophages and occasional islands of cells consisting largely of eosinophils or lymphocytes. Analysis of lung lavage fluid revealed the presence of a leukocytic infiltrate consisting of lymphocytes, neutrophils and eosinophils. Mice expressing IL-4 had greater baseline airway resistance but did not demonstrate hyperreactivity to methacholine. Thus, the expression of IL-4 selectively within the lung elicits an inflammatory response characterized by epithelial cell hypertrophy, and the accumulation of macrophages, lymphocytes, eosinophils, and neutrophils without resulting in an alteration in airway reactivity to inhaled methacholine.

The c-maf proto-oncogene is involved in tissue-specific expression of interleukin-4 (IL-4) in CD4+ T helper (Th) 2 cells. The expression of c-Maf occurs only in differentiating and mature Th2 cells and not in Th1 cells. Induction of c-Maf along with IL-4 occurs in Th2 cells on signal transmission through the T cell receptor. Ectopic expression of c-Maf, which transactivates the IL-4 promoter in Th1, nonlymphoid and B cells, maps to the c-Maf response element (MARE) and a region downstream of the MARE.

To better understand the role of class II major histocompatibility complex molecules in both normal and autoimmune responses, we have produced a series of I-Ab transgenic mice. One of these transgenic constructs, designated NOD.PD, has the sequence of the NOD [Beta] chain (A[[Beta].sup.g7]) except at positions 56 and 57, where Pro-Asp replaces His-Ser. Several NOD.PD transgenic lines have been produced. One line of these mice carried a very high number of copies (>50) of the NOD.PD transgene. As has been described in other mice carrying high copy numbers of I-Ab transgenes, B-cell development was abnormal. The steady state numbers of mature B cells (Ig[M.sup.+]/Ig[D.sup.hi]) in the periphery were greatly reduced in transgenic mice compared to nontransgenic littermates. Surprisingly, rather than being accompanied by a generalized hypogammaglobulinemia, this B-cell deficiency was accompanied by elevated concentrations of IgG1 and IgE in the serum. Conversely, the levels of IgG2a were reduced in transgenic mice compared to nontransgenic littermates. Because this isotype pattern was characteristic of interleukin (IL)-4-induced class-switching, we then investigated the role of IL-4 in causing the observed phenotype. We crossed the high copy number transgenic mice with an IL-4-deficient strain of mice. As expected, the elevated levels of IgE in high copy number transgenic mice were eliminated when the IL-4 gene was inactivated. However, the reduction in the number of B cells was not ameliorated. These data indicate that the primary defect caused by the transgene was to reduce the number of B cells in these mice. This reduction was accompanied by a secondary increase in IL-4 production, which drove the remaining B cells toward the production of IgG1 and IgE.

Interleukin-4 (IL-4), a pleiotropic cytokine, stimulates a dose-dependent increase in the number of mouse primordial germ cells in culture. Results from bromodeoxyuridine incorporation assays suggest that IL-4 acts as a survival factor rather than as a mitogen for primordial germ cells in this system. Studies on the embryonic expression patterns of IL-4 and its receptors, using RT-PCR and ELISA, show that IL-4 and its receptors are present at the correct time and place to influence PGC numbers in vivo.

The monoclonal antibodies (Mab) 25D2 and 35F2 bind to human interleukin 4 (huIL-4) receptor and produce a 54-fold decrease in its affinity for the extracellular domain of its receptor alpha-subunit (shuIL-4R-alpha). Size exclusion chromatography and Western blot analysis indicate that Mab 25D2 binds to huIL-4 and inhibits the interaction between huIL-4 and secondary receptor subunit, IL-2R-gamma. Mab 25D2 induces the neutralization effect through a dual mechanism, influencing the function of the two subunits simultaneously.

Interleukin-4 (IL-4), a pleiotropic cytokine, is a major regulator of the immune system and is considered crucial for the development of T helper cell type 2 ([T.sub.H]2) responses. The susceptibility [...]

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The binding subunit of the interleukin-13 (IL-13) receptor which is also a part of the interleukin-4 (IL-4) receptor has been cloned and characterized. Data indicate that the IL-13 receptor alpha chain is the main binding subunit of the IL-13 receptor and could also be a component of IL-4 receptors. Murine NR4 was observed to encode IL-13Ralpha. while IL-13Ralpha expression was found to allow transduction of a proliferative signal by IL-13.

A study conducted of the activation of a signal-transducing factor (STF-IL4) and an interleukin 4 (IL-4) -induced phosphotyrosine substrate (4PS) shows that the activation of STF-IL4 does not require the presence of 4PS/insulin receptor substrate-1 (IRS-1). However the activation of both STF-IL4 and ISL-1/4PS involves the same region of IL-4 receptor and may involve common factors. In 32D cells phosphorylated 4PS is absent but STF-IL4 is activated. The activation of STF-Il4 does not cause the expression of any inducible c-myc.

A new site-specific conjugation method to develop interleukin 4 (IL4) toxins that have enhanced cytotoxic and binding activities involves the substitution of an additional seventh cysteine residue at the 105, 68, 28, 70, 97 or 38 positions without affecting the binding affinity or conjugation of Pseudomonas exotoxin (PE35) to site-specific IL4 positions. PE35 conjugation to IL4 causes a 10-fold increase in the cytotoxic and binding activities of IL4-toxins. The improvement in the activities of conjugated IL4-toxins is more than in recombinant IL4 that has toxin fusions in its C-terminus.