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J C Cambier

Publications and source records attributed to J C Cambier.

At least 127 records · Page 7Linked to original sources
PubMed1989

Altered I-A protein-mediated transmembrane signaling in B cells that express truncated I-Ak protein.

Recent evidence suggests that the major histocompatibility complex class II molecules of B lymphocytes function as signal-transducing receptors during the generation of T lymphocyte-dependent humoral immune responses. By analogy with other receptors, we postulate that perturbation of the class II molecules is coupled to the generation of intracellular second messengers through interactions involving the transmembrane and/or cytoplasmic domains of the class II molecules. We report a series of experiments that assess which amino acids of the class II molecule I-Ak are required for coupling it to the signal-transduction pathway. We prepared a series of B-lymphocyte transfectants that express I-Ak molecules with COOH-terminal truncations of either the Ak alpha or Ak beta chain or both. The ability of each transfected class II molecule to transduce a signal after being bound by monoclonal antibody was found by monitoring the translocation of protein kinase C from the cytosol to the "nuclear compartment" of the transfected B lymphocyte. Results indicate that the Ak beta chain plays the dominant role in signal transduction and that the 6 cytoplasmic amino acids of Ak beta chain most proximal to the inner plasma membrane are of greatest importance in coupling I-Ak molecules to the molecules of the signaling cascade.

Amino Acid Sequence↗
PubMed1989

Transmembrane signaling in T lymphocyte dependent B lymphocyte activation.

The precise number and nature of signaling events required for T helper cell dependent activation of quiescent B lymphocytes remains obscure. Here we discuss the extant literature, as well as recent unpublished findings from our own laboratory, which address the issue. Based on available information, we suggest that activation of quiescent B cells into proliferative cycle requires signaling mediated by antigen, IL4 and T cell associated by alphabetaTCR and CD4 and transduced through B cell membrane immunoglobulins, IL4 receptors and Ia respectively. Further, these signals must be received in sequence and are transduced by distinct biochemical mechanisms.

Animals↗
PubMed1988

Evaluation of methods for the isolation of plasma membranes displaying guanosine 5'-triphosphate-dependence for the regulation of adenylate cyclase activity: potential application to the study of other guanosine 5'-triphosphate-dependent transduction systems.

The GTP-dependence for stimulatory and inhibitory regulation of plasma membrane adenylate cyclase activity was measured in plasma membrane fractions isolated from a variety of cell types (platelets, lymphocytes, PC12 cells, GH3 cells, NBP2 cells, and hepatocytes). This report shows that the isolation of plasma membranes for the study of GTP-dependent adenylate cyclase activity was, for some cells, enhanced by the exposure of the cells to glycerol prior to cell lysis. The isolation of plasma membranes from other cells, which did not appear to be sensitive to glycerol pretreatment, was enhanced by the removal of heavy particulate matter prior to fractionation of the cell lysate. The regulation of enzyme activity by various agents was found to be dependent upon the presence of (exogenous) GTP to varying degrees, indicating variable contamination of membrane preparations with GTP. It is concluded that (i) exposure of platelets and lymphocytes to glycerol prior to cell lysis decreases subsequent contamination of the plasma membrane preparation with GTP, and (ii) although glycerol pretreatment of other cells does not ensure the subsequent isolation of plasma membrane adenylate cyclase activity displaying high requirements for (exogenous) GTP, it is a reasonable first approach to be used during the development of procedures for the isolation of plasma membranes.

Adenylyl Cyclases↗
PubMed1988

Increased plasma membrane permeability to Ca2+ in anti-Ig-stimulated B lymphocytes is dependent on activation of phosphoinositide hydrolysis.

The biochemical basis of Ca2+ mobilization after anti-Ig binding to B cell Ag-R has been further characterized by flow cytometric analysis of indo-1-loaded B cells. The ability to distinguish intracellular Ca2+ release from extracellular Ca2+ influx by using an extracellular calcium depletion-repletion approach has allowed us to study the relationship between the mobilization of Ca2+ from these sources. Studies involving manipulation of the Ca2+ gradient across the plasma membrane indicate that a significant portion of the Ca2+ mobilization response is preserved even when the normal inwardly directed Ca2+ gradient is reversed. In the presence of an extracellular calcium concentration ([Ca2+]o) of 10 microM, the response to anti-Ig is not blocked by the organic Ca2+ channel blockers. This response is not reduced by further depletion of [Ca2+]o by EGTA Ca2+-binding buffers. Thus, the Ca2+ response that occurs when [Ca2+]o less than or equal to 10 microM represents intracellular calcium release. Analysis of B cells stimulated with anti-Ig in low Ca2+ medium ([Ca2+]o = less than 10 microM) followed by repletion of [Ca2+]o to 1 to 5 mM reveals that a significant increase in permeability of the plasma membrane to Ca2+ develops in the stimulated cells. The resultant Ca2+ influx is nimodipine (20 microM) sensitive. Both intracellular Ca2+ release and Ca2+ influx are reduced in parallel as the concentration of anti-Ig stimulus is decreased, suggesting that Ca2+ influx may be coupled to the release of intracellular stores. Neomycin blocks anti-Ig-stimulated formation of inositol trisphosphate, which mediates release of Ca2+ from the endoplasmic reticulum. It also blocks the anti-Ig-induced release of intracellular Ca2+ stores as well as Ca2+ influx, indicating that both responses may be dependent upon phosphatidylinositol 4,5-bisphosphate hydrolysis.

