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At least 793 records · Page 44Linked to original sources
PubMed1995

Identification of signaling motifs within human Fc gamma RIIa and Fc gamma RIIb isoforms.

To assess the functional capacity of the heterogeneous Fc gamma RII (CD32) family and to identify critical regions for functioning, we generated a panel of B-cell transfectants. The Fc gamma R-negative B-cell line IIA1.6 was transfected with wild-type or mutant human Fc gamma RIIa and IIb molecules. Solely Fc gamma RIIa-expressing IIA1.6 cells were capable of phagocytosing opsonized Staphylococcus aureus bacteria, and cross-linking of Fc gamma RIIa triggered a rapid induction of tyrosine phosphorylation after 20 seconds. Analysis of Fc gamma RIIa mutants identified the immunoreceptor tyrosine-based activation motif (ITAM; previously described as ARH-1 motif) within the IIa cytoplasmic tail to be critical for B-cell activation. In contrast, Fc gamma RIIb isoforms triggered tyrosine phosphorylation on cross-linking with much slower kinetics (> 3 minutes) than Fc gamma RIIa. Furthermore, solely Fc gamma RIIb molecules proved capable of downregulating [Ca2+]i and interleukin-2 production on co-cross-linking with sIgG in IIA1.6. The Fc gamma RIIb-mediated functions were absent in Fc gamma RIIb mutants in which the tyrosine or leucine within the YSLL motif in a conserved 13-aa region (now known as immunoreceptor tyrosine-based inhibitor motif [ITIM]) were changed into phenylalanines. In conclusion, these data show the presence of functionally critical motifs within Fc gamma RII cytoplasmic tails. Fc gamma RIIa contains an ITAM involved in B-cell activatory functions, whereas the downregulatory activity of Fc gamma RIIb isoforms is linked to an ITIM.

Amino Acid Sequence↗
PubMed1995

Analysis of antigen receptor signaling in B cells from mice with a retrovirus-induced acquired immunodeficiency syndrome.

MAIDS is a retrovirus-induced immunodeficiency syndrome in mice that has similarities to human AIDS. Because the functional defects in B cells from retroviral immunodeficiency syndromes have not been characterized in detail, we examined early and late parameters of B cell responses to IgM cross-linking in B cells from MAIDS and normal mice. Splenic B cells from mice with MAIDS have defective in vitro proliferative responses to LPS and anti-IgM-mediated stimuli, as well as to PMA plus calcium ionophore, indicating a generalized defect in proliferative response potential independent of specific receptor-mediated signaling. When early signaling parameters were analyzed in response to IgM cross-linking, it was found that calcium flux in B cells from MAIDS mice was significantly reduced; this reduction was not accounted for by quantitative differences in cell-surface IgM expression and therefore indicates a defect in early signal transduction through the IgM receptor. The tyrosine phosphorylation response to IgM cross-linking was also markedly deficient; tyrosine phosphorylation of Ig-alpha, Ig-beta, and an undefined protein of 80 kDa was detected in MAIDS B cells after anti-IgM stimulation, at levels substantially less than those observed in normal B cells. Multiple other tyrosine phosphorylation events observed in normal B cells, including phosphorylation of GTPase-activating protein, P13-kinase, and syk kinase, were not detected in MAIDS B cells in response to IgM cross-linking. The defect in tyrosine phosphorylation seemed to correlate with reduced surface IgM levels on a subpopulation of MAIDS B cells. B cells from mice expressing the MAIDS retrovirus-induced immunodeficiency thus reflect defects in early signaling through the Ag-specific IgM receptor as well as a generalized defect in proliferative responsiveness.

Animals↗
PubMed1994

Reconstitution of antibody-dependent phagocytosis in fibroblasts expressing Fc gamma receptor IIIB and the complement receptor type 3.

