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J C Cambier

Publications and source records attributed to J C Cambier.

At least 145 records · Page 8Linked to original sources
PubMed1986

Single cell analysis of calcium mobilization in anti-immunoglobulin-stimulated B lymphocytes.

A rapid increase in the concentration of intracellular free calcium ([Ca++i]) in B cells after mIg crosslinking has been documented previously by fluorimetric analysis of cell populations loaded with the fluorescent Ca++ indicator Quin 2. Although providing a valuable indication of Ca++ mobilization in the population as a whole, it has not been possible to determine whether only a subpopulation of the cells or the entire population exhibits this response. In this report, we describe the marriage of flow cytometry and Quin 2 technology, which permits discrimination of Ca++ mobilization by subpopulations of cells, as well as in whole populations. We have determined that the entire mIg+ population exhibits a synchronous increase in [Ca++i] rapidly after stimulation. Furthermore, all members of the population appear to undergo an approximately equal response.

Aminoquinolines↗
PubMed1986

Supernatant from a cloned helper T cell stimulates most small resting B cells to undergo increased I-A expression, blastogenesis, and progression through cell cycle.

Helper T cell clone 52.3 supernatant (52.3 SN) was previously shown to be able to stimulate gradient-purified murine resting B cells in the absence of any additional stimulus. However, the proportion of cells that were accounting for the thymidine uptake and the Ig production was unknown. In this paper, we have studied induced changes that can be measured at the single cell level, and have thus determined the frequency of resting B cells that respond to 52.3 SN. Results indicate that 52.3 SN induces an increased I-A expression and a cell size enlargement on virtually all resting B cells. A significant proportion (30%) of these cells later becomes large blasts. Acridine orange staining revealed that in the presence of 52.3 SN a large fraction of the resting B cells undergoes the G0 to G1 transition. Furthermore, 52.3 SN is able to induce at least 20% of the cells to continue through the cell cycle into S phase as indicated by propidium iodide staining of DNA. Finally, a fraction of the 52.3 SN-stimulated cells differentiate to Ig-producing cells. Our present results suggest that resting B cells express functional receptors for some lymphokines and that these lymphokines can act in the absence of membrane Ig occupancy. Our findings further support the existence of a B cell-activating factor acting in a MHC-unrestricted manner and responsible for the entry of resting B cells into cell cycle. The relationship between this factor and other lymphokines is discussed.

Acridine Orange↗
PubMed1986

B cell activation. VII. Independent and synergistic effects of mobilized calcium and diacylglycerol on membrane potential and I-A expression.

Although cross-linking of murine B cell membrane Ig (mIg) has been shown to induce a rapid increase in intracellular free calcium [Ca++)i), both the source and the function of the Ca++ in lymphocyte activation is unclear. Toward elucidation of its function, we investigated the relationship between the initial (Ca++)i response and other cell physiologic changes that occur early after mIg cross-linking, apparently as a linear cascade, leading to increased membrane I-A expression. Results suggest that the (Ca++)i response results from polyphosphoinositol hydrolysis induced by mIg cross-linking. The (Ca++)i response cannot be induced by activation of protein kinase C (PKC) with phorbol diesters (e.g., PMA) or synthetic diacylglycerol (DAG), suggesting that this response precedes the PKC activation. However, inhibition of phosphatidylinositol turnover by exposure of cells to dbcAMP during anti-Ig stimulation significantly inhibits the (Ca++)i response, suggesting that phosphatidylinositol turnover may be causally related to Ca++ mobilization. The ability of exogenous phospholipase C to induce the (Ca++)i response also supports this conclusion. Of the products of mono- and poly-phosphatidylinositol hydrolysis, the inositol phosphates (InsP, InsP2, InsP3) are implicated as promoters of Ca++ mobilization, because exogenous synthetic diacylglycerol is without effect on (Ca++)i. In light of recent evidence obtained with other systems, we suggest that InsP3 is responsible for mIg cross-linking-induced Ca++ mobilization from intracellular stores in B lymphocytes. Both depolarization and increased I-A expression are induced by increasing (Ca++)i with the Ca++ ionophores A23187 and ionomycin. These events can also be induced by the activation of PKC with high doses of PMA. When suboptimal doses of both A23187 and PMA are present, these reagents synergize in the induction of depolarization. This suggests that one role for the initial rise in (Ca++)i is to act with the DAG liberated from PtdIns turnover, possibly by enhancing translocation of cytosolic PKC to the plasma membrane, and thereby promote changes in ion transport that are apparent as a decrease in the membrane potential.

