Kinetics of membrane immunoglobulin capping on murine B lymphocytes. Effects of phospholipid fatty acid replacement

Detailed analyses regarding the effects of temperature and phospholipid fatty acid replacement on the capping of membrane immunoglobulin (mIg) have been performed using a recently described flow cytometric procedure (Cuchens, M. A., and Buttke, T. M. (1984) Cytometry 5, 601-609). Purified murine B cells were incubated for 12-20 h in the presence of bovine serum albumin-complexed 80 microM stearic (18:0), oleic (cis-18:1), or linoleic (cis, cis-18:2) free fatty acids. Unmodified and free fatty acid-treated cells were stained with fluorescein-conjugated rabbit anti-mouse Ig and subjected to pulse-shape (width) analyses to follow the kinetics of mIg capping. In both unmodified and free fatty acid-treated cells, capping of mIg occurred at all temperatures between 17 and 37 degrees C, but the rate of cap formation was temperature dependent. Arrhenius plots of mIg capping were linear, with activation energies ranging from 14 to 23 kcal/mol depending on the saturated/unsaturated fatty acid ratio of B cell phospholipids. Ligand-induced redistribution of mIg thus appears to be sensitive to changes in membrane acyl chain composition.     

Temperature-mediated processes in teleost immunity: the effects of temperature on membrane immunoglobulin capping on channel catfish B lymphocytes

1. In order to better understand ligand-induced redistribution of membrane receptors and lymphocyte activation in ectothermic vertebrates, flow cytometry was used to monitor the effects of both in vivo acclimation temperature and in vitro assay temperatures on the kinetics of monoclonal antibody-induced membrane immunoglobulin (mIg) capping on channel catfish lymphocytes. 2. It was observed that the kinetics of mIg capping were dependent on in vitro assay temperatures, in vivo acclimation temperatures, and the length of time of in vivo acclimation. In the latter situation in vivo acclimation of fish to 27, 22 and 17 degrees C was considered complete after 3 weeks, while acclimation to 12 degrees C required a minimum of 5 weeks. 3. The energies of activation required for mIg capping ranged from 33 to 24 kcal/mol; lower energies of activation were observed with lower temperature acclimation. 4. It was also noted that the lower energies of activation were associated with concomitant decreases in cellular phospholipid saturated/unsaturated fatty acid ratios. 5. It appears that channel catfish B cell mIg capping, presumably a requisite for immune function, can be significantly affected by environmental temperatures; most likely such effects are attributable to changes in plasma membrane viscosities.     

Membrane Ig-cytoskeletal interactions. I. Flow cytofluorometric and biochemical analysis of membrane IgM-cytoskeletal interactions

Membrane IgM (mIgM) and mIgD are the receptors for Ag on the surface of B lymphocytes, mIg is soluble in detergent; however, when mIg is cross-linked with anti-Ig, the mIg becomes associated with the cytoskeletal matrix and is rendered detergent-insoluble. By a novel flow cytofluorometric assay and by biochemical analysis, it has been shown that anti-isotype-specific antibodies induce mIgM and mIgD to associate with the cytoskeleton of B lymphocytes in an isotype-specific fashion. The detergent solubility of other prominent B lymphocyte surface proteins, such as class I and class II MHC proteins were unaffected by cross-linking of mIg. A panel of mu-specific mAb was analyzed for their ability to induce mIgM-cytoskeletal association. Although all mAb bound mIgM, only three out of seven rendered mIgM cytoskeletally associated. Further analysis revealed a strict correlation in the capacity of mu-specific mAb to induce capping and to induce the association of mIgM with the cytoskeleton.     

Membrane Ig cross-linking regulates phosphatidylinositol 3-kinase in B lymphocytes.