In this study, we test the hypothesis that co-expression of both the complement receptor type 3 (CR3; CD11b/CD18) and Fc gamma receptor type IIIB (Fc gamma RIIIB) (CD16) are sufficient to mediate Ab-dependent phagocytosis. To explore the roles of these receptors in a simple and well-defined in vitro system, stable transfectants of fibroblasts expressing either CR3, Fc gamma RIIIB, or the combination of CR3 and Fc gamma RIIIB were generated. Cells not expressing either receptor, but exposed to the transfection protocol, were used as controls. Cell surface expression of CR3 and/or Fc gamma RIIIB were confirmed by using both flow cytometry and epifluorescence microscopy. The cell lines were analyzed for their ability to bind and internalize opsonized erythrocytes. Cells expressing both CR3 and Fc gamma RIIIB were able to both bind and phagocytose IgG-coated erythrocytes. In contrast, cells expressing CR3 only were able to phagocytose yeast, but not to bind nor phagocytose IgG-coated erythrocytes. Similarly, cells expressing Fc gamma RIIIB only were able to bind IgG-coated erythrocytes, but not to phagocytose either the erythrocytes or yeast. These studies demonstrate that, although CR3 does not participate in Ab-dependent recognition, it can complement the function of Fc gamma RIIIB to effect Ab-dependent phagocytosis. These studies also suggest that one mechanism for glycosylphosphatidylinositol-linked proteins to mediate intracellular functions is through interactions with transmembrane proteins.

3T3 Cells↗
PubMed1994

Evidence for protein tyrosine kinase involvement in ligand-induced TCR/CD3 internalization and surface redistribution.

Triggering of the TCR/CD3 complex can lead to its internalization and modulation from the cell surface. In the present study, we address the question of the dependence of internalization on protein tyrosine kinase (PTK) activation. With use of an activating anti-clonotypic (anti-Ti) mAb on a CTL clone, we have shown that the PTK inhibitors genistein and tyrphostin 25 delayed anti-Ti-induced internalization, but did not affect fluid phase protein uptake or transferrin receptor cycling. Confocal microscopy with use of fluorescent anti-Ti mAb revealed that the inhibition of TCR internalization corresponded to the induction of large patches that were localized in cell membrane areas depleted of polymerized actin, the formation of which was dependent on the combined action of the anti-Ti mAb and the PTK inhibitors. In contrast to the effect of these PTK inhibitors, depletion of Src-like PTKs by T cell pretreatment with herbimycin A led to an increased rate of anti-Ti-induced internalization. Internalization induced by the monovalent Fab fraction of anti-Ti mAb was similarly affected by the PTK inhibitors, although the extent of induced internalization was less by approximately one-half. An analysis of substrates phosphorylated in kinase assays on TCR/CD3 immunoprecipitates of the CTL, which were activated by anti-Ti mAb in both the absence and presence of genistein, identified protein bands in which phosphorylation or association with CD3 was inhibited in the presence of genistein. Thus, a genistein-sensitive PTK activity seems to control ligand-induced TCR/CD3 complex redistribution and internalization.

Actins↗
PubMed1993

Surface transport and internalization of the membrane IgM H chain in the absence of the Mb-1 and B29 proteins.

The micron IgH chain is one component of the membrane IgM complex, the endocytic and signal transducing receptor for Ag on the surface of B lymphocytes. It is transported to the cell surface in association with three other B cell-specific proteins, an L chain and the products of the mb-1 and B29 genes. The roles played by these various proteins in mediating the functions of the complex are unclear. To analyze micron function in the absence of other lymphoid-specific proteins, we first attempted to express micron on the surface of nonlymphoid cells. Deletion of the CH1 domain was sufficient to allow surface expression of this protein in transfected COS cells as well as in mouse L cells. To determine whether this extracellularly truncated micron was capable of endocytosis, we used an assay to detect the internalization of anti-mu antibody bound to the surface of transfected cells expressing the protein. Under both cross-linking and non cross-linking conditions, the CH1-deleted micron protein was internalized in endocytic vesicles. We conclude from these observations that a) the CH1 domain of micron contains a retention signal, the elimination of which allows surface transport of this protein, and b) micron by itself is capable of at least one of the functions of the membrane IgM complex.

Amino Acid Sequence↗
PubMed1993

The TAPA-1 molecule is associated on the surface of B cells with HLA-DR molecules.

TAPA-1 is a transmembrane protein that has been shown to be involved in cell growth and cellular adhesion. Our studies were aimed at determining the mechanisms of the biologic phenomena mediated by TAPA-1, which include the identification of proteins that are associated with it on the surface of lymphocytes. We and others have previously shown that Leu-13, a leukocyte Ag, is one such molecule and that in B cells TAPA-1 is associated with the CD19 Ag. Herein we identify an additional molecule, HLA-DR, that is noncovalently associated on the surface of B cells with TAPA-1. This association was first detected by immunoprecipitation by anti-TAPA-1 and by anti-HLA-DR antibodies in the presence of mild detergents. The initial observation was confirmed by 2-dimensional SDS-PAGE and by direct identification of TAPA-1 in anti-HLA-DR immunoprecipitates by Western blot analysis. The association of the two molecules on the surface of a human B cell line was shown by cocapping experiments. In addition, antibodies to both molecules can induce cellular adhesion and an antiproliferative effect. Because the tissue distribution of these two molecules only partially overlaps, with TAPA-1 being expressed on most cell types and MHC class II expressed on a more restricted group of tissue, it is possible that the TAPA-1 molecule provides a basic function that can augment a cell type specific activity. In B cells the association of TAPA-1 with CD19 and HLA-DR may increase cellular interaction and play a supporting role in the transmission of specific signals.