Animals↗
PubMed1985

Hyper-Ia antigen expression on B cells from B6-lpr/lpr mice correlates with manifestations of the autoimmune state.

To investigate the state of activation of B cells from mice with the lpr gene defect, membrane Ia antigen (mIa) expression was analyzed on B cells from B6-lpr/lpr (lpr) and control B6- +/-/+/- mice. B cells from lpr mice exhibited marked increases in levels of mIa as determined by flow cytometry using a monoclonal anti-I-Ab,d reagent. This increase, which was progressive with age, suggests that phenotypic alteration of B-cell mIa expression is a consequence of lpr gene action. Since B-cell activation manifest by elevated mIa expression may promote productive interactions with helper T cells, these observations suggest an important role for B-cell abnormalities in the etiology of lpr-induced autoimmune disease.

Animals↗
PubMed1985

Identification and characterization of a hapten-modifiable TEPC 15 cross-reactive idiotype in swine.

Rabbits and swine immunized with TEPC 15 IgA, goats immunized with T15-positive IgM and swine immunized with affinity-pure swine anti-phosphorylcholine (PC) all produce antibodies which recognize a hapten-inhibitable idiotypic determinant on swine anti-PC. The similarity in reactivity and order of inhibitability with various PC analogs of the heterologous (swine anti-TEPC 15) and isologous (swine anti-swine anti-PC) reagents indicates that they recognize a related idiotype and suggest it may be the predominant idiotype expressed on swine anti-PC antibodies. The heterologous and isologous anti-idiotypic reagents generated in this study recognize swine and mouse anti-PC but not normal swine IgM, IgG or MOPC 460. Only reactions with swine anti-PC and mouse T15-positive anti-PC proteins are hapten-inhibitable. The greater inhibitory capacity of trimethylammonium and acetylcholine than PC suggests that the idiotope(s) recognized on swine anti-PC by the anti-idiotypic reagents is integral rather than peripheral to the anti-PC binding site. The nearly exclusive IgM anti-PC response of swine to Streptococcus pneumoniae R36A and PC-Brucella have so far hindered attempts to study the isotypic distribution of the idiotype.

Animals↗
PubMed1985

B cell activation. VI. Effects of exogenous diglyceride and modulators of phospholipid metabolism suggest a central role for diacylglycerol generation in transmembrane signaling by mIg.

Previous evidence indicates that in vitro activators of protein kinase C, such as phorbol myristate acetate (PMA), are able to induce early activation events in murine B cells, including membrane depolarization and increased I-A antigen expression. These same events are induced by specific antigen and anti-receptor antibody. This evidence suggests that protein kinase C activation may be an important intermediary event in mIg-mediated transmembrane signaling. Previously, investigators have suggested that protein kinase C activation is regulated by a novel second messenger, diacylglycerol (DG), and DG is generated by phosphatidylinositol (PI) hydrolysis after receptor-ligand interaction in many systems. In view of this concept, we examined the effects of nonspecific activators and inhibitors of DG production and DG itself on membrane potential and levels of I-A antigen expression in murine B cells. Our results indicate that exposure to DG, or induction of DG production by treatment of B cells with exogenous phospholipase C, results in depolarization and increased I-A antigen expression similar to that induced by anti-receptor antibody and specific antigen. Furthermore, we demonstrate that depolarization and increased I-A expression induced by anti-receptor antibody is blocked under conditions in which DG production is inhibited. As expected, based on its direct activation of protein kinase C, PMA stimulation is unaffected by this inhibition. These results support our earlier hypothesis that occupancy of antigen receptors on B cells is linked to subsequent activation events by PI hydrolysis, DG generation, and protein kinase C activation.

Animals↗
PubMed1984

Interleukin-induced increase in Ia expression by normal mouse B cells.