Cross-linking of the B cell AgR results in activation of mature B cells and tolerization of immature B cells. The initial signaling events stimulated by membrane immunoglobulin (mIg) cross-linking are tyrosine phosphorylation of a number of proteins. Among the targets of mIg-induced tyrosine phosphorylation are the tyrosine kinases encoded by the lyn, blk, fyn, and syk genes, the mIg-associated proteins MB-1 and Ig-beta, phospholipase C-gamma 1 and -gamma 2, as well as many unidentified proteins. In this report we show that mIg cross-linking also regulates phosphatidylinositol 3-kinase (PtdIns 3-kinase), an enzyme that phosphorylates inositol phospholipids and plays a key role in mediating the effects of tyrosine kinases on growth control in fibroblasts. Cross-linking mIg on B lymphocytes greatly increased the amount of PtdIns 3-kinase activity which could be immunoprecipitated with anti-phosphotyrosine (anti-tyr(P) antibodies. This response was observed after mIg cross-linking in mIgM- and mIgG-bearing B cell lines and after cross-linking either mIgM or mIgD in murine splenic B cells. Thus, regulation of PtdIns 3-kinase is a common feature of signaling by several different isotypes of mIg. This response was rapid and peaked 2 to 3 min after the addition of anti-Ig antibodies. The anti-Ig-stimulated increase in PtdIns 3-kinase activity associated with anti-Tyr(P) immunoprecipitates could reflect increased tyrosine phosphorylation of PtdIns 3-kinase, increased activity of the enzyme, or both. In favor of the first possibility, the tyrosine kinase inhibitor herbimycin A blocked the increase in ant-Tyr(P)-immunoprecipitated PtdIns 3-kinase activity as well as the anti-Ig-induced tyrosine phosphorylation. Moreover, this response was not secondary to phospholipase C activation but rather seemed to be a direct consequence of mIg-induced tyrosine phosphorylation. Activation of the phosphoinositide pathway by a transfected M1 muscarinic acetylcholine receptor expressed in WEHI-231 B lymphoma cells did not increase the amount of PtdIns 3-kinase activity which could be precipitated with anti-Tyr(P) antibodies. Similarly, inhibition of the phosphoinositide pathway did not abrogate the ability of mIg cross-linking to stimulate this response. Thus, mIg-induced tyrosine phosphorylation regulates PtdIns 3-kinase, an important mediator of growth control in fibroblasts and potentially an important regulatory component in B cells as well.     

IL-4 promotes Stat6-dependent survival of autoreactive B cells in vivo without inducing autoantibody production

Persistent cross-linking of hen egg lysozyme (HEL)-specific B cell membrane Ig (mIg) in double transgenic mice that express soluble HEL as a self Ag (HEL-Ig mice) decreases B cell mIgM expression, responsiveness, and life span. Because in vitro treatment with IL-4 inhibits T cell apoptosis through a Stat6-independent mechanism, increases mIg expression, and suppresses activation-induced B cell death, we studied IL-4 effects on B cell mIg expression, survival, and Ab secretion in Stat6-sufficient and deficient HEL-Ig mice. IL-4 treatment nearly normalized B cell number and greatly increased the percentage of mature B cells in HEL-Ig mice, but failed to normalize mIgM expression or spontaneous LPS-induced IgM secretion. IL-4 effects on B cell survival and maturation were CD4(+) T cell independent, but Stat6 dependent, and did not involve receptor editing. IL-4 had to be present while B cells were generated to have a detectable effect on autoreactive B cell survival; however, the survival of B cells generated in the presence of IL-4 was substantially increased even after IL-4 was withdrawn. These observations suggest that: 1) activation-induced B cell death and anergy are independent processes; 2) B cells that survive to maturity develop increased resistance to Ag-induced deletion; and 3) IL-4 promotes B and T cell survival through different mechanisms.     

