Cytokine receptor-mediated trafficking of preformed IL-4 in eosinophils identifies an innate immune mechanism of cytokine secretion (original) (raw)
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Traffic, 2005
Secretion of interleukin-4 (IL-4) by leukocytes is important for varied immune responses including allergic inflammation. Within eosinophils, unlike lymphocytes, IL-4 is stored in granules (termed specific granules) and can be rapidly released by brefeldin A (BFA)-inhibitable mechanisms upon stimulation with eotaxin, a chemokine that activates eosinophils. In studying eotaxin-elicited IL-4 secretion, we identified at the ultrastructural level distinct vesicular IL-4 transport mechanisms. Interleukin-4 traffics from granules via two vesicular compartments, large vesiculotubular carriers, which we term eosinophil sombrero vesicles (EoSV), and small classical spherical vesicles. These two vesicles may represent alternative pathways for transport to the plasma membrane. Loci of both secreted IL-4 and IL-4-loaded vesicles were imaged at the plasma membranes by a novel EliCell assay using a fluoronanogold probe. Three dimensional electron tomographic reconstructions revealed EoSVs to be folded, flattened and elongated tubules with substantial membrane surfaces. As documented with quantitative electron microscopy, eotaxin-induced significant formation of EoSVs while BFA pretreatment suppressed eotaxin-elicited EoSVs. Electron tomography showed that both EoSVs and small vesicles interact with and arise from granules in response to stimulation. Thus, this intracellular vesicular system mediates the rapid mobilization and secretion of preformed IL-4 by activated eosinophils. These findings, highlighting the participation of large tubular carriers, provide new insights into vesicular trafficking of cytokines.
IL-4 Induces Differentiation and Expansion of Th2 Cytokine-Producing Eosinophils
The Journal of …, 2004
Innate effector cells that produce Th2-type cytokines are critical in Th2 cell-mediated immune responses. However, it is not known how these cells acquire the ability to produce Th2 cytokines. IL-4 is a potent inducer that directs differentiation of naive CD4(+) T cells into CD4(+) Th2 effector cells. To determine whether IL-4 can induce differentiation and expansion of Th2 cytokine-producing innate cells, we used mice whose il-4 gene was replaced by a knock-in green fluorescence protein (gfp) gene. We found that, directly ex vivo, IL-4 increased the number of GFP(+) cells in the airway and the lung tissue in an Ag-specific manner. The majority of GFP(+) cells were eosinophils, suggesting that IL-4 plays a pivotal role in expanding IL-4-producing eosinophils in vivo. IL-4-producing eosinophils showed some unique features compared with IL-4-producing CD4(+) T cells. They exhibited biallelic expression of the il-4 gene when stimulated and were more dominant IL-4- and IL-5-producing ce...
American Journal of Respiratory Cell and Molecular Biology, 1998
Similar to interleukin-3 (IL-3), IL-5, and granulocyte macrophage colony-stimulating factor (GM-CSF), IL-4 can be secreted by several cell types involved in allergic inflammatory reactions, and therefore can affect eosinophil function similarly. In this study, we investigated the presence of an IL-4 receptor (IL-4R) on human eosinophils. When two different monoclonal antibodies (mAbs) against the IL-4R ␣-chain (IL-4R ␣) were used, fluorescent-activated cell sorter analysis revealed the presence of an IL-4R ␣ on both eosinophils of normal donors and atopic dermatitis patients. In addition, the expression of the IL-2R ␥-chain, a functional component of the IL-4R in some cell types, was demonstrated. The IL-4R ␣ appeared to be expressed constitutively, and stimulation with cytokines IL-2, IL-3, IL-5, GM-CSF, and interferon-␥ did not further increase IL-4R ␣ expression. Evidence for an IL-4R ␣ was further substantiated by mRNA analysis. Both Northern blot analysis and reverse transcriptase/polymerase chain reaction revealed the presence of mRNA for the IL-4R ␣ in eosinophils from normal individuals and AD patients. Furthermore, we demonstrated that both IL-4 and IL-13 were capable of inducing PI-3 kinase activity in human eosinophils. Because this activation could be inhibited by an IL-4R ␣ mAb, we conclude that both cytokines can activate human eosinophils through binding to a receptor complex comprising the IL-4R ␣ and-yet to be identified-associated proteins. In addition, the involvement of IL-4 in functional responses was studied. IL-4 appeared to "prime" eosinophils to respond chemotactically toward regulated on activation, normal T cells expressed and secreted, but did not affect platelet-activating factor-induced chemotaxis. Taken together, these data show the presence of a functional IL-4R on human eosinophils.