The constitutive culture supernatant (SN) of the macrophage tumor line P388D1 (P388 SN) and the concanavalin A (Con A)-induced culture supernatant of the T cell hybridoma FS6-14.13 (FS6 Con A SN) were shown to contain nonspecific factors capable of inducing increased Ia expression by normal resting B cells in a dose-dependent manner. In six consecutive experiments the relative increase in Ia expression induced by P388 SN was 4.9 +/- 0.9, with FS6 Con A SN 10.7 +/- 1.5, and with a combination of both preparations 13.0 +/- 1.7. This increase in Ia expression was observed to occur in virtually all the B cells, reaching maximum levels within 24 h of culture. The interleukin-induced increase in B cell Ia expression occurred in the absence of ancillary signals provided by ligand-receptor Ig cross-linking and despite the fact that virtually all the control B cells, cultured in the absence of factors, remained in G0. These results suggest that functional receptors for at least some interleukins are expressed on normal resting B cells and their effects can be manifest in the absence of additional activating signals. The increased Ia expression induced by the nonspecific factor preparations was shown to be correlated with enhanced antigen-presenting capacity by the B cells to T cell hybridomas. The nature of the interleukins responsible for these effects remains to be definitively determined, however, the activity of FS6 Con A SN was shown to correlate with B cell growth factor activity and increased B cell Ia expression was not observed using interleukin 2 (IL-2) or interferon-gamma, prepared by recombinant DNA technology.

Animals↗
PubMed1984

B cell activation. VIII. Membrane immunoglobulins transduce signals via activation of phosphatidylinositol hydrolysis.

Considerable evidence indicates that cross-linking of B cell surface Ig results in a "first signal" in B cell activation. We have shown that transduction of this signal is manifest by changes in plasma membrane potential leading to increased expression of surface I-A antigen. In previous studies, we have provided evidence that suggests that this signal is transduced via phosphatidylinositol (PI) hydrolysis liberating diacylglycerol (DAG), which subsequently activates protein kinase C. These biochemical events are aspects of a transmembrane signal transduction mechanism that is common in nature and utilizes the PI metabolic cycle for generation of "second messenger" diacylglycerol. Here we report direct evidence that treatment of B cells with various antibodies to surface Ig results in activation of the PI cycle. Results suggest that the increased phospholipid metabolism that occurs in B cells in response to anti-Ig involves only those phospholipids in the PI cycle and is a consequence of turnover of existing lipid rather than de novo synthesis. Furthermore, we show that PI cycle activation requires cross-linking of membrane Ig and is inhibitable by increased intracellular cyclic AMP. These findings are particularly important in view of previous studies that have shown identical requirements for and inhibitability of induction of B cell membrane depolarization and increased I-A expression. Thus, these results are consistent with our previous hypothesis that early B cell activation events initiated by receptor Ig occupancy are mediated via PI hydrolysis, diacylglycerol generation, and protein kinase C activation.

Animals↗
PubMed1984

B cell activation. IV. Induction of cell membrane depolarization and hyper-I-A expression by phorbol diesters suggests a role for protein kinase C in murine B lymphocyte activation.

Analysis of the effects of phorbol diesters on mouse B lymphocyte kinase C activity, membrane potential, mI-A expression, and cell cycle state are reported. Results indicate that the phorbol diesters PMA and 4 beta-PDD, which are potent tumor promoters, activate partially purified B cell protein kinase C and stimulate B cell membrane depolarization and increased mI-A expression. The analog 4 alpha-PDD has none of these effects. Similarly, none of the phorbol diesters tested promoted G0 to G1 transition of B lymphocytes. Results are consistent with the possibility that the transmembrane signal transduction mediated by cell membrane immunoglobulin, which results in membrane depolarization and increased I-A antigen expression, operates via activation of protein kinase C.

Animals↗
PubMed1984

mIgM:mIgD ratios on B cells: mean mIgD expression exceeds mIgM by 10-fold on most splenic B cells.

The relative frequency of mIgM and mIgD molecules on B cell surfaces is important in determining, in large part, the isotype involvement in antigen binding and signal transduction. Although it is generally assumed that on most mature B cells, mIgM and mIgD occur in roughly equal quantities, no formal analysis of this question has been reported. In this report, we describe such an analysis based on the quantitation of anti-Fab or anti-kappa specific immunofluorescence of splenic B cells before or after capping with rabbit anti-IgD or anti-IgM antibodies or both. The results indicate that, whereas mean expression of IgD exceeds IgM on splenocytes by threefold, members of the major B cell subpopulation (60 to 70% of cells) express 10-fold more IgD than IgM.

Animals↗
PubMed1984

B cell activation. V. Differentiation signaling of B cell membrane depolarization, increased I-A expression, G0 to G1 transition, and thymidine uptake by anti-IgM and anti-IgD antibodies.

