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J C Cambier

Publications and source records attributed to J C Cambier.

At least 55 records · Page 3Linked to original sources
PubMed1998

Interleukin-4 overcomes the negative influence of cyclic AMP accumulation on antigen receptor stimulated B lymphocytes.

Activation of protein kinase A (PKA) in B lymphocytes prior to the ligation of the B cell antigen receptor (BCR) results in a profound inhibition of BCR induced proliferation. The major effect of increased PKA activity in B lymphocytes was the induction of apoptosis leading to a reduced BCR induced growth response. The growth promoting cytokine IL-4 rescued B lymphocytes from PKA mediated negative effects. IL-4 protected BCR stimulated cells from PKA mediated inhibition primarily by preventing apoptosis and growth arrest. PKA-activation caused a downregulation of anti-IgM induced expression of Bcl-xL protein, that was restored by IL-4. Previous studies have shown that PKA-activation blocks BCR induced phospholipase Cgamma-activation and calcium mobilization. IL-4 was unable to overcome the block in anti-IgM mediated calcium mobilization due to PKA-activation. B cell apoptosis induced by PKA-activation was also seen in CD72 stimulated cells, although CD72 mediated B-lymphocyte proliferation was not affected. PKA mediated block in phospholipase gamma-activation and calcium mobilization were not due to alterations in the activation of tyrosine kinases lyn, blk and syk. Moreover, BCR mediated tyrosine phosphorylation of PLC gamma2 and CD19 were also unaffected by cAMP accumulation. These observations are in contrast to the ability of PKA to drastically reduce the activity of ZAP-70 and syk in T lymphocytes and neutrophils, respectively. The IL-4 mediated protection appears to be due to a change in late events in BCR signaling, which are important for Bcl-xL expression.

Animals↗
PubMed1997

Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization.

Genetic ablation of the B cell surface glycoprotein CD19 severely impairs the humoral immune response. This requirement is thought to reflect a critical role of CD19 in signal transduction that occurs upon antigen C3dg coligation of antigen receptors with CD19 containing type 2 complement receptors (CR2). Here we show that CD19 plays a key accessory role in B cell antigen receptor signaling independent of CR2 coligation and define molecular circuitry by which this function is mediated. While CD19 is not required for antigen-mediated activation of receptor proximal tyrosines kinases, it is critical for activation of phosphatidylinositol 3-kinase (PI3-kinase). PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515. Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses. Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

Androstadienes↗
PubMed1997

Delivery of B cell receptor-internalized antigen to endosomes and class II vesicles.

B cell receptor (BCR)-mediated antigen processing is a mechanism that allows class II-restricted presentation of specific antigen by B cells at relatively low antigen concentrations. Although BCR-mediated antigen processing and class II peptide loading may occur within one or more endocytic compartments, the functions of these compartments and their relationships to endosomes and lysosomes remain uncertain. In murine B cells, at least one population of class II- containing endocytic vesicles (i.e., CIIV) has been identified and demonstrated to be distinct both physically and functionally from endosomes and lysosomes. We now demonstrate the delivery of BCR-internalized antigen to CIIV within the time frame during which BCR-mediated antigen processing and formation of peptide-class II complexes occurs. Only a fraction of the BCR-internalized antigen was delivered to CIIV, with the majority of internalized antigen being delivered to lysosomes that are largely class II negative. The extensive colocalization of BCR-internalized antigen and newly synthesized class II molecules in CIIV suggests that CIIV may represent a specialized subcellular compartment for BCR-mediated antigen processing. Additionally, we have identified a putative CIIV-marker protein, immunologically related to the Igalpha subunit of the BCR, which further illustrates the unique nature of these endocytic vesicles.

Animals↗
PubMed1997

B cell antigen receptor desensitization: disruption of receptor coupling to tyrosine kinase activation.

Antigen binding to the B cell receptor (BCR) induces receptor desensitization, a condition characterized by cellular unresponsiveness to subsequent Ag stimulation despite the continued ability to bind Ag. To better understand the molecular mechanism of this unresponsiveness, we have used complementary lymphoma (K46 mu) and Ig transgenic (3-83 mu delta) mouse models to study regulation of BCR signaling. Our findings in the lymphoma model show that an initial Ag encounter renders receptors unresponsive to subsequent Ag challenge, as measured by their inability to mobilize Ca2+ and to mediate phosphorylation of receptor-proximal kinases, including Lyn, Blk, and Syk. Most importantly, the Ig alpha and Ig beta components of desensitized receptors are not phosphorylated, and receptor-associated kinases are not activated upon Ag challenge. The molecular defect does not appear to result from Lyn inactivation, sequestration, or repression, since Lyn from desensitized cell lysates is activated in vitro by synthetic doubly phosphorylated immunoreceptor tyrosine-based activation motif peptides. A similar deficit in Ag-induced receptor phosphorylation was observed in desensitized B cells from 3-83 mu delta transgenic mice. These studies indicate that Ag receptor desensitization reflects an inability to initiate activation of receptor-associated kinases that normally phosphorylate receptor Ig alphabeta subunits, leading to signal propagation.