High anti-TNP plaque-forming cell potential of residual mIg+ cells in a T cell population

In the course of experiments designed to study the immune response of purified populations of B lymphocytes to thymus-independent (TI) antigens, a variety of cell purification procedures were followed. In using anti-immunoglobulin-coated dishes to separate lymphocytes bearing membrane immunoglobulin (mIg) from mIg- lymphocytes, it was found that the nonadherent fraction, which was predominantly mIg-, complement receptor negative, and nonresponsive to the B cell mitogen lipopolysaccharide, gave very substantial anti-TNP plaque-forming cell responses to 2 TI antigens. These responses could be inhibited by incubation of such cells in the presence of anti-mu and thus appeared to be attributable to mIg+ cells. The evidence suggests the existence of a population of B lymphocytes that constitute a minor component of mIg+ cells having a high potential to make in vitro antibody responses. Users of techniques that utilize anti-Ig as a tool for separating B and T lymphocytes should carefully assess the extent to which residual B lymphocytes in the mIg- population contribute to antibody responses being studied.     

Analysis of H2-O influence on antigen presentation by B cells

HLA-DM (DM; in mouse H2-DM) promotes the exchange of MHC class II-associated peptides, resulting in the accumulation of stable MHC class II-peptide complexes. In naive (but not germinal center) B cells, a large part of DM is tightly associated with HLA-DO (DO; in mouse H2-O), but the functional consequence of this association for Ag presentation is debated. Here, we have extended previous studies by examining the presentation of multiple epitopes after Ag internalization by fluid phase endocytosis or receptor-mediated uptake by membrane Ig (mIg) receptors. We find that the effects of H2-O are more complex than previously appreciated; thus, while only minor influences on Ag presentation could be detected after fluid phase uptake, many epitopes were substantially affected after mIg-mediated uptake. Unexpectedly, the presentation of different epitopes was found to be enhanced, diminished, or unaffected in the absence of H2-O, depending on the specificity of the mIg used for Ag internalization. Interestingly, epitopes from the same Ag did not necessarily show the same H2-O dependency. This finding suggests that H2-O may control the repertoire of peptides presented by B cells depending on the mIg-Ag interaction. The absence of DO/H2-O from germinal center B cells suggests that this control may be released during B cell maturation.     

A Possible role of the nucleus in cytochalasin B-Induced capping

The possible influence of the nucleus on Cytochalasin B (CB)-induced capping of antibodies to surface antigens on L cells SV40-3T3 and NRK La 334 cells was studied. The cap formation induced by CB, was generally localized opposite the nucleus which was displaced against the cell periphery. To be able to observe the nuclear membrane in relation to the capping process we have taken advantage of an antiserum specific for antigens in the nuclear membrane but lacking reactivity to the plasma membrane and intranuclear antigens. This approach indicated that the CB-induced capping caused an accumulation of nuclear membrane antigens in the area of the nucleus facing the cap. The CB-induced local accumulation of nuclear membrane antigens required intact cells and could not be induced by binding of antibodies to the nuclear membrane followed by exposure to CB. Whatever the basis for the CB-induced altered reactivity of the anti-nuclear membrane antibodies (folding of the nuclear periphery, for example) this result indicated that the nuclear membrane was affected by CB capping. The possible role of the nucleus in the CB-induced capping process was further investigated in enucleated cells. The results obtained indicate that such cells both when enucleated in suspension and adherent to a surface did not exhibit CB capping. This disappearance of CB capping did probably not reflect decreased cell viability, previous exposure of the cells to CB during the enucleation procedure or a decreased capacity of the enucleated cells to bind CB.     