Interleukin 4 signals through two related pathways
Proceedings of the National Academy of Sciences, 1995
The interleukin 4 (IL-4) signaling pathway involves activation, by tyrosine phosphorylation, of two distinct substrates, a signal-transducing factor (STF-IL4) and the IL-4-induced phosphotyrosine substrate (4PS). It is not known whether the IL-4-mediated activation of these substrates occurs via related or distinct signaling pathways. We report that 32D cells, an IL-3-dependent myeloid progenitor cell line in which no phosphorylated 4PS is found, activate high levels of STF-IL4 in response to IL-4. Consistent with the known requirement for 4PS or insulin receptor substrate 1 (IRS-1) in IL-4-mediated mitogenesis, activation of STF-IL4 in 32D cells is not sufficient for IL-4-inducible c-myc expression. In addition, we have examined the ability of 32D cells transfected with different truncation mutants of the human IL-4 receptor to activate Jak-3 kinase and STF-IL4 in response to human IL-4. As in the case of4PS/IRS-1, we have found that activation of both Jak-3 and STF-IL4 requires the presence of the IL-4 receptor region comprising aa 437-557. The finding that the same region of the IL-4 receptor is required for the induction of both 4PS/IRS-1 and STF-IL4 suggests that the IL-4-stimulated activation of these two substrates might involve common factors.
Regulation of the Expression of the Soluble and Membrane Forms of the Murine IL-4 Receptor
Cellular Immunology, 1997
switch to the IgG1 and IgE isotypes; the differentiation of precursor T helper cells into mature Th2 cells; and The actions of interleukin-4 (IL-4) in vivo are likely inhibitory effects on the production of proinflammatory to be positively influenced by the expression of memcytokines by monocytes (3-8). High-affinity receptors brane IL-4 receptors (mIL-4R) on target cells and negatively by the concentration of soluble IL-4 receptors for IL-4 (IL-4R) are expressed on a wide variety of he-(sIL-4R) in the extracellular environment. Inasmuch mopoietic and nonhemopoietic cells and are composed as the two forms of the mouse IL-4R are differentially of a 140-kDa ligand-binding chain (IL-4Ra) in associaencoded by alternatively spliced mRNA transcripts, tion with the common g chain, a signal transducing the purpose of this work was to determine how their subunit shared by several cytokine receptor complexes expression is regulated by IL-4 and T cell activation including IL-2R, IL-7R, IL-9R, and IL-15R (9-14). In and whether there is preferential expression of one addition, a second class of IL-4R has been reported to type of transcript over the other. In this study, the be formed by the interaction of the IL-4Ra chain with expression of sIL-4R and mIL-4R transcripts was anathe IL-13Ra chain (15, 16). lyzed by a semiquantitative RT-PCR method in resting The IL-4Ra chain is produced naturally both as a and mitogen-activated splenic cells. Irrespectively of membrane-associated and as a truncated, soluble form the state of cell activation, IL-4 up-regulated the levels (mIL-4R and sIL-4R, respectively) (17-19). The two ILof both types of mRNA with similar kinetics and dose-4R forms are encoded by distinct mRNA species that response curves. In contrast, ConA failed to enhance arise from alternative splicing of a primary transcript the steady-state levels of sIL-4R or mIL-4R transcripts despite increased expression at the protein level, sug-of the IL-4R gene (7). The difference between the two gesting that sIL-4R expression is also regulated at lev-mRNAs is the splicing out of sequences corresponding els other than transcription. Western blot analysis of to exon 8 in the