Animals↗
PubMed1997

Syk, but not Lyn, recruitment to B cell antigen receptor and activation following stimulation of CD45- B cells.

B cell Ag receptor (BCR) signaling occurs via tyrosine phosphorylation of CD79a and CD79b ITAMs, leading to recruitment and activation of Lyn and Syk tyrosine kinases and subsequent downstream events. CD45 expression is required for BCR triggering of certain of these downstream events, such as calcium mobilization and p21ras activation. However, the site in the BCR signaling cascade at which CD45 impinges is poorly defined. To address this question, we have studied CD45 function in the CD45-deficient (CD45-) and CD45-reconstituted (CD45+) J558L mu m3 plasmacytoma. In both CD45+ and CD45- cells, Ag stimulation led to CD79a and CD79b tyrosine phosphorylation as well as Syk tyrosine phosphorylation, recruitment to the receptors, and activation. In contrast to CD45+ cells, Lyn exhibited high basal tyrosine phosphorylation in the CD45- cells and was not further phosphorylated upon Ag stimulation. Mapping studies indicated that the observed constitutive phosphorylation of Lyn reflects phosphorylation of its C-terminal tyrosine, Y508, at high stoichiometry. Constitutively Y508-phosphorylated Lyn was neither recruited to the BCR nor activated upon Ag stimulation. Moreover, CD79a-ITAM phosphopeptides failed to bind Lyn from the CD45- cells. Thus, Y508 phosphorylation of Lyn occurs in the absence of cellular CD45 expression and appears to render the kinase unable to associate with the phosphorylated receptor complex via its Src homology 2 domain and to participate in signal propagation. Surprisingly, in view of previous findings implicating Src family kinases in ITAM phosphorylation, the data indicate that Ag-induced CD79a and CD79b tyrosine phosphorylation and Syk recruitment and activation can occur in the absence of CD45 expression and, hence, Src-family kinase activation.

Animals↗
PubMed1997

Molecular targets of CD45 in B cell antigen receptor signal transduction.

Expression of the phosphotyrosine phosphatase CD45 is essential for B cell Ag receptor (BCR)-mediated p21ras activation and calcium mobilization. To examine the molecular basis of this requirement, we analyzed signaling events following BCR ligation in CD45-deficient (CD45-) and CD45-reconstituted (CD45+) variants of J558Lmicrom3 cells. Ag stimulation resulted in tyrosine phosphorylation of cellular proteins in both cells. However, the spectrum of proteins phosphorylated in the CD45+ cells was qualitatively and/or quantitatively distinct from that in the CD45- cells. Among the protein tyrosine kinases examined, the Src family kinases Fyn and Blk were inducibly tyrosine phosphorylated and activated by receptor ligation only in CD45+ cells. While Ag-induced Btk tyrosine phosphorylation occurred in both cells, its activation was greatly diminished in the CD45- cells. Analysis of specific effector molecules revealed that tyrosine phosphorylation of Shc, but not rasGAP or Vav, correlated with the unique ability of BCR ligation to trigger p21ras activation in CD45+ cells. BCR-mediated Shc phosphorylation and recruitment of Grb2 depended on CD45 expression. Thus, Shc tyrosine phosphorylation may be the primary CD45-dependent mechanism by which Ag receptors are coupled to the p21ras pathway in J558Lmicrom3. In addition, phospholipase Cgamma1 (PLCgamma1) and PLCgamma2 were tyrosine phosphorylated upon Ag stimulation in CD45- cells, despite much reduced inositol trisphosphate production and lack of calcium mobilization. These findings suggest that CD45 may modulate events other than PLCgamma phosphorylation, which regulate phosphoinositide hydrolysis and the calcium mobilization response following BCR ligation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Qualitatively distinct signaling through T cell antigen receptor subunits.

