Interleukin-3 stabilizes CD124/IL-4α surface expression in mast cells via Tyk2 and STAT6

Interleukin (IL)-4 signals can modulate mast cells, which express the IL-4Rα chain. The IL-4Rα can heterodimerise with the common γ-chain and utilizes JAK1 and JAK2 for signal transduction, while complexes of IL-4Rα with IL-13Rα1 subunit mediates signals via JAK2 and Tyk2. Here, we report that IL-3 is an essential factor for the continuous expression of the IL-4Rα chain on mast cells, which did not express the IL-13Rα1 chain. We demonstrate that the signals induced by IL-3 important for IL-4Rα expression are mediated by Tyk2 and STAT6 activation and the subsequent maintenance of HSP90 levels. In line with that, inhibition of either Tyk2, STAT6 or HSP90 impaired the IL-3-induced IL-4Rα upregulation. Consequently, the IL-3 maintained IL-4Rα surface expression via Tyk2 is essential for the costimulatory effect of IL-4 on the IL-33-induced production of IL-6 and IL-13.     

IgA肾病患者IL—2的产生及IL—2受体的表达

本文对IgA肾病(IgAN)外周血淋巴细胞白细胞介素2(IL-2)的产生、受体的表达及免疫球蛋白的产生进行了研究。结果发现:外周血淋巴细胞产生IL-2的活性明显增高,IL-2受体表达亦明显增强并伴有免疫球蛋白产生增多,提示IgAN存在着细胞免疫功能的紊乱。     

Agonist-induced T cell receptor down-regulation: molecular requirements and dissociation from T cell activation

T cell receptor (TCR) down-regulation is a consequence of specific receptor engagement and plays an important role in modulating the T cell response. We have investigated the role of protein kinase C (PKC) and protein tyrosine kinases (PTK) in the induction of TCR down-regulation. We report that the mutation of S126 in the CD3-γ chain that is known to inhibit phorbol-12-myristate 13-acetate-induced TCR down-regulation does not affect down-regulation induced by a specific agonist. In addition, agonist-induced TCR down-regulation is not affected by blockade or depletion of PKC, neither by blockade or lack of PTK, while the same treatments efficiently interfere with T cell activation. These results demonstrate that TCR down-regulation is induced by early events which follow specific engagement by an agonist and can be dissociated from those required for full T cell activation.     

抗白细胞介素—6受体单克隆抗体的制备

用白细胞介素－６（ＩＬ－６）依赖性人多发性骨髓瘤细胞株ＸＧ－１作为免疫原，我们成功地制备获得一株分泌抗人ＩＬ－６受体单克隆抗体的杂交瘤，命名为ＳＩ１５。夹心ＥＬＩＳＡ和位点竞争试验表明，ＳＩ１５和抗人ＩＬ－６受体单克隆抗抗Ｍ１９识别ＩＬ－６受体上的同一位点，对ＩＬ－６受体的生物活性无影响。     

Binding Sites of Interleukin-3-Mimetic Monoclonal Autoantibodies Derived from a MRL/lpr Mouse

MRL/lpr mouse-derived interleukin-3 (IL-3)-mimetic monoclonal antibodies were examined for their binding sites. One of these five antibodies (B10, F8, F9, F12, H 11), F9 interacted with the IL-3 receptor, as if it were an anti-idiotypic antibody; the IL-3-mimetic activity of F9 was blocked by a neutralizing rat monoclonal anti-IL-3 antibody. IL-3 mRNA was not detected in hybridoma F9, as analyzed by the SI protection assay, Thus, the activity neutralized by the rat antibody is of the F9 antibody itself but not the IL-3 type. Such blocking was not observed with the IL-3-mimetic activity of the other MRL/lpr-derived monoclonal antibodies. On the other hand, the binding of all these monoclonal antibodies to IL-3-depen-dent cells was inhibited by each other and vice versa, as analyzed by two-color flow cytometry. This indicates that the binding sites of the five monoclonal antibodies are located so close to each other that the binding of one would interfere with the binding of any one of the others (since the binding experiment was done on ice, it is unlikely that the inhibition is due to down-modulation of the receptors). Taken together the results obtained by the enzyme digestion study, we discussed that all five IL-3-mimetic monoclonal antibodies are directed to the IL-3 receptor, but only F9 binds to the portion directly responsible for the binding of IL-3 and the other antibodies (B10, F8, F12, H11) bind to different portions, respectively, which are adjacent or overlapping to the binding site of F9.     