membrane IL-4R-specific mRNA (20). supernatants of IL-4-and ConA-stimulated spleen cells Retention of exon 8 in the sIL-4R-specific transcript substantiated the presence of sIL-4R molecules deresults in the addition of six novel amino acids followed rived by translation of sIL-4R-specific transcripts, thus by premature translational termination immediately confirming the importance of this mechanism for the upstream of the transmembrane domain region generation of sIL-4R molecules in normal cells. These (17, 20). The sIL-4R protein, therefore, consists of the results indicate that the sIL-4R-and mIL-4R-specific extracytoplasmic domain of the mIL-4R followed by a transcripts are normally regulated in a parallel manshort C-terminal peptide sequence not present in the ner and further suggest that expression of both forms membrane form. of the IL-4R is controlled at multiple levels (i.e., tran-Whereas the function of the mIL-4R in ligand bindscriptional and posttranscriptional). ᭧ 1997 Academic Press ing and subsequent signaling appears obvious, the role of the sIL-4R is less clear. Since the sIL-4R binds IL-INTRODUCTION 4 with high affinity, it can act as a natural antagonist of IL-4 activity, competing with the mIL-4R on target Interleukin-4 (IL-4) is a pleiotropic cytokine procells for the binding of IL-4. Indeed, recombinant sILduced by the Th2 subset of CD4 / T cells, mast cells, 4R inhibits IL-4-dependent activities, both in vitro (21) and basophils (1, 2). Among its many activities are the and in vivo, in murine allograft rejection (22) and IgE activation and differentiation of B lymphocytes, includproduction after injection of anti-IgD antibodies (23) or ing the induction of immunoglobulin heavy-chain class allergens (24). Moreover, the administration of recombinant sIL-4R has also been reported to confer resistance in otherwise susceptible BALB/c mice against in-1 To whom correspondence and reprint requests should be addressed.
PLoS ONE, 2012
Background: Previous work from our laboratory demonstrated that IL-4Ra expression on a myeloid cell type was responsible for enhancement of Th2-driven eosinophilic inflammation in a mouse model of allergic lung inflammation. Subsequently, we have shown that IL-4 signaling through type I IL-4 receptors on monocytes/macrophages strongly induced activation of the IRS-2 pathway and a subset of genes characteristic of alternatively activated macrophages. The direct effect(s) of IL-4 and IL-13 on mouse eosinophils are not clear. The goal of this study was determine the effect of IL-4 and IL-13 on mouse eosinophil function. Methods: Standard Transwell chemotaxis assay was used to assay migration of mouse eosinophils and signal transduction was assessed by Western blotting. Results: Here we determined that (i) mouse eosinophils express both type I and type II IL-4 receptors, (ii) in contrast to human eosinophils, mouse eosinophils do not chemotax to IL-4 or IL-13 although (iii) pre-treatment with IL-4 but not IL-13 enhanced migration to eotaxin-1. This IL-4-mediated enhancement was dependent on type I IL-4 receptor expression: cCdeficient eosinophils did not show enhancement of migratory capacity when pre-treated with IL-4. In addition, mouse eosinophils responded to IL-4 with the robust tyrosine phosphorylation of STAT6 and IRS-2, while IL-13-induced responses were considerably weaker. Conclusions: The presence of IL-4 in combination with eotaxin-1 in the allergic inflammatory milieu could potentiate infiltration of eosinophils into the lungs. Therapies that block IL-4 and chemokine receptors on eosinophils might be more effective clinically in reducing eosinophilic lung inflammation.