T cell antigen receptors (TCR) contain several subunits including CD3gamma, delta, and epsilon, and TCRzeta and eta which are capable of mediating signal transduction. It is unclear whether the signaling function of these subunits is completely redundant. To assess the relative signaling capabilities of TCR subunits, we compared proximal events in signal transduction by wild-type TCR complexes and TCR devoid of functional zeta subunits, as well as chimeric receptors containing the cytoplasmic domains of TCRzeta or CD3epsilon. Results demonstrate that in BW5147 wild-type TCR, tail-less zeta TCR, CD3epsilon, and TCRzeta transduce signals leading to tyrosine phosphorylation of similar sets of cellular substrates, including the receptor subunits, Fyn, ZAP-70, and phospholipase Cgamma1 (PLCgamma1). Surprisingly, unlike wild-type TCR, tail-less zeta TCR, and CD3epsilon, TCRzeta was incapable of transducing signals resulting in inositol triphosphate (IP3) generation or intracellular free calcium ([Ca2+]i) mobilization. These data indicate that tyrosine phosphorylation of PLCgamma1 is not sufficient to drive IP3 production and [Ca2+]i mobilization. Most importantly, data presented indicate that TCRzeta and CD3epsilon engage partially distinct signaling pathways.

Amino Acid Sequence↗
PubMed1997

Differential association of phosphatases with hematopoietic co-receptors bearing immunoreceptor tyrosine-based inhibition motifs.

A novel family of inhibitory co-receptors has been recently defined according to the presence in their intracytoplasmic domain of immunoreceptor tyrosine-based inhibition motifs (ITIM). In particular, this family includes a low-affinity receptor for IgG, Fc gammaRIIB, which is widely expressed on hematopoietic cells, as well as killer cell inhibitory receptors (KIR) for major histocompatibility complex (MHC) class I proteins, expressed on both T and natural killer (NK) lymphocytes. Fc gammaRIIB and KIR inhibitory function depends upon the tyrosine phosphorylation of their respective ITIM. Phosphorylated Fc gammaRIIB and KIR ITIM bind the tandem SH2 tyrosine phosphatases, SHP-1 and SHP-2. Recently, Fc gammaRIIB has been shown to associate with a polyphosphate inositol 5-phosphatase, SHIP, which appears to be involved in its inhibitory function. Using cell lysate adsorption to phosphorylated ITIM peptides and surface plasmon resonance, we demonstrate here that, in contrast to Fc gammaRIIB, KIR (CD158b: p58.2) do not bind to SHIP, and only recruit SHP-1 and SHP-2. In addition, we show that point mutation of the amino acid residue in position tyrosine-2 of Fc gammaRIIB and KIR ITIM abolihes their binding to SHP-1 and SHP-2, but leaves intact the association of SHIP with Fc gammaRIIB ITIM. These data contribute to the structural definition of ITIM and document a differential recruitment of phosphatases by distinct ITIM. These findings also reveal that diverse strategies of inhibition are used by distinct members of the ITIM-bearing co-receptor family.

Amino Acid Sequence↗
PubMed1997

Fc gammaRIIB1 inhibition of BCR-mediated phosphoinositide hydrolysis and Ca2+ mobilization is integrated by CD19 dephosphorylation.

The B cell receptor for immunoglobulin G, Fc gammaRIIB1, is a potent transducer of signals that block antigen-induced B cell activation. Coligation of Fc gammaRIIB1 with B lymphocyte antigen receptors (BCR) causes premature termination of phosphoinositide hydrolysis and Ca2+ mobilization and inhibits proliferation. This inhibitory signal is mediated in part by phosphorylation of Fc gammaRIIB1 and recruitment of phosphatases; however, the molecular target(s) of effectors is unknown. Here we report that Fc gammaRIIB1 inhibition of BCR signaling is mediated in part by selective dephosphorylation of CD19, a BCR accessory molecule and coreceptor. CD19 dephosphorylation leads to failed CD19 association with phosphatidylinositol 3-kinase, and this in turn leads to termination of inositol-1,4,5-trisphosphate production, intracellular Ca2+ release, and Ca2+ influx. The results define a molecular circuit by which Fc gammaRIIB signals block phosphoinositide hydrolysis.

Animals↗
PubMed1997

Co-receptor and accessory regulation of B-cell antigen receptor signal transduction.

The development and function of the immune system is precisely regulated to assure the generation of protective immune responses while avoiding autoimmunity. This regulation is accomplished by the engagement of a multitude of cell-surface receptors which transduce signals that activate or regulate cell differentiative and proliferative pathways. In some cases biologic responses reflect the integration of signals generated by co-aggregation of multiple receptors by complex ligands. For example, B-cell responses to antigen receptor aggregation can be modulated by co-aggregation of receptors for immunoglobulin G (Fc gamma RIIB1), complement components (CR2), and alpha 2, 6-sialoglycoproteins (CD22). Here we review our recent studies of molecular mechanisms underlying co-receptor modulation of B-cell antigen receptor signaling. Our results define interesting circuitry involving interactions among the B-cell antigen receptor, CD19 and Fc gamma RIIB1. CD19 may function as an important integrator of positive and negative signals that regulate B-cell antigen receptor signal output.