Association of the p56lck protein tyrosine kinase with the FCγRIIIA/CD16 complex in human natural killer cells

The multimeric FcγRIIIA (CD16) complex is expressed on the surface of natural killer (NK) cells and is composed of a 50–70-kDa transmembrane glycoprotein Fcγ receptor (CD16), the T cell receptor (TCR)-ζ chain, and the FcεRIγ chain. Cross-linking FcγRIIIA initiates the rapid tyrosine phosphorylation of multiple substrates including the ζ, subunit and causes subsequent cell activation and antibody-dependent cellular cytotoxicity (ADCC). The subunits of the FcγRIIIA complex lack intrinsic protein tyrosine kinase (PTK) activity, suggesting that receptor-induced tyrosine phosphorylation events are mediated by a nonreceptor PTK. We report here that the human FcγRIIIA is complexed with p56^lck, a src-family PTK previously found associated with the CD4 and CD8 receptors on T cells. Upon engagement of the CD16 receptor, p56^lck is rapidly (within 30 s) and transiently phosphorylated on tyrosine residues. Several FcγRIIIA-associated proteins are identified in immune complex kinase assays including the TCR-ζ, subunit, a p70–90 ζ-associated protein (ZAP), p50a (acidic) and p50b (basic), and p56^lck. We demonstrate that the src-family protein tyrosine kinase inhibitor, herbimycin A, blocks increased intracellular calcium levels and ADCC caused by CD16 cross-linking on NK3.3 cells. Likewise cross-linking CD16 with the protein tyrosine phosphatase CD45, abrogates CD16-induced calcium mobilization. These data suggest that p56^lck is physically associated with FcγRIIIA(CD16) and functions to mediate signaling events related to the control of NK cellular cytotoxicity.     

Human mast cells release Interleukin-8 and induce neutrophil chemotaxis on contact with activated T cells

BACKGROUND: Mast cells have recently been shown to control neutrophil recruitment during T-cell mediated cutaneous DTH reaction in vivo through TNF-alpha and MIP-2, the functional murine analogue of human IL-8. Although the nature of signals transmitted from T cells which activate mast cells has not yet been defined, we hypothesized that a direct cross-talk (i.e. heterotypic adhesion) between these two cell populations exists, as has previously been reported. AIMS: The present study was aimed at gaining insight into the functional role of mast cell-T cell contact in expression and release of IL-8, and its effect on neutrophil chemotaxis. METHODS: The IL-8 gene expression was identified by Affymetrix GeneChip arrays, validated by RT-PCR and the protein measured by ELISA. Chemotaxis was evaluated by using a modified Boyden chamber assay. RESULTS: Mast cells were found to express and release significantly higher concentrations of IL-8 on incubation with membranes obtained from activated, as compared to resting T cells. Supernatants obtained from these activated mast cells induced significant neutrophil chemotaxis that was inhibited by neutralizing mAb to IL-8. CONCLUSIONS: Thus, activated T cells, on heterotypic adhesion to mast cells, deliver the necessary signals for the latter to release cytokines and chemokines necessary for cell migration to sites of antigen challenge, thereby facilitating T-cell mediated inflammatory processes.     