Cross-linkage of Ly-6A/E induces Ca2+ translocation in the absence of phosphatidylinositol turnover and mediates proliferation of normal murine B lymphocytes

Ly-6A/E is a phosphatidylinositol (PI)-linked membrane protein whose expression is induced or upregulated on normal murine T and B cells by IFN-gamma. Cross-linkage of Ly-6A/E expressed on normal murine T cells stimulates Ca2+ translocation, and in the presence of a protein kinase C (PKC) activator, lymphokine secretion, and cellular proliferation. Utilizing an anti-Ly-6A/E mAb, we studied the effect of cross-linking Ly-6A/E on IFN-gamma-treated resting B cells, for Ca2+ translocation, PI turnover, and cellular proliferation. Since these events are known to be stimulated by cross-linkage of B cell membrane (m)Ig, we compared the changes mediated through these respective membrane proteins. We show that cross-linkage of B cell Ly-6A/E stimulates a large, rapid, and sustained increase in the concentration of intracellular free calcium ([Ca2+]i) comparable in magnitude, though somewhat delayed, relative to that observed after cross-linking of mIg. Cross-linkage of B cell Ly-6A/E does not, however, stimulate detectable PI turnover, in contrast to PI turnover induced by ligation of mIg. Both the Ly-6A/E- and mIg-mediated increase in [Ca2+]i occur through mobilization of internal Ca2+ stores as well as entry of Ca2+ into the cell from the extracellular compartment. Ly-6A/E-mediated Ca2+ translocation appears to be under the regulation of PKC in that short term pretreatment of B cells with the PKC activator, PMA, inhibits the Ly-6A/E- as well as the mIg-mediated increase in [Ca2+]i, whereas prolonged exposure to PMA, under conditions that lead to depletion of PKC, results in an augmentation in Ca2+ translocation after ligation of either Ly-6A/E or mIg. Co-capping studies indicate that Ly-6A/E and mIg cap independently in the B cell membrane, thus suggesting that the Ly-6A/E-induced effects on Ca2+ translocation are not mediated through simultaneous modulation of mIg. Anti-Ly6A/E, by itself, does not stimulate an increase in [3H]thymidine incorporation by IFN-gamma-treated resting B cells, but induces a striking increase in the presence of PMA. By contrast, anti-Ig by itself stimulates significant increases in [3H]thymidine incorporation that is inhibited by PMA. Thus, Ly-6A/E is a potent mediator of B cell activation that may use a signal transduction system in quiescent B cells that is distinct from that of the Ag receptor.     

Impaired lymphocyte functions in heterozygous rabbits recovering from neonatal allotype suppression

Lymphocytes from heterozygous rabbits suppressed for an allotypic determinant on kappa light chains by exposure to maternally derived antibodies specific for the paternal gene product were analyzed for their capacity to express membrane-bound and secreted immunoglobulin (Ig). Individual cells displaying allotypic membrane Ig (mIg) were enumerated by a rosette test, while Ig-secreting cells were assessed by means of a hemolytic plaque assay. In a group of suppressed rabbits varying in age from 3 to 19 months, the proportion of cells with mIg of the paternal type was markedly higher than that of cells secreting that type of Ig. The same high proportion of lymphocytes displaying mIg of the suppressed type was observed whether lymphocytes from blood, spleen, or lymph nodes of suppressed rabbits were examined. In contrast, similar analyses performed with cells of normal heterozygous rabbits showed no discrepancy between mIg expression and secretion of either allotype. Lymphocytes synthesizing Ig of the paternal type were also defective in responses to lipopolysaccharide (LPS), which stimulates differentiation to Ig secretion in normal B lymphocytes. These results support the idea that B lymphocytes capable of synthesizing the suppressed type of Ig have functional impairments affecting secretion and responses to environmental stimuli.     

Memory B cell survival and function in the absence of secreted antibody and immune complexes on follicular dendritic cells

Ag, in the form of immune complexes retained on follicular dendritic cells, has been implicated in the development and maintenance of B cell memory. We addressed this question using a H chain transgenic (Tg) mouse model that lacks secreted Ig (mIg), and thus does not deposit Ag-containing immune complexes. We compared the ability of the mIg strain and a control Tg strain, which secretes IgM, to develop and maintain long-lived memory cells. After immunization, there was an increase of Ag-specific B cells in both strains that was maintained for at least 20 wk. We labeled the long-lived Ag-specific cells with BrdU and found that this population was similarly maintained. In addition, both Tgs were able to maintain a functional memory response as measured by secondary germinal center reactions. Our studies indicate that localization of Ag on follicular dendritic cells is not necessary for development and maintenance of B cell memory.     