Animals↗
PubMed1988

Early changes in quiescent B cell physiology subsequent to cognate and bystander interaction with helper T cells.

We have assessed early changes in quiescent B cells following cognate and bystander interaction with cloned helper T cells. Variables monitored include Ia expression, blastogenesis, G0 to G1 transition, and progression through cycle. We have also assessed the antigen specificity, Ia restriction, and dependence on membrane immunoglobulin crosslinking of both generation and delivery of effectors that mediate B cell responses. The results demonstrate that antigen presentation by quiescent B cells to T cells resulting in the generation of effectors that activate B cells is Ia-restricted and dependent on antigen and an (mIgM and mIgD) crosslinking signal. However, once generated, T cell effector functions act independently of Ia haplotype to promote Ia expression, blastogenesis, and G0 to G1 transition by most small B cells. Although these responses can be mediated by T cell supernatants, further progression of B cells through S, G2 and M is only efficient when Th cells are present in cultures. Thus, results suggest that one or more Ia unrestricted, labile and/or cell-associated factors, not active in most conventional T cell supernatants, are necessary to stimulate proliferation of small B cells.

Animals↗
PubMed1987

B lymphocyte activation. Transmembrane signal transduction by membrane immunoglobulin in isolated cell membranes.

We report the development of cell-free systems in which ligation of B cell membrane immunoglobulin leads to demonstrable mono- and polyphosphatidylinositol hydrolysis. Membranes were prepared by differential centrifugation of sonicates of normal murine B cells. Incubation of these membranes with 32P-adenosine triphosphate in the presence of Mg2+ effected the radiolabeling of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and phosphatidylinositol 4-phosphate (PtdInsP). Alternately, membranes were labeled with exogenous 3H(inositol)-PtdInsP2 in sodium cholate. Stimulation of labeled membranes with anti-immunoglobulin, but not anti-Ia or anti-H2 antibodies, resulted in hydrolysis of phosphatidylinositol, PdtInsP, PtdInsP2 and generation of inositol phosphates indicative of activation of a phospholipase C. The response was rapid, being detectable within 30 sec of stimulation, and independent of Ca2+ and guanosine 5'-triphosphate. Optimal responses were dependent on the presence of a cytosolic factor presumed to be phospholipase C. Development of these systems represents an important step towards reconstitution of membrane immunoglobulin-mediated transmembrane signaling in artificial membranes.

Animals↗
PubMed1987

Transmembrane signaling through B cell MHC class II molecules: anti-Ia antibodies induce protein kinase C translocation to the nuclear fraction.

Among receptors of immunologic importance, antigen receptors on B cells (mIg) and T cells (Ti/T3), as well as receptors for IL 2 and IL 3, appear to utilize a transmembrane signal transduction mechanism that involves protein kinase C (PKC) translocation from the cytosol to the plasma membrane. Here we report evidence that B cell surface type II major histocompatibility molecules (I-A and I-E) can act as signal transducing molecules, as evidenced by the ability of soluble ligands that bind these molecules to stimulate rapid translocation (less than 2 min) of cytosolic PKC. This phenomenon differs qualitatively from translocation induced by anti-Ig antibodies and all other ligands thus far studied, in involving an apparent translocation of protein kinase C to the nucleus. Data suggest that I-A/E molecules may play an active role in T cell-dependent B cell activation. They also provide an explanation for the previously observed suppressive effects of soluble I-A and I-E binding ligands on B cell activation in demonstrating that these ligands (anti-IA/E) deplete the cytosolic reservoir of PKC normally translocated to the plasma membrane after anti-Ig or LPS stimulation.

Animals↗
PubMed1987

Neither interleukin 2 nor gamma interferon directly promote growth or differentiation of mouse B cells.

The roles of interleukin 2 (IL-2) and gamma interferon (IFN-gamma) as direct mediators of B-cell growth and differentiation were analysed. Products of cloned genes were used in both cases. The use of flow cytometric assays coupled with density fractionation of responding splenic B-cell populations enabled both the characterization of B cells responding to various stimuli and the estimation of their frequency. B cells responding to non-IL-2 related lymphokines promoting growth and differentiation were restricted to low buoyant density fractions. In addition, these cells expressed densities of IL-2 receptor determinants comparable to those found on T cells, although, IL-2 did not support their growth or differentiation. The inability to demonstrate any direct effect of either IL-2 or IFN-gamma on B cells in any state of activation suggests that their physiological roles are mediated through additional cell types.