白细胞介素-3的分子生物学

白细胞介素-3(Interleukin-3,IL-3)是T细胞分泌的一种蛋白质因子,它除与T细胞的增殖与分化有关外、最重要的生物功能是刺激骨髓早期多能祖细胞增殖,促进多种造血祖细胞的增殖与分化,故又有多能集落刺激因子(Pluripotent CSF)之称。最近发现某些白血病的发生可能与IL-3基因的缺失有关、因此,阐明IL-3基因的结构及其表达的调节,对研究某些严重血液病,如再生障碍性贫血,急性放射病及某些白血病的发生机制,具有重要意义。     

Role of c-kit receptor tyrosine kinase in the development, survival and neoplastic transformation of mast cells

The c-kit gene is allelic with the dominant spotting ( W ) locus on mouse chromosome 5 and encodes a receptor tyrosine kinase. The llgand for c-klt receptor is stem cell factor (SCF), which is the principal growth factor for mast cells. The loss-of-function mutations of c-kit receptor affect the development of mast cells, thereby resulting in a depletion of mast cells. The abundant expression of c-ktt receptor is indispensable for the survival of mast cells. In addition, the galn-of-function mutations of c-kit receptor were found in several tumor mast cell lines. When these galn-of-function mutations were introduced to cells of murine interleukin (IL)-3-dependent cell lines, the expression of c-kit receptor with constitutive tyrosine kinase activity not only abrogated the IL-3 requirement of the cells, but also caused them to become tumorlgenic in nude athymic mice. The gain-of-function mutations of c-kit receptor appear to result in the malignant transformation of mast cells. Taken together, the signals from the c-ktt receptor are essential for the development, survival, and malignant transformation of mast cells.     

Masitinib, a c-kit/PDGF receptor tyrosine kinase inhibitor, improves disease control in severe corticosteroid-dependent asthmatics

Background:  Masitinib is a tyrosine kinase inhibitor targeting stem cell factor receptor (c-kit) and platelet-derived growth factor (PDGF) receptor, which are expressed on several cell types including mast cells and bronchial structural cells, respectively. We hypothesized that c-kit and PDGF receptor inhibition may decrease bronchial inflammation and interfere with airway remodeling, which are crucial features of severe asthma.
Objectives:  The primary endpoint was the percent change from baseline in oral corticosteroids after 16 weeks of treatment. Change in asthma control (asthma control questionnaire), exacerbation rate, pulmonary function tests, rescue medication requirement and safety were secondary endpoints.
Methods:  A 16-week randomized, dose-ranging (3, 4.5, and 6 mg/kg/day), placebo-controlled study was undertaken in 44 patients with severe corticosteroid-dependent asthma who remained poorly controlled despite optimal asthma management.
Results:  At 16 weeks of treatment, a comparable reduction in oral corticosteroids was achieved with masitinib and placebo (median reduction of ­78% and ­57% in the masitinib and placebo arms, respectively). Despite this similar reduction, the Asthma Control Questionnaire score was significantly better in the masitinib arm as compared to placebo with a reduction by 0.99 unit at week 16 ( P  < 0.001) vs 0.43 unit in the placebo arm. Masitinib therapy was associated with more transient skin rash and edema.
Conclusions:  Masitinib, a c-kit and PDGF-receptor tyrosine kinase inhibitor, may represent an innovative avenue of treatment in corticosteroid-dependent asthma. These preliminary results warrant further long-term clinical studies in severe asthma (ClinicalTrials.gov Identifier: NCT00842270).     

Isolation and characterization of a novel HS1 SH3 domain binding protein, HS1BP3

We have isolated a novel gene, HS1BP3, which encodes an HS1 binding protein. Analysis of HS1BP3 cDNA indicates several potentially important segments, including a PX domain, a leucine zipper, immunoreceptor tyrosine-based inhibitory motif-like motifs and proline-rich regions. HS1BP3 associates with HS1 proteins in vivo as confirmed by immunoprecipitation in B and T cell lines. HS1BP3 preferentially associates with the HS1 SH3 domains rather than with other SH3 molecules, suggesting a role of HS1BP3 as an HS1 signaling mediator. Overexpression of mutant HS1BP3 protein in T cell lines results in decreased IL-2 production. Our data suggest a novel role for HS1BP3 in lymphocyte activation.     