Cell, 1989
Receptors for interleukin-4 (11-4) are expressed at low levels on a wide variety of primary cells and cultured cell lines. Fluorescence-activated sorting of CTLG2 cells resulted in the isolation of a subclone, CTLL 19.4, which expressed lo6 IL-4 receptors per cell. These cells were used for the purification of IL-4 receptor protein and to prepare a hybrid-subtracted cDNA probe for isolation of cDNA clones. Three classes of IL-4 receptor cDNA were identified. The first encoded a 140 kd membrane bound IL-4 receptor containing extracellular, transmembrane, and cytoplasmic domains. The second class lacked the cytoplasmic region, and the third encoded a secreted form of the receptor. All cDNA clones expressed in COS-7 cells had IL-4 binding properties comparable to the natlve IL-4 receptor. The soluble form of the IL-4 receptor blocked the ability of IL-4 to induce CTLL cell proliferation and may represent a regulatory molecule specific for IL-4-dependent immune responses.
Transient Inhibition of Interleukin 4 Signaling by T Cell Receptor Ligation
2010
Interleukin (IL)-4 and IL-12 together with T cell receptor (TCR) engagement are crucial for the differentiation of CD4 ϩ T cells into T helper (Th)2 or Th1 cells, respectively. Although IL-4 receptors (IL-4Rs) but not IL-12Rs are expressed on naive CD4 ϩ T cells, IL-4 has no apparent advantage over IL-12 in driving naive T cell differentiation when the cells are primed with both IL-4 and IL-12 in vitro. It was found that IL-4-induced phosphorylation of Janus kinases 1 and 3, IL-4R ␣ , signal transducer and activator of transcription 6, and insulin receptor substrate 2 was strikingly but transiently inhibited by TCR ligation both in conventional and TCR transgenic T cells. TCR engagement also blocked the expression of an IL-4-inducible gene. Signals induced by other cytokines, including IL-2, IL-6, and interferon ␣ , but not by insulin-like growth factor 1, were also blocked by TCR engagement. The capacity of various inhibitors to reverse TCR-mediated inhibition of IL-4 signaling suggested that activation of the Ras-mitogen-activated protein kinase pathway and of the calcineurin pathway contribute to desensitizing IL-4R. IL-4 responsiveness returned at about the time ( ف 12 h) that IL-12mediated signaling was first observed. Thus, through different mechanisms, neither IL-4R nor IL-12R has any clear advantage in polarizing cells; rather, the availability of cytokine is probably the limiting factor in this process.
IL-4 upregulates FcϵRI α-chain messenger RNA in eosinophils
Journal of Allergy and Clinical Immunology, 1995
By using the reverse transcription polymerase chain reaction and Southern blot hybridization, we demonstrated that FceRl e~-chain (FceRIe 0 messenger RNA was expressed in eosinophils purified from the peripheral blood of patients with allergic rhinitis and that this expression was enhanced by studies in which flow cytometty or immunostaining was used did not reveal the expression of FceRIc~ protein on eosinophils from peripheral blood. Neither IL-4 alone nor the combination oflL-4 and other cytokines could induce detectable FceRIc~ protein; nevertheless, they do express FceRIc~ mRNA. Double-labeling immunostaining on ctyostat sections of nasal mucosa clearly demonstrated that some FceRIc~-positive cells were eosinophil cationic protein-positive, which confirms their eosinophilic nature. Not all the eosinophil cationic protein-positive cells express an FceRIa signal. Considering that FceRIe~ mRNA was detectable in four of five samples of eosinophils from those patients with nasal allergy and that only one of five eosinophil samples from normal subjects expressed FceRIc~ mRNA, the level of FceRI expression may be correlated with the activation of eosinophils. B seems vety likely that some other unidentified factors are required for the process from the expression of FceRIc~ mRNA to that of FceRI as a protein. (J ALLERGY CLIN IMMUNOL 1995; 96.'1161-9.) The high-affinity IgE receptor (FceRI) was considered to be present only on basophils and mast cells, but its presence also on Langerhans' cells, dendritic cells, 1 monocytes, 2 and eosinophils 3 has been recently reported. The expression of FceRI on eosinophils is important in that it suggests the possibilities of direct contact of antigen and eosinophils, followed by subsequent degranulation, and the function of antigen presentation of eo-From ~Department