Tyrosine phosphorylation of phospholipase C-gamma 2 upon cross-linking of membrane Ig on murine B lymphocytes.

When membrane Ig (mIg) on the surface of B lymphocytes is cross-linked using anti-Ig antibodies, the enzyme phospholipase C (PLC) is activated to cleave inositol phospholipids. Tyrosine kinase inhibitors have been reported to inhibit this event. Therefore, we investigated the effect of cross-linking of mIg on the state of tyrosine phosphorylation of PLC activity in two murine B cell lines and in normal resting mouse B cells. Proteins from lysates of stimulated or unstimulated cells were immunoprecipitated with an antiphosphotyrosine antibody and subsequently assayed for PLC activity. Treatment of the B cell line WEHI-231 with anti-IgM led within 15 to 30 s to a 10- to 20-fold increase in tyrosine-phosphorylated PLC activity. Inositol trisphosphate generation by WEHI-231 cells stimulated under the same conditions demonstrated similar kinetics. Normal resting B cells treated with anti-IgM or anti-IgD demonstrated 2.5- and 4-fold increases, respectively, of tyrosine-phosphorylated PLC activity. To identify the isozyme of PLC that was phosphorylated, we immunoprecipitated PLC-gamma 1 or PLC-gamma 2 with specific antibodies and assessed the amount of tyrosine phosphorylation of these proteins by antiphosphotyrosine immunoblotting. Treatment of WEHI-231 or Bal17 cells with anti-IgM induced an increase in PLC-gamma 2 tyrosine phosphorylation over background levels. There was no detectable tyrosine phosphorylation of PLC-gamma 1 in treated or untreated WEHI-231 cells, whereas anti-IgM-treated Bal17 cells did exhibit low but detectable levels of tyrosine phosphorylation of PLC-gamma 1. In normal resting mouse B cells, there was no detectable PLC-gamma 1, but PLC-gamma 2 was abundant. These observations suggest that PLC-gamma 2 is a significant substrate for the mIg-activated protein tyrosine kinase and may be responsible for mediating mIg stimulation of inositol phospholipid hydrolysis in murine B cells.     

Interactions between lymphocyte membrane molecules. II. Characterization of an interaction between B lymphocyte surface IgD and Fc IgG receptors that differs from the surface IgM-Fc IgG receptor interaction

The B lymphocyte surface membrane receptors IgD (sigD) and Fc IgG receptors (Fc gamma R) were evaluated for interactions by means of immunofluorescence. Ligand-(F(ab')2 anti-delta) induced capping of sIgD resulted in co-capping of Fc gamma R if the latter were occupied during the capping process by soluble antigen-antibody complexes (which themselves provided insufficient cross-linking to result in capping), but not if the Fc gamma R were occupied by monomeric IgG or unoccupied. Capping of Fc gamma R by highly cross-linked complexes did not cause co-capping of sIgD occupied by monomeric F(ab') anti-delta. The interaction between sIgD and Fc gamma R was specific in that cross-reactions between ligands were excluded and ligand-induced capping of sIgD did not cause co-capping of ligand-occupied sIgM or I-A antigens. The sIgD-Fc gamma R interaction occurred on only approximately 60% of B lymphocytes, and this B cell subpopulation did not correlate with other B cell subpopulations (CBA/N strain B cells and B cells bearing either large or small amounts of sIgD). The sIgD-Fc gamma R interaction differed from the sIgM-Fc gamma R interaction in that co-redistribution of the Fc gamma R was occupied by monomeric IgG and involved nearly all B lymphocytes. The qualitative and quantitative differences between the sIgD-Fc gamma R and sIgM-Fc gamma R interactions suggest a mechanism whereby the two antigen receptors could provide different signals to the B lymphocyte.     