Antigens, CD↗
PubMed1996

Regulation of the catalytic subunit (p34PSK-J3/cdk4) for the major D-type cyclin in mature B lymphocytes.

We examined the expression of the cyclin-dependent kinase 4, p34PSK-J3/cdk4 protein, in small dense, activated, and proliferating primary B lymphocytes. A small steady state level of cdk4 synthesis was detected in resting B cells. Stimulation of resting B cells with mitogenic amounts of F(ab')2 fragments of goat anti-mouse IgM (anti-Ig) resulted in increased synthesis of cdk4 protein during the mid to late G1 phase of the cell cycle; LPS or the combination of phorbol ester and calcium ionophore also elevated cdk4 levels. Resting B cells that we rendered competent by treatment with IL 4 or low doses of anti-Ig or, alternatively, were activated by phorbol ester or ionomycin alone also exhibited heightened cdk4 protein levels. Subsequent analysis of potential cdk4 regulatory subunit D-type cyclins revealed that cyclin D2, not cyclin D1 or D3, is expressed in primary mature B lymphocytes. The induction of cyclin D2 synthesis in response to mitogenic anti-Ig paralleled cdk4 expression; however, IL-4 or low dose anti-Ig alone did not increase the rate of de novo cyclin D2 synthesis above that of resting B cells. The significance of the lack of cyclin D2 regulation by competence-inducing growth factors was demonstrated, in that only mitogenic factors that stimulated DNA synthesis 1) led to the formation of stable cyclin D2/cdk4 holoenzyme complexes during G1 phase progression, and 2) afforded the isolation of anti-cyclin D2 or anti-cdk4 immunoprecipitates that phosphorylated retinoblastoma. These findings suggest a role for these proteins during the mid to late G1 phase progression and possibly the G1/S phase transition in primary mature B lymphocytes.

Animals↗
PubMed1996

Inhibition of CD4 cross-linking-induced lymphocytes apoptosis by vesnarinone as a novel immunomodulating agent: vesnarinone inhibits Fas expression and apoptosis by blocking cytokine secretion.

Evidence is accumulating that T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals show accelerated cell death through apoptosis. We have recently demonstrated that the cross-linking of CD4 molecules (CD4XL) results in death of normal peripheral T cells through apoptosis and imbalanced cytokine secretion (ie, induction of tumor necrosis factor-alpha [TNF-alpha] and interferon-gamma [IFN-gamma] in the absence of interleukin-2 [IL-2] or IL-4 secretion). These upregulated cytokines (TNF-alpha/IFN-gamma) largely contributed to upregulation of the apoptosis-inducing cell surface molecule, Fas (APO-1/CD95) and apoptosis induction. The present study investigated the effect of vesnarinone as a novel immunomodulating agent on CD4XL-induced T-cell apoptosis. The addition of vesnarinone to peripheral blood mononuclear cells (PBMC) significantly inhibited CD4XL-induced lymphocyte apoptosis. This apoptosis-inhibitory effect of vesnarinone was associated with the blocking of CD4XL-induced TNF-alpha IFN-gamma secretion and of Fas antigen upregulation. However, vesnarinone did not block effects of exogenously supplemented TNF-alpha/IFN-gamma on Fas induction. These data suggest that vesnarinone inhibits CD4XL-induced TNF-alpha/IFN-gamma secretion, thereby blocking subsequent Fas upregulation and apoptosis induction. Given the potent pathogenic role of imbalanced cytokine secretion observed in HIV-infection, an agent such as vesnarinone may be of therapeutic value in slowing disease progression.

Adjuvants, Immunologic↗
PubMed1996

Reciprocal regulation of mucosal surface IgA+ B cells by Ig receptor cross-linking and CD40 ligand.