Although studies have suggested that B cell membrane-associated IgM and IgD may function differently in the generation of humoral immune responses, this difference has proven difficult to define precisely. Toward a better understanding of the function of these antigen receptors, we compared the ability of F(ab')2 fragments of antibodies specific for mouse Fab, IgM, and IgD to induce early changes in B cell physiology that are indicative of cell activation. Specifically, we assessed the ability of membrane IgM (mIgM) or membrane IgD (mIgD) or both in combination to transduce signals that result in membrane depolarization, increased I-A expression, G0 to G1 transition, and thymidine uptake. Results indicate that at low levels of anti-receptor antibody (0.01 to 0.1 microgram/ml) both isotypes transduce signals for the induction of membrane depolarization and increased I-A expression by a significant proportion of B cells, i.e., Ig+ cells. Higher concentrations of ligand (5 to 25 micrograms/ml) are required to induce a significant proportion of cells to undergo G0 to G1 transition and increase thymidine uptake. At optimal concentrations, anti-IgM and anti-IgD induce membrane depolarization and increased I-A expression by 80 to 90% of B cells. Although mIgM also transduces signals sufficient for G0 to G1 transition by 80 to 90% of B cells, mIgD transduces signals sufficient for G0 to G1 by only about 40% of B cells. A similar difference exists for the induction of thymidine uptake. Thus, results suggest that membrane Ig may transduce two different signals depending upon the amount of ligand that they bind. Although in some B cells mIgD can transduce both signals resulting in membrane depolarization and progression into G1, in most cells mIgD transduces only the first signal, resulting in membrane depolarization and increased I-A expression.

Animals↗
PubMed1983

B cell activation. III. B cell plasma membrane depolarization and hyper-Ia antigen expression induced by receptor immunoglobulin cross-linking are coupled.

We report investigation of the relationship between ligand-induced B cell plasma membrane depolarization and increased expression of membrane-associated, I-A subregion encoded (mI-A) antigens. Results demonstrate that equal frequencies of B cells are stimulated to undergo membrane depolarization and to increase mI-A expression in response to mitogen, anti-Ig, and thymus-independent (TI) or thymus-dependent (TD) antigens. Further, a cause-and-effect relationship between these two events is suggested by results that demonstrate that inhibition of anti-Fab--induced depolarization by valinomycin also inhibits the subsequent increase in mI-A antigen expression and "passive" (non-ligand-mediated) depolarization of murine B cells by K+ results in hyper-mI-A antigen expression. Based upon these results we hypothesize that antigen-mediated receptor cross-linking results in signal transduction via membrane depolarization, which is resultant in increased mI-A antigen synthesis and cell surface expression. This increase in mI-A antigen density may render the B cell more receptive to subsequent interaction with I-region-restricted helper T cells.

Animals↗
PubMed1983

Sorting of B lymphoblasts based upon cell diameter provides cell populations enriched in different stages of cell cycle.

Here we report analysis and correlation of changes in cell size and cycle state resulting from exposure of murine B lymphocytes to the mitogens lipopolysaccharide (LPS) and dextran sulfate (DxSO4). Cell cycle changes are assessed by flow cytofluorometric analysis of acridine orange stained cells. Cell diameters are determined by flow cytometric analysis of the pulse-width (time of flight) of the axial light extinction signal. Results indicate that within 12 h of exposure of B cell populations to these mitogens, cells displaying increased diameter and containing increased RNA can be detected. Under these conditions, increased RNA content is considered indicative of G0 to G1 transition or entry into cell cycle (Darzynkiewicz et al., 1976). Progressive increases in cell size and transition through G1, S, G2, and M occur in parallel during 48 h of culture with mitogens. Sorting of cells based upon size followed by cell cycle analysis reveals a direct correlation between cell size and cycle phase. Specifically, cells 4.5-5.5 microns in diameter are in primarily G0. Cells 5.5-7.0 microns in diameter are in early G1. Populations of cells 7.0-10 microns in diameter are comprised of late G1 and S phase cells. Populations of cells 10-12 microns in diameter consist of S, G2, and M phase cells. The importance of this correlation is discussed in view of needs to more rigorously define B cell populations for investigations of biochemical events of and accessory cell requirements for activation of B lymphocytes.

Acridine Orange↗
PubMed1983

B cell activation. I. Anti-immunoglobulin-induced receptor cross-linking results in a decrease in the plasma membrane potential of murine B lymphocytes.