Animals↗
PubMed1986

Anti-Ig induces release of inositol 1,4,5-trisphosphate, which mediates mobilization of intracellular Ca++ stores in B lymphocytes.

Evidence from a variety of laboratories indicates that crosslinking of B cell mIg induces a rapid increase in intracellular free calcium (Ca++i). This mobilized Ca++ appears to act in concert with diacylglycerol (DAG; also released upon mIg cross-linking) to optimally activate Ca++/phospholipid-dependent protein kinase C, which plays a pivotal role in B cell activation. Here we report analysis of the source of this mobilized calcium and the mechanism responsible for its release into the cytosol. We observed the cross-linking of mIg induces the release of inositol 1,4,5-trisphosphate (InsP3), presumably as a result of action of phospholipase C on plasma membrane phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The release of InsP3 and the elevation of Ca++i are coincidental, suggesting that they may be causally related. Finally, we demonstrate that submicromolar doses of InsP3 induce release of Ca++ from permeabilized cells that had preaccumulated 45Ca++ in the endoplasmic reticulum. On the basis of these findings we suggest that mIg cross-linking leads to mobilization of Ca++, in part by causing hydrolysis of PtdInsP2, yielding InsP3, which in turn causes release of calcium from the endoplasmic reticulum.

Animals↗
PubMed1986

Abortive activation of B lymphocytes by monoclonal anti-immunoglobulin antibodies.

Toward a better understanding of the signaling role of antigen-mIg binding in the generation of humoral immune responses, we have assessed the effects of soluble, monoclonal anti-Ig antibodies on various cell physiologic parameters known to change during B cell activation. These parameters include membrane potential, I-A antigen expression, narrow angle light scattering properties (size), and cell cycle state. Results indicate that all monoclonal antibodies that bind cell to surface IgM or IgD, or both, induce virtually all small B cells to undergo membrane depolarization and increased I-A expression. Only a small subset of these antibodies, exemplified by b-7-6 anti-mu, induce all small B cells to enter G1. An increasingly smaller proportion of these cells traverse each subsequent cell cycle phase, with 10% of cells reaching G2 or M phases by 60 hr of culture. The kinetics of this response to b-7-6 are considerably slower than those of the response induced by LPS. Finally, analysis of Percoll density-fractionated cells revealed that although B blasts made by b-7-6 stimulation of small cells remain b-7-6 responsive, natural B blasts isolated from the spleen are refractory to monoclonal anti-Ig stimulation as indicated by membrane depolarization, increased IA expression, blastogenesis, and [3H]thymidine uptake.

Animals↗
PubMed1986

Desferoxamine blocks IL 2 receptor expression on human T lymphocytes.

Thymidine uptake by PHA-stimulated human lymphocytes is reduced in the presence of 100 microM or greater concentrations of the iron-chelating agent desferoxamine (DF). We assessed expression of IL 2 receptor, 4F2 and Ia antigens, IL 2 production, and cell cycle progression by blood mononuclear cells (MNC) stimulated by PHA in the presence or absence of DF to determine whether the lack of T cell proliferation was a manifestation of inhibition of an earlier activation event. Tac antigen expression on PHA-stimulated MNC was inhibited by DF throughout 8 days of culture, and those cells which were positive had a low density of Tac antigen as compared with controls without DF. Expression of other activation antigens, 4F2 and Ia, was not impaired by DF. The supernatants of the DF-containing and control cultures contained equivalent IL 2 activity, as measured on the HT-2 cell line. Cell cycle analysis of these cultures shows that the addition of DF at the beginning of culture blocks most cells from undergoing G0 to G1 transition, whereas later addition of DF arrests the progression of the T cell blasts through the cell cycle. Separation of cells cultured with PHA and DF into Tac+ and Tac- subsets showed that progression from G0 to G1 was restricted to the former subset. These results suggest that interference with IL 2 receptor expression might contribute to the block in mitogen-induced proliferation caused by DF.

Antigens, Surface↗
PubMed1986

Translocation of protein kinase C during membrane immunoglobulin-mediated transmembrane signaling in B lymphocytes.

Previous studies have implicated a role for protein kinase C (PKC) in transmembrane signal transduction by B cell surface immunoglobulin (Ig). Specifically, the pharmacologic PKC activator phorbol myristate acetate mimics the biologic effects of mIg cross-linking ligands, and cross-linking of membrane Ig (mIg) induces polyphosphoinositide hydrolysis generating diacylglycerol, a potent activator of PKC. Studies described here additionally implicate PKC in mIg-mediated signaling by demonstrating rapid translocation of activatable PKC (PKCa) from cytosol to Triton-soluble membrane fractions after cross-linking of cell surface IgM or IgD. This response, which is also induced by phorbol myristate acetate and lipolysaccharide, is detectable within 1 min of mIg cross-linking and is followed within 4 min by additional translocation of PKCa to a Triton-insoluble particulate compartment. The ability of dbcAMP plus theophylline to inhibit polyphosphoinositide hydrolysis, PKCa translocation, and the B cell's subsequent biological response suggests that these events may be causally related.

Animals↗