gp49B1 inhibits IgE-initiated mast cell activation through both immunoreceptor tyrosine-based inhibitory motifs, recruitment of src homology 2 domain-containing phosphatase-1, and suppression of early and late calcium mobilization.
We define by molecular, pharmacologic, and physiologic approaches the proximal mechanism by which the immunoglobulin superfamily member gp49B1 inhibits mast cell activation mediated by the high affinity Fc receptor for IgE (FcepsilonRI). In rat basophilic leukemia-2H3 cells expressing transfected mouse gp49B1, mutation of tyrosine to phenylalanine in either of the two immunoreceptor tyrosine-based inhibitory motifs of the gp49B1 cytoplasmic domain partially suppressed gp49B1-mediated inhibition of exocytosis, whereas mutation of both abolished inhibitory capacity. Sodium pervanadate elicited tyrosine phosphorylation of native gp49B1 and association of the tyrosine phosphatases src homology 2 domain-containing phosphatase-1 (SHP-1) and SHP-2 in mouse bone marrow-derived mast cells (mBMMCs). SHP-1 associated transiently with gp49B1 within 1 min after coligation of gp49B1 with cross-linked FcepsilonRI in mBMMCs. SHP-1-deficient mBMMCs exhibited a partial loss of gp49B1-mediated inhibition of FcepsilonRI-induced exocytosis at concentrations of IgE providing optimal exocytosis, revealing a central, but not exclusive, SHP-1 requirement in the counter-regulatory pathway. Coligation of gp49B1 with cross-linked FcepsilonRI on mBMMCs inhibited early release of calcium from intracellular stores and subsequent influx of extracellular calcium, consistent with SHP-1 participation. Because exocytosis is complete within 2 min in mBMMCs, our studies establish a role for SHP-1 in the initial counter-regulatory cellular responses whereby gp49B1 immunoreceptor tyrosine-based inhibition motifs rapidly transmit inhibition of FcepsilonRI-mediated exocytosis. (+info
The vitronectin receptor and its associated CD47 molecule mediates proinflammatory cytokine synthesis in human monocytes by interaction with soluble CD23.
The vitronectin receptor, alphavbeta3 integrin, plays an important role in tumor cell invasion, angiogenesis, and phagocytosis of apoptotic cells. CD47, a member of the multispan transmembrane receptor family, physically and functionally associates with vitronectin receptor (VnR). Although vitronectin (Vn) is not a ligand of CD47, anti-CD47 and beta3 mAbs suppress Vn, but not fibronectin (Fn) binding and function. Here, we show that anti-CD47, anti-beta3 mAb and Vn, but not Fn, inhibit sCD23-mediated proinflammatory function (TNF-alpha, IL-12, and IFN-gamma release). Surprisingly, anti-CD47 and beta3 mAbs do not block sCD23 binding to alphav+beta3+ T cell lines, whereas Vn and an alphav mAb (clone AMF7) do inhibit sCD23 binding, suggesting the VnR complex may be a functional receptor for sCD23. sCD23 directly binds alphav+beta3+/CD47(-) cell lines, but coexpression of CD47 increases binding. Moreover, sCD23 binds purified alphav protein and a single human alphav chain CHO transfectant. We conclude that the VnR and its associated CD47 molecule may function as a novel receptor for sCD23 to mediate its proinflammatory activity and, as such, may be involved in the inflammatory process of the immune response. (+info
Terreic acid, a quinone epoxide inhibitor of Bruton's tyrosine kinase.
Bruton's tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development. Btk mutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using an in vitro assay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects of btk mutant mast cells in high-affinity IgE receptor-stimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of Btk in mast cells and other immune cell systems. Another insight obtained from this study is that the screening method used to identify TA is a useful approach to finding more efficacious Btk inhibitors. (+info
Predictive value of CD19 measurements for bacterial infections in children infected with human immunodeficiency virus.