In the present study, we analyze the role of Ig receptor cross-linking in T cell-dependent stimulation of both preswitch (surface IgM+ (sIgM+/sIgD+) B cells and postswitch (sIgA+) B cells. We demonstrate that purified sIgA+ B cells pretreated with anti-IgA-dextran at low concentrations (10 and 100 ng/ml) exhibited an increased response to activated T cells, whereas pretreatment with higher doses (1 and 10 micrograms/ml) led to a profound suppression of IgA secretion (> or = 90%). The suppressive effect of anti-IgA-dextran was accentuated in the presence of IL-2 and attenuated in the presence of IL-4. Anti-IgA-dextran pretreatment had no effect on sIgA+ B cell survival. sIgM+/sIgD+ B cells pretreated with anti-IgD-dextran or anti-IgM-dextran did not show significant inhibition. The increased susceptibility of sIgA+ B cells, but not of sIgM+/sIgD+ B cells, to Ig cross-linking-mediated suppression was confirmed in cross-linking studies with the same Ab (anti-kappa-dextran). Importantly, anti-IgA-dextran-mediated suppression could be reversed by stimulation of sIgA+ B cells with fibroblasts expressing CD40L; such a reversal required persistent exposure to cells expressing high levels of CD40L. These studies imply that Ig receptor cross-linking renders postswitch sIgA+ B cells unresponsive to subsequent stimulation via activated T cells, but this unresponsiveness is overcome by a persistent high level CD40L signal.

Animals↗
PubMed1996

Physical and functional association of Fc mu receptor on human natural killer cells with the zeta- and Fc epsilon RI gamma-chains and with src family protein tyrosine kinases.

We recently reported that Fc mu R on NK cells is a signal transducing protein that stimulates a rapid increase in the level of cytoplasmic free calcium upon binding of IgM. This study was designed to examine signal transduction via the Fc mu R on NK cells and to characterize intracellular second messengers activated by IgM. Immunoprecipitation of IgM-bound Fc mu R by IgM-specific Ab coimmunoprecipitated the zeta- and Fc epsilon RI gamma-chains. Furthermore, engagement and clustering of Fc mu R by polyclonal IgM induced tyrosine phosphorylation of the zeta- and Fc epsilon RI gamma-chains, indicating their functional association with the Fc mu R-induced signal transduction cascade. Ligand-induced clustering of the Fc mu R also induced activity of src family kinases, Lck, Fyn, Lyn, and Src, as well as their physical interaction with the receptor. Triggering via Fc mu R also induced the activity of Syk and Zap-70, tyrosine kinases demonstrated to associate with zeta and Lck. Phospholipase C-gamma 1 and phosphatidylinositol 3-kinase were identified as substrates phosphorylated on tyrosine, as down-stream components of the signaling pathway activated in NK cells by polyclonal IgM. Although the Fc mu R on NK cells has not yet been biochemically characterized, our results suggest that the zeta- and Fc epsilon RI gamma-chains are functional subunits of this as well as other important cell surface receptors and that the Fc mu R is coupled either directly or indirectly to nonreceptor tyrosine kinases, which phosphorylate and thereby activate regulatory enzymes such as phospholipase C-gamma 1 and phosphatidylinositol 3-kinase.

Calcium↗
PubMed1996

Human blood basophils produce interleukin-13 in response to IgE-receptor-dependent and -independent activation.

Interleukin-13 (IL-13) is a recently discovered immunoregulatory cytokine. The cellular sources of IL-13 and the regulation of its expression are largely unknown. Here we show that human basophils produce IL-13 in response to IgE-receptor (IgER) crosslinking, IL-3, IL-3 plus C5a, but not C5a alone. Human basophils express IL-13 in a restricted manner since, apart from IL-4, no other cytokines encoded on the cytokine gene cluster (IL-3, IL-5, and granulocyte macrophage-colony-stimulating factor [GM-CSF]), are induced. Highest levels of IL-13 are formed after IgE-independent activation leading to a prolonged secretion of IL-13. The response to IgER-cross-linking is more transient preferentially inducing IL-4, IL-3 is a unique cytokine regulating IL-13 production by human basophils: Among a large number of cytokines tested, only IL-3 is capable of directly inducing IL-13 expression. Furthermore, although some IL-13 is produced in response to C5a in the presence of IL-5, GM-CSF, IGF-1 or IL-1 beta, IL-3 is by far the most effective. IL-13 production was blocked by actinomycin D and cycloheximide and conditions leading to IL-13 release also lead to the induction of IL-13 mRNA. This study supports an important immunoregulatory role of human blood basophils, owing to their capacity to simultaneously express IL-13 and IL-4 in a restricted manner.

Basophils↗