c-Jun NH2-terminal kinase (JNK)-dependent nuclear translocation of apoptosis-inducing factor (AIF) following engagement of membrane immunoglobulin on WEHI-231 B lymphoma cells

WEHI-231 B lymphoma cells have been employed for analysis of antigen-induced B cell unresponsiveness because these cells undergo cell cycle arrest in G1, accompanied by induction of apoptosis. In the present study, we examined the requirement for toxic small molecules apoptosis-inducing factor (AIF) and cytochrome c, and subsequent caspase activation in apoptotic cell death in WEHI-231 and CH31 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Pan-caspase inhibitor BD-fmk blocked mIg-mediated increase in cells with sub-G1 DNA content, whereas it did not affect mIg-mediated loss of mitochondrial membrane potential and phosphatidylserine exposure on B cell membrane. Dominant-negative form of c-Jun NH2-terminal kinase1 (JNK1) blocked the translocation of AIF into the nuclei and cytosol from the mitochondria in the WEHI-231 and CH31 cells following mIg engagement, whereas constitutively active form of JNK1 enhanced it. This AIF translocation was also blocked by Bcl-xL, but not by BD-fmk. Moreover, AIF-deficient clones via small interfering RNA (siRNA)-mediated method showed small increase in loss of mitochondrial membrane potential. After mIg engagement, the AIF-deficient clones displayed an enhanced sensitivity to mIg-mediated apoptosis, concomitant with translocation of a residual AIF into the nuclei, compared with control clone. Our findings are compatible with the notion that AIF has dual role, with a proapoptotic function in the nuclei and a distinct anti-apoptotic function in the mitochondria. These observations would be valuable for analysis of B cell unresponsiveness and hopefully for treatment of diseases involving B cell dysfunction.     

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.     

Membrane immunoglobulin: anti-immunoglobulin interactions mediate the phosphorylation of actin associated proteins in the B-lymphocyte

The binding of membrane immunoglobulin (mIg) by anti-Ig antibodies is known to initiate a mitogenic signal in B lymphocytes. Because in many instances growth control appears to be correlated with phosphokinase activity, as well as with alterations in cytoskeletal architecture, we asked the question whether antibodies binding to mIg would also lead to the specific phosphorylation of lymphocyte actin-associated proteins. Utilizing a myosin affinity technique, we directly examined phosphoproteins that were associated with actin in the chicken B cell. We found that in a few instances the level of phosphorylation was indeed modulated by mIg:anti-Ig interactions. These actin-binding phosphoproteins may be important control elements in the lymphocyte cytoskeleton.     

Increased membrane immunoglobulin capping of B cells from C57Bl/6 lpr/lpr and C57Bl/6 nu/nu mice

When the capping of membrane immunoglobulin on spleen B cells from normal C57Bl/6 mice (B6) is taken as reference, a faster capping rate is found for cells of age-matched B6 mice which are congenic at the lymphoproliferation (lpr) or nude (nu) loci. Though both congenic strains can be characterized by an abnormal T-lineage cell content, the nature of the abnormality itself is very different since B6 nudes lack thymus-processed/influenced lymphocytes whereas B6 mice with the lpr phenotype suffer from an invasion of all lymphoid organs with cells of a particular T-cell subset. Moreover, the more "normal" capping rate of B cells from the double congenic B6 mice (nu/nu, lpr/lpr) is intriguing. Since other mice homozygous at the lpr locus (MRL-1) or at the nu locus (BALB/c nude) also cap faster than their congenic controls (MRL-n and BALB/c, respectively), the observed effects do not appear to depend on a peculiarity of the B6 genetic background. If the faster capping of B cells of nu congenic and of lpr congenic mice had a common origin, it might be that T cells would control in some way the mobility of B-cell membrane immunoglobulins: both congenic mice have in their spleen a very low proportion of mature T cells together with a very high proportion of prethymic/thymic immature T-cell types, either of which might affect B-cell behavioral responses to membrane immunoglobulin clustering.     