We investigated the predictive value of CD19 cell percentages (CD19%) for times to bacterial infections, using data from six pediatric AIDS Clinical Trials Group protocols and adjusting for other potentially prognostic variables, such as CD4%, CD8%, immunoglobulin (IgA) level, lymphocyte count, prior infections, prior zidovudine treatment, and age. In addition, we explored the combined effects of CD19% and IgG level in predicting time to infection. We found that a low CD19% is associated with a nonsignificant 1.2-fold increase in hazard of bacterial infection (95% confidence interval: 0.97, 1.49). In contrast, a high IgG level is associated with a nonsignificant 0.87-fold decrease in hazard of infection (95% confidence interval: 0.68, 1.12). CD4% was more prognostic of time to bacterial infection than CD19% or IgG level. Low CD19% and high IgG levels together lead to a significant (P < 0. 01) 0.50-fold decrease in hazard (95% confidence interval: 0.35, 0. 73) relative to low CD19% and low IgG levels. Similarly, in a model involving assay result changes (from baseline to 6 months) as well as baseline values, the effect of CD19% by itself is reversed from its effect in conjunction with IgG. In this model, CD19% that are increasing and high are associated with decreases in hazard of infection (P < 0.01), while increasing CD19% and increasing IgG levels are associated with significant (at the P = 0.01 level) fourfold increases in hazard of infection relative to stable CD19% and decreasing, stable, or increasing IgG levels. Our data suggest that CD19%, in conjunction with IgG level, provides a useful prognostic tool for bacterial infections. It is highly likely that T-helper function impacts on B-cell function; thus, inclusion of CD4% in such analyses may greatly enhance the assessment of risk for bacterial infection. (+info
Fc receptor beta subunit is required for full activation of mast cells through Fc receptor engagement.
The high-affinity IgE receptor (Fc epsilonRI) and the low-affinity IgG receptor (Fc gammaRIII) on mast cells are the key molecules involved in triggering the allergic reaction. These receptors share the common beta subunit (FcRbeta) which contains an immunoreceptor tyrosine-based activation motif and transduces the signals of these receptors' aggregation. In rodents, FcRbeta is essential for the cell surface expression of the Fc epsilonRI. In humans, the FcRbeta gene was reported to be one of the candidate genes causing atopic diseases. However, the role of FcRbeta in vivo still remains ambiguous. To elucidate the functions of FcRbeta, we developed the mice lacking FcRbeta [FcRbeta(-/-)]. The FcRbeta(-/-) mice lacked the expression of the Fc epsilonRI on mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) was not induced in FcRbeta(-/-) mice as was expected. In these mice, the expression of IgG receptors on mast cells was augmented but the IgG-mediated PCA reaction was attenuated. Although with bone marrow-derived cultured mast cells from FcRbeta(-/-), adhesion to fibronectin and Ca2+ flux upon aggregation of IgG receptors were enhanced, mast cells co-cultured with 3T3 fibroblasts exhibited impaired degranulation on receptor aggregation. These observations indicate that FcRbeta accelerates the degranulation of mature mast cells via the IgG receptor in connective tissues. (+info
Affinity modulation of very late antigen-5 through phosphatidylinositol 3-kinase in mast cells.
Adhesiveness of integrins is up-regulated rapidly by a number of molecules, including growth factors, cytokines, chemokines, and other cell surface receptors, through a mechanism termed inside-out signaling. The inside-out signaling pathways are thought to alter integrin affinity for ligand, or cell surface distribution of integrin by diffusion/clustering. However, it remains to be clarified whether any physiologically relevant agonists induce a rapid change in the affinity of beta1 integrins and how ligand-binding affinity is modulated upon stimulation. In this study, we reported that affinity of beta1 integrin very late Ag-5 (VLA-5) for fibronectin was rapidly increased in bone marrow-derived mast cells by Ag cross-linking of FcepsilonRI. Ligand-binding affinity of VLA-5 was also augmented by receptor tyrosine kinases when the phospholipase Cgamma-1/protein kinase C pathway was inhibited. Wortmannin suppressed induction of the high affinity state VLA-5 in either case. Conversely, introduction of a constitutively active p110 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) increased the binding affinity for fibronectin. Failure of a constitutively active Akt to stimulate adhesion suggested that the affinity modulation mechanisms mediated by PI 3-kinase are distinct from the mechanisms to control growth and apoptosis by PI 3-kinase. Taken together, our findings demonstrated that the increase of affinity of VLA-5 was induced by physiologically relevant stimuli and PI 3-kinase was a critical affinity modulator of VLA-5. (+info
Differential roles of N- and C-terminal immunoreceptor tyrosine-based inhibition motifs during inhibition of cell activation by killer cell inhibitory receptors.