Fcγ receptor-mediated phagocytosis requires tyrosine kinase activity and is ligand independent

Receptors for the invariant chain of immunoglobulins (FcR) define the cellular response to specific antigens. FcγR recognize IgG and so elicit a variety of effector functions including phagocytosis. We are interested in the structural determinants for FcγR-mediated phagocytosis, specifically FcγRI(p135) and FcγRIIa isoforms. The low-affinity receptor, FcγRIIa, is found on macrophages and its cytoplasmic domain contains a tyrosine activation motif which has previously been shown to regulate endocytosis. In contrast, FcγRI has no known signaling motifs, though a functional interaction has recently been demonstrated with the γ chain of the high-affinity receptor for IgE, FcεRI. This accessory molecule has a cytoplasmic tyrosine activation motif implicated in signal transduction. Here we demonstrate that although FcγRI transiently expressed on COS-7 cells is able to rosette opsonized SRBC, it cannot phagocytose them. If the cytoplasmic domain of either γ chain or FcγRIIa replaces that of FcγRI in a chimeric receptor, efficient phagocytosis occurs. This particle ingestion is sensitive to the tyrosine kinase inhibitor genistein. Chimeric receptors where the extracellular domain of either FcγRI or FcγRIIa is replaced with that of CD2, a T cell antigen, indicate that FcγR-mediated phagocytosis is ligand independent. We conclude that phagocytosis is dependent upon close particle apposition, tyrosine kinase activity, and that the process is ligand independent.     

Recombinant Human Mullerian Inhibiting Substance Inhibits Epidermal Growth Factor Receptor Tyrosine Kinase

Abstract

Autophosphorylation of the epidermal growth factor (EGF) receptor in A-431 cells and plasma membrane fractions was inhibited by partially purified recombinant human Mullerian Inhibiting Substance (MIS). Immunoprecipitation of the EFG receptor using anti-EGF receptor or anti-phosphotyrosine antibodies, and phosphoamino acid analysis of this receptor, demonstrated that MIS specifically inhibited EGF-induced tyrosine phosphorylation. Inhibition of EGF receptor autophosphorylation by MIS in membrane preparations was not affected by increasing concentrations of EGF, manganese or [γ-(32)P] ATP. Thus, it is unlikely that MIS competes for EGF binding sites or sequesters substrate. Immunoabsorption of MIS with anti-human MIS antibody blocked the MIS inhibition of EGF receptor autophosphorylation, indicating that the inhibition was due to MIS. Our data suggest that MIS regulates the activity of the EGF receptor tyrosine kinase in A-431 cells.     

IL-37 a New IL-1 Family Member Emerges as a Key Suppressor of Asthma Mediated by Mast Cells

In 1986, we reported a multiple biological effect of IL-1 including immunological, inflammatory, and tumor killing activity. Since then other IL-1 family cytokines have been discovered, some with inflammatory and other with anti-inflammatory activity. In this review article, we speculate on the possible inhibitory effect of IL-37 in the light of new findings.

IL-37, formerly termed IL-1 family member 7 (IL-1F7), binding IL-18 receptor α chain, acts as a cytokine with intracellular as well as extracellular functionality and as a natural inhibitor of immune responses and inflammation. IL-37 inhibits many pro-inflammatory cytokine and increases anti-inflammatory cytokines such as IL-10.

Asthma pathogenesis involves multiple cell types including mast cells, which are important cellular constituents of the human innate and adaptive immunity. IL-37 has an impact on inflammatory cytokines generated by mast cells and is beneficial for and protective in asthma. However, the precise mechanism(s), safety, and tolerability of IL-37 are unclear and still remain a mystery.