Animals↗
PubMed1996

Human and mouse killer-cell inhibitory receptors recruit PTP1C and PTP1D protein tyrosine phosphatases.

NK cells express cell surface receptors for MHC class I proteins (KIR). Engagement of these receptors inhibits NK cell cytotoxic programs. KIR can be expressed on T cells, and their engagement also results in inhibition of effector functions initiated by the CD3/TCR complex. While human KIR genes belong to the Ig gene superfamily, mouse KIR belong to a family of dimeric lectins. Despite these distinct evolutionary origins, we show here that both HLA-Cw3-specific human p58.183 receptors and H-2D d/k-specific mouse Ly49A receptors recruit the same protein tyrosine phosphatases, PTP1C and PTP1D, upon phosphorylation of critical intracytoplasmic tyrosine residues. These results document a common pathway by which diverse KIR can down-regulate NK and T cell activation programs, and further define the sequence of the immunoreceptor tyrosine-based inhibitory motif (ITIM), initially described in FcgammaRIIB1, and expressed in both human and mouse KIR.

Amino Acid Sequence↗
PubMed1996

Distinct mechanisms mediate SHC association with the activated and resting B cell antigen receptor.

Ligation of the B cell antigen receptor (BCR) complex initiates tyrosine phosphorylation of the receptor's transducer components, Ig-alpha and Ig-beta and tyrosine kinase-dependent accumulation of GTP-bound, activated p21ras. The mechanism of receptor coupling to p21ras activation and the roles of Ig-alpha and Ig-beta are unknown. The results reported here indicate that the resting, nonphosphorylated BCR associates with the Grb-2/Sos-linker SHC via the Ig-alpha immunoreceptor-based tyrosine activation motif (ITAM). Ig-alpha specificity of this interaction is determined by the sequence DCSM found in Ig-alpha, but not Ig-beta. Tyrosine phosphorylation of Ig-alpha and Ig-beta ITAM allows recruitment of SHC, which now binds directly to both Ig-alpha and Ig-beta via a phosphotyrosine/SH2 interaction. In confirmation of recent studies by Saxton et al. (J. Immunol. 1994. 153: 623) receptor ligation leads to tyrosine phosphorylation of SHC and to the formation of a phospho-SHC/Grb2/Sos complex. In view of previous studies which demonstrated p21ras co-capping with ligated BCR, the data presented here suggest that Ig-alpha/beta- and SHC tyrosine phosphorylation-dependent recruitment of the Grb2/Sos complex to the receptor can occur and may provide a mechanism by which the nucleotide exchange activity of Sos could mediate activation of BCR-localized p21ras.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Bacterial superantigens induce V beta-specific T cell receptor internalization.

Staphylococcal enterotoxins can cause toxic shock syndrome and autoimmune diseases. Circulating T cells from these diseases have a very wide range of expression in particular T cell receptor (TCR) beta chain variable regions (V beta). One possibility for this wide range of TCR V beta expression is that during acute infection with organisms secreting superantigens (SAg) these potent molecules might modulate TCR expression. To test this hypothesis, we investigated the potential effects of SAg on TCR V beta cell surface expression. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with staphylococcal SAg. Toxic shock syndrome toxin-1 (TSST-1) induced downregulation of V beta 2 expression, whereas staphylococcal enterotoxin (SE) B induced V beta 3-and V beta 12-specific downregulation. TSST-1 did not interfere with anti-V beta 2 mAb binding. Therefore, this downregulation was not due to steric hindrance of Ab binding by TSST-1. TSST-1 induced V beta 2 downregulation was time-, dose- and temperature-dependent. CD3 expression decreased in parallel with reduction of V beta expression. CD4 and CD8 expression were only slightly decreased. CD2, CD25 and HLA-DR expression were upregulated following TSST-1 stimulation of T cell lines. To investigate the fate of TCR after toxin stimulation, V beta 8+ Jurkat T cells were incubated with SEE which is known to stimulate V beta 8+ T cells, and analysed with fluoresence microscopy, and immunoprecipitation and Western blotting. After SEE stimulation, there was an increase in V beta 8 molecules found in the cytoplasm which correlated with loss of cell surface V beta 8 molecules, suggesting internalization of cell surface V beta 8 molecules was induced by SEE stimulation. Shedding of V beta 8 molecules into the culture supernatant was not detected. These data demonstrate that SAg mediated downregulation of TCR expression occurs primarily as the result of TCR internalization. This downregulation phenomenon may have physiological and pathological consequences in patients infected with Staphylococcus aureus.