The MB-1/B29 heterodimer couples the B cell antigen receptor to multiple src family protein tyrosine kinases.

The B cell Ag receptor complex is comprised of membrane (m)IgM or mIgD noncovalently associated with one or more heterodimers, each containing one subunit of MB-1 (IgM alpha or IgD alpha) and one of B29 (Ig beta or Ig gamma). It is known that cross-linking of the B cell Ag receptor results in protein tyrosine kinase activation. Recent reports from other laboratories have demonstrated that mIg coprecipitates with multiple src family protein tyrosine kinases, including blk, lyn, and fyn. However, the mechanism by which these kinases are physically coupled to the Ag receptor has not been confirmed. It has been hypothesized that the mIg-associated proteins MB-1 and B29 provide a physical link between the Ag receptor (mIg) and one or more protein tyrosine kinases. In this study, we confirm previous findings demonstrating that the B cell Ag receptor coprecipitates with the MB-1/B29 heterodimer as well as the protein tyrosine kinases blk, lyn, and fyn under mild detergent conditions (1% digitonin). Additionally, we demonstrate that in detergent conditions (1% Nonidet P-40 (NP-40)) which disrupt the association between mIg and the MB-1/B29 heterodimer, no protein tyrosine kinase activity can be detected in association with mIg. These findings indicated that NP-40 effectively dissociates the B cell Ag receptor from ancillary signal transducing proteins. MB-1 and B29 were however, found to coprecipitate with blk, lyn, and fyn isolated from B cell lysates containing 1% NP-40. No significant difference was observed in the stoichiometry of association between the kinases and the MB-1/B29 heterodimer in the presence of 1% NP-40 when compared to 1% digitonin. It was further determined that in resting B cells, only a small fraction (approximately 1-3%) of the MB-1/B29 heterodimers appear to be complexed with protein tyrosine kinases. Finally, based on preclearing experiments, it appears that individual heterodimers may associate with a single species of protein tyrosine kinase. These data support the hypothesis that the MB-1/B29 heterodimer couples the antigen receptor to protein tyrosine kinases, thereby providing a physical link that facilitates Ag receptor-mediated regulation of kinase activity.     

Two isoforms of human membrane-bound alpha Ig resulting from alternative mRNA splicing in the membrane segment

Antibodies specific for membrane-bound Ig (mIg) but not for their secreted forms would be useful not only for studying the function of mIg but also for modulating B cell activities in vivo. We have proposed that the extracellular portions of the membrane anchor peptides of mIg can be used as antigenic sites for isotype-specific targeting of B cells. Clones containing the genes of human Ig alpha 1 or alpha 2 subclasses were isolated from a genomic DNA library. The gene segments encoding the membrane peptides and their flanking regions were amplified by polymerase chain reaction, subcloned into plasmid pUC19, and the DNA sequences were determined. Human alpha 1 and alpha 2 genes, like murine alpha gene, each has only one membrane exon. The sequences of the human alpha 1 and alpha 2 genes are almost identical in the membrane peptide-coding region. The mRNA from a human mIgA-expressing B cell line, DAKIKI, was isolated, its cDNA prepared, and the segments spanning the membrane peptide-coding region and a part of the constant domain 3 amplified by polymerase chain reaction. DNA sequences revealed that there are two isoforms of alpha 1-chain, resulting from the alternative splicing of the third constant domain of H chain to two acceptor sites in the membrane exon. One isoform has a segment of 32 and the other 26 amino acid residues in the extracellular portion of the membrane peptide. These segments may serve as isotype-specific antigenic epitopes for antibody targeting of mIgA-bearing B cells.