Abbreviations: GBP (Guanylate Binding Proteins); HMGB1 (High Mobility Group Box protein 1); NLRP (Nucleotide-like Receptor Pyrin domain 1); ASC (Apoptosis-associated Speck-like protein containing CARD, Caspase Recruitment Domain); FGF2 (Fibroblast Growth Factor 2).     

The role of tyrosine phosphorylation in the interaction of cellular tyrosine kinases with the T cell receptor ζ chain tyrosine-based activation motif

Immunoglobulin receptor family tyrosine-based activation motifs (ITAM) define a conserved signaling sequence, EX₂ YX₂L/IX₇YX₂L/I, that mediates coupling of the T cell antigen receptor (TCR) to protein tyrosine kinases (PTK). In the present study, we explored the role of phosphorylation of the two ITAM tyrosine residues in the interactions of the motif with the PTK ZAP-70 and p59fyn. The data show that the phosphorylation of a single tyrosine within the motif enables binding of p59fyn, whereas phosphorylation of both tyrosines within the motif is required for maximal binding of the PTK ZAP-70. Quantitative binding experiments show that nanomolar concentrations of the doubly phosphorylated ζ1-ITAM are sufficient for ZAP-70 recruitment, whereas micromolar levels of singly phosphorylated ITAM are necessary for p59fyn binding. ZAP-70 binds with low efficiency to a singly phosphorylated ITAM, but shows preferential binding to the C-terminal phosphotyrosine in the ITAM, whereas p59fyn binds selectively to the N-terminal phosphotyrosine. The present data thus show that there is the potential for a singly phosphorylated ITAM to couple to cellular PTK. Moreover, the data suggest a mechanism for heterogeneity in signal transduction responses by the TCR, since ITAM could differentially couple the TCR to downstream signaling events depending on their phosphorylation state.     

Feedback regulation of IFN‐α/β signaling by Axl receptor tyrosine kinase modulates HBV immunity

Hepatitis B virus (HBV) is known to cause age‐dependent infection outcomes wherein most infections during young age result in chronicity. The mechanism underlying the differential outcome remains elusive. By using hydrodynamic injection of the replication‐competent pAAV‐HBV, we established a mouse model in which HBV persistence was generated in 4–5 w/o C57BL/6 young mice, but not in adult mice over 10 w/o. HBV‐tolerant young mice expressed higher interferon (IFN)‐α/β levels in hepatocytes and intrahepatic plasmacytoid DCs (pDCs) than adult mice after pAAV‐HBV injection. Excessive IFN‐α/β expression in young mice was associated with induction of the Axl regulatory pathway and expansion of intrahepatic Treg cells. In line with these findings, augmented IFN‐β expression increased Axl expression in the liver and HBV persistence in adult mice, whereas IFN‐α/β signaling blockage decreased Axl expression and HBV persistence in young mice. Accordingly, Axl overexpression decreased HBV clearance of adult mice whereas Axl silencing enhanced HBV clearance of young mice. In vitro, IFN‐β priming of pDCs and Axl‐overexpressing macrophages enhanced Treg‐cell differentiation. These findings suggest that age‐dependent HBV chronicity is attributed to IFN‐β‐Axl immune regulation, which is selectively induced in young mice by excessive IFN‐α/β production at early stage of HBV infection.     

Evidence for a Glucocorticoid Receptor in Human Leukocytes

The glucocorticoid uptake in vitro by human periferal leukocytes was studied. The uptake showed 2 main components, one saturable and one non-saturable. The saturable component was compared with the uptake by the specific glucocorticoid receptor in rabbit granulocytes. The similarities with the rabbit receptor in structural specificity, time course of uptake at 37° C, sensitivity to metabolic inhibition by PCMS and the physiological concentration for half saturation indicate that the saturable component corresponds to a specific glucocorticoid receptor. Cells from chronic lymphatic leukemia and chronic myeloic leukemia were also studied. Only the former had a saturable glucocorticoid uptake.