Antibodies, Monoclonal↗
PubMed1996

The SHIP phosphatase becomes associated with Fc gammaRIIB1 and is tyrosine phosphorylated during 'negative' signaling.

Immune-complex mediated co-ligation of antigen and Fc receptors on B-cells leads to abortive antigen receptor (BCR) signaling and provides a mechanism for feedback regulation of the immune response. A phosphotyrosine-containing 13 amino acid sequence (ITIM) found in the FcgammaRIIB1 cytoplasmic tail mediates this inhibition and specifically associates with the phosphotyrosine phosphatase SHP1. In vitro binding studies demonstrate that the phosphorylated ITIM binds unidentified proteins of 70 and 160 kD in addition to SHP1. Here we report the identification of p70 as SHP2 and p160 as the SH2 containing phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase SHIP. SHIP is inducibly tyrosine phosphorylated following BCR-FcgammaRIIB1 co-ligation. Further, we observe SHIP association with tyrosine phosphorylated FcgammaRIIB1 in intact cells following BCR-FcgammaRIIB1 co-ligation. To a much lesser but significant degree, tyrosine phosphorylation of SHIP is also observed upon BCR ligation. These observations suggest that SHIP may play an important role in FcgammaRIIB1 dependent and independent regulation of BCR signaling.

Animals↗
PubMed1996

Selective in vivo recruitment of the phosphatidylinositol phosphatase SHIP by phosphorylated Fc gammaRIIB during negative regulation of IgE-dependent mouse mast cell activation.

We demonstrated previously that the low-affinity IgG receptors Fc gammaRIIB, which are coexpressed with the high-affinity IgE receptors Fc epsilonRI in mouse mast cells, can inhibit IgE-induced release of inflammatory mediators and cytokines by these cells. Inhibition was found to require the coaggregation of the two receptors and to depend on the presence of a tyrosine-based inhibition motif (ITIM) in the intracytoplasmic domain of Fc gammaRIIB. We report here that the coaggregation with Fc gammaRIIB does not prevent Fc epsilonRI from triggering activation signals in BMMC and induces the tyrosine phosphorylation of Fc gammaRIIB. Phosphorylated ITIM peptides bound in vitro to three SH2 domain-containing phosphatases present in BMMC lysates: the phosphotyrosine phosphatases SHP-1 and SHP-2. and the inositolphosphate phosphatase SHIP. Using BMMC generated from the SHP-1-deficient motheaten mice, SHP-1 was found to be dispensable for inhibition of mast cell activation. When analyzed for in vivo association, SHIP coprecipitated with phosphorylated Fc gammaRIIB, whereas SHP-1 or SHP-2 did not. These observations altogether indicate that Fc epsilonRI actively participates in its own regulation and that the mechanisms by which Fc gammaRIIB inhibit cell activation might be different in mast cells and in B-cells.

Animals↗
PubMed1995

Role of the Syk autophosphorylation site and SH2 domains in B cell antigen receptor signaling.

To explore the mechanism(s) by which the Syk protein tyrosine kinase participates in B cell antigen receptor (BCR) signaling, we have studied the function of various Syk mutants in B cells made Syk deficient by homologous recombination knockout. Both Syk SH2 domains were required for BCR-mediated Syk and phospholipase C (PLC)-gamma 2 phosphorylation, inositol 1,4,5-triphosphate release, and Ca2+ mobilization. A possible explanation for this requirement was provided by findings that recruitment of Syk to tyrosine-phosphorylated immunoglobulin (Ig) alpha and Ig beta requires both Syk SH2 domains. A Syk mutant in which the putative autophosphorylation site (Y518/Y519) of Syk was changed to phenylalanine was also defective in signal transduction; however, this mutation did not affect recruitment to the phosphorylated immunoreceptor family tyrosine-based activation motifs (ITAMs). These findings not only confirm that both SH2 domains are necessary for Syk binding to tyrosine-phosphorylated Ig alpha and Ig beta but indicate that this binding is necessary for Syk (Y518/519) phosphorylation after BCR ligation. This sequence of events is apparently required for coupling the BCR to most cellular protein tyrosine phosphorylation, to the phosphorylation and activation of PLC-gamma 2, and to Ca2+ mobilization.

Amino Acid Sequence↗