Killer cell inhibitory receptors (KIRs) inhibit NK and T cell cytotoxicity when recognizing MHC class I molecules on target cells. They possess two tandem intracytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs) that, when phosphorylated, each bind to the two Src homology 2 domain-bearing protein tyrosine phosphatases SHP-1 and SHP-2 in vitro. Using chimeric receptors having an intact intracytoplasmic KIR domain bearing both ITIMs (N + C-KIR), a deleted domain containing the N-terminal ITIM only (N-KIR), or a deleted domain containing the C-terminal ITIM only (C-KIR), we examined the respective contributions of the two ITIMs in the inhibition of cell activation in two experimental models (a rat mast cell and a mouse B cell line) that have been widely used to analyze KIR functions. We found that the two KIR ITIMs play distinct roles. When coaggregated with immunoreceptor tyrosine-based activation motif-bearing receptors such as high-affinity IgE receptors or B cell receptors, the N + C-KIR and the N-KIR chimeras, but not the C-KIR chimera, inhibited mast cell and B cell activation, became tyrosyl-phosphorylated, and recruited phosphatases in vivo. The N + C-KIR chimera recruited SHP-1 as expected, but also SHP-2. Surprisingly, the N-KIR chimera failed to recruit SHP-1; however, it did recruit SHP-2. Consequently, the N-terminal ITIM is sufficient to recruit SHP-2 and to inhibit cell activation, whereas the N-terminal and the C-terminal ITIMs are both necessary to recruit SHP-1. The two KIR ITIMs, therefore, are neither mandatory for inhibition nor redundant. Rather than simply amplifying inhibitory signals, they differentially contribute to the recruitment of distinct phosphatases that may cooperate to inhibit cell activation. (+info
Clinico-biological implications of increased serum levels of interleukin-8 in B-cell chronic lymphocytic leukemia.
BACKGROUND AND OBJECTIVE: Constitutive cellular expression and serum release of biologically active interleukin-8 (IL-8) has been reported in B-cell chronic lymphocytic leukemia (CLL). Given the autocrine role played by IL-8 in the process of cell accumulation characteristic of this disease we tried to investigate clinico-biological implications of increased serum levels of this cytokine in an unselected series of B-cell CLL patients. DESIGN AND METHODS: Serum levels of IL-8 were determined at the time of diagnosis in 58 previously untreated B-CLL patients using an immunoenzyme assay. Results were correlated with main clinico-hematologic features as well as with the risk of disease progression. Finally, we looked for associations between IL-8 and molecules directly involved in apoptosis, such as intracellular bcl-2 and soluble APO-1/Fas. RESULTS: Increased serum levels of IL-8 were found in 15 out of 58 (25.8%) B-cell CLL patients. Serum levels of IL-8 did not reflect clinico-biological features representative of tumor mass such as clinical stage, histopathologic pattern of bone marrow (BM) involvement, b2-microglobulin, sCD23 and sCD27 titers. Interestingly, circulating levels of IL-8 paralleled those of intracellular bcl-2 (r = 0.522; p = 0.01), thus confirming that the antiapoptotic effect of IL-8 can be exerted through a bcl-2 dependent pathway. Levels of IL-8 did not match those of soluble Apo-1/Fas (r = -0.013; p = 0.943). Finally, stage A patients with levels of IL-8 above the median value (i.e. 4.5 pg/mL) were more likely to progress to a more advanced clinical stage than those with levels below the median value (p < 0.05). INTERPRETATION AND CONCLUSIONS: IL-8 is an interesting marker in B-cell CLL, closely involved in the pathogenesis of disease. Furthermore, it is useful for predicting the pace of disease progression in early clinical stages. (+info