We report analyses of the effect of anti-Fab antibodies on plasma membrane potential of mouse B lymphocytes. Results indicate that divalent fragments of anti-Fab antibodies mitogenic for B cells stimulate membrane depolarization detectable by cytofluorometric analysis of 3,3'-dipentyloxacarbocyanine iodide-stained cells. Depolarization is detectable within 5 min of exposure to ligand and maximal within 1 h of exposure when greater than or equal to 80% of splenic B cells exhibit decreased membrane potential. The ineffectiveness of monovalent Fab antibody fragments in inducing this event suggests that receptor immunoglobulin cross-linking is essential. Frequencies of cells induced to enter cell cycle, as assessed by acridine orange cell cycle analysis, are equal to those induced to depolarize by lipopolysaccharide plus dextran sulfate or anti-Fab, which suggests a relationship between these events. However, membrane depolarization is itself an insufficient signal to promote subsequent thymidine uptake, as evidenced by the fact that doses of anti-Fab that are suboptimal for thymidine uptake induce maximal depolarization. These results suggest that cross-linking of surface immunoglobulin on B cells may provide an initial signal for activation but is itself insufficient to drive B cell proliferation.

Animals↗
PubMed1983

B lymphocyte activation: entry into cell cycle is accompanied by decreased expression of IgD but not IgM.

To gain insight into the function of cell surface IgM (sIgM) and IgD (sIgD), the expression of these markers on B lymphocytes was quantitated during activation and progression through the cell cycle. Specifically, analysis and correlation of changes in cell cycle state, sIgM and sIgD expression and cell size following exposure of murine B cells to mitogenic levels of lipopolysaccharide and dextran sulfate is reported. As assessed by flow cytometric acridine orange analysis, a large proportion of normal splenic B cells respond within 48 h of exposure to these mitogens by entry into the cell cycle. This response is accompanied by an increase in cell size as determined by flow cytometric "time of flight" measurement. Flow cytometric immunofluorescence analysis reveals a simultaneous alteration in sIg expression. Specifically, cells leaving G0 and transiting G1 increase in diameter from 5 microns to 6 microns and lose greater than 80% of sIgD while sIgM remains constant. Progression through the remainder of the cell cycle is accompanied by a further increase in mean cell diameter to approximately 12 microns while sIgM and sIgD levels remain at G1 levels. The abrupt loss of sIgD as cells transit G1 suggests that an active process mediates this decrease.

Animals↗
PubMed1983

B cell activation. II. Receptor cross-linking by thymus-independent and thymus-dependent antigens induces a rapid decrease in the plasma membrane potential of antigen-binding B lymphocytes.

We report the specific induction of B cell plasma membrane depolarization with the use of thymus-dependent and -independent antigens. We have utilized various trinitrophenol-carrier conjugates for the stimulation of isolated trinitrophenol-binding mouse B cells. Membrane depolarization was assessed by flow cytometric analysis of 3-3'-pentyloxacarbocyanine iodide (DiOC5[3])-stained cells. Entry into the cell cycle was determined by flow cytometric analysis of acridine orange-stained cells. The results indicate that polyvalent antigens, but not free hapten, induce B cell membrane depolarization by a large proportion of antigen-binding cells within 2 hr of stimulation. Although all polyvalent antigens induce membrane depolarization, only thymus-independent antigens induce the subsequent G0 to G1 transition, suggesting that the membrane Ig cross-linking signal alone, although sufficient to induce membrane depolarization and subsequent increased IA expression, is insufficient to drive the entry of B cells into the cell cycle. The G0 to G1 transition appears to be dependent on a second signal, perhaps mediated by the thymus-independent carrier or antigen-specific, Ia-restricted T cell helper.

Animals↗
PubMed1983

Constitutive production of a factor supporting B lymphocyte differentiation by a T cell hybridoma.

To investigate the role of soluble T cell products during B cell differentiation more fully, we have produced T cell hybridomas by the fusion of normal helper T cells with the T cell lymphoma BW5147. In this report we describe the production of one such hybrid, 14G3, the subclone 14G3.1F2, and the functional activity of the constitutive product. The hybrid supernatant acts exclusively in antigen-nonspecific, but antigen-dependent, promotion of B cell differentiation. It is optimally effective in the presence of small amounts of EL4 supernatant. It does not itself contain any detectable IL 2 or BCGF or interferon activity, however. 14G3.1F2 activity is probably an important component of the conventional TRF preparations produced by mixed lymphocyte populations, and will be useful in further dissection of the contributions of different soluble T cell products to B cell differentiation.

Animals↗