Anti-mu antibody stimulates the phosphatidylinositol cycle and immunoglobulin secretion in a human lymphoblastoid B-cell line, LA350

Within 5 min of the binding of anti-mu antibody (anti-mu) to surface IgM on LA350, a human lymphoblastoid B-cell line, there was a significantly enhanced incorporation of 32P into the phosphatidic acid (PA) and phosphatidylinositol (PI) fractions of cellular phospholipids and the magnitude of the early increase in PA was twice as great as that in PI. This anti-mu-enhanced incorporation of 32P into PA and PI required the binding of a divalent form of antibody (IgG or F(ab')2), was blocked by coincubation with micromolar concentrations of soluble IgM, was decreased by incubation of cells at temperatures lower than 37 degrees C, and was inhibited by coincubation with millimolar concentrations of dibutyryl cyclic AMP and theophylline. Similar incorporation studies with [3H]inositol demonstrated a selective and significant increase in labeling of PI. In LA350 labeled with [3H]inositol for 30 hr (equilibrium) and acutely stimulated by anti-mu, specific hydrolysis of phosphorylated PI (PI 4,5-bisphosphate) was measured by the significantly increased release at 15 min of radioactive inositol 1,4,5-trisphosphate, inositol 1,4 bisphosphate, and inositol 1-phosphate. The release of these inositol phosphates was significantly augmented by coincubation with 0.01 M LiCl which prevented their simultaneous enzymatic degradation. All of these findings are consistent with an activation of a linked series of metabolic events known as the PI cycle. In similar cell cultures anti-mu significantly stimulated the secretion of IgM by LA350 as measured at 48 hr in a reverse hemolytic plaque assay. Two other IgM-bearing human lymphoblastoid B-cell lines which gave no evidence of turnover of 32P in PA and PI in response to binding by anti-mu likewise failed to enhance their secretion of IgM. We conclude that the binding of surface IgM on LA350 by anti-mu results in the generation of a transmembrane signal which causes a rapid activation of the PI cycle which itself may play a role in the subsequent increase in IgM secretion.     

Early events in B-cell activation: anti-Lyb2, but not BSF-1, induces a phosphatidylinositol response in murine B cells

Previous studies have indicated that the murine surface antigen Lyb2 is involved in an activation pathway that apparently does not involve the surface immunoglobulin receptor. As sIg has been shown to transduce its activation signal through the breakdown of phosphatidylinositol (PI), and since activation via Lyb2 does not involve sIg, it was of interest to determine if binding to Lyb2 generates a PI response. We have demonstrated that an allele-specific monoclonal antibody to Lyb2 (anti-Lyb2 mab), which has previously been shown to drive B cells into S, also activated PI metabolism in these cells. This activation occurred in a dose-dependent and allele-specific manner. Antibodies to other B-cell surface molecules such as Ia did not induce a PI response. The effect of anti-Lyb2 mab was always less in magnitude than that induced by anti-IgM, but the effects of the two antibody preparations were most comparable in larger, presumptively preactivated cells. To explore the issue that Lyb2 may represent a receptor for a growth factor, possibly the early-acting B-cell growth factor BSF-1, we studied the PI response to BSF-1 and the effect of BSF-1 on Lyb2-induced PI turnover. BSF-1 neither induced a PI response nor inhibited competitively the response induced by anti-Lyb2 mab.     

Differential effects of the stimulation of complement receptors CR1 (CD35) and CR2 (CD21) on cell proliferation and intracellular Ca2+ mobilization of chronic lymphocytic leukemia B cells.

The regulatory role of CR1 and CR2 on B cell activation and proliferation has been investigated by using B cells from patients with chronic lymphocytic leukemia. The chronic lymphocytic leukemia B cells are clonal expansions of B lymphocytes frozen at specific stages of activation. They displayed two patterns of response upon surface Ig (sIg) cross-linking in terms of in vitro proliferation and intracellular free Ca2+ mobilization: cells from patient F (first pattern) proliferated in the presence of mitogenic anti-mu antibodies, whereas cells from patient A (second pattern) did not respond to sIg cross-linking but proliferated in the presence of low m.w. B cell growth factor and IL-2. Coculture of A or F cells with C3b-bearing SRBC led to a two- to four-fold increase in thymidine incorporation in cultures containing low m.w. B cell growth factor but not in cultures containing rIL-2. This enhanced proliferation was inhibited by F(ab')2 polyclonal rabbit antihuman CR1 antibodies. Only cells which proliferated in the presence of anti-mu (cells F) responded to cross-linking of sIg with a rise in intracellular Ca2+. No increase in calcium mobilization was observed after co-cross-linking of CR1 and sIg on A and F cells with mAb or polyclonal anti-CR1 antibodies. Co-cross-linking of CR2 with sIg only led to an enhanced intracellular Ca2+ rise in F cells but not in A cells. The lack of CR2-mediated synergy in Ca2+ rise in A cells indicates that the synergy occurs only if there is a proper coupling of sIg to phospholipase C. CR1-induced proliferation of B cells does not involve the signaling pathways of sIg. These results provide additional evidences for the role of C3 fragments in modulation of human B cell activation.     

Differential responsiveness of immature- and mature-stage murine B cells to anti-IgM reflects both FcR-dependent and -independent mechanisms.

Mature and immature B cells differ in their responses to antigen receptor crosslinking. Whereas mature B cells enter cell cycle in response to such stimulation, immature B cells exhibit proliferative unresponsiveness and undergo induced tolerance following surface immunoglobulin (sIg) engagement. Previous studies evaluating antigen receptor-mediated negative signaling have utilized intact goat anti-immunoglobulin (anti-Ig) antibodies as polyclonal ligands based upon observations that the Fc portion of these reagents does not interact with and mediate negative signaling through the FcR on mature B cells. Thus, the negative effects of goat anti-Ig on immature B cells have been attributed solely to signals mediated via their antigen receptors. In the studies reported here we show that the activation unresponsiveness inherent to immature B cells is FcR independent. However, we also show that immature B cells are sensitive to FcR-mediated inhibition and that these effects can be mediated by intact goat antibodies at concentrations that promote positive activation signals in mature B cells. Our results demonstrate that inhibition of immature B cell LPS responses by anti-Ig antibodies, used in previous studies as an in vitro model for B cell tolerance induction, is an FcR-mediated phenomenon. We show that developmentally associated anti-Ig-mediated inhibition of LPS requires the use of intact antibodies, and that this inhibition can be blocked by the anti-FcR monoclonal antibody 2.4G2. Flow cytometric analysis of FcR-positive B cells indicates that both mature and immature B cells express equivalent levels of FcR gamma. Therefore, the sensitivity of immature, but not mature, cells to intact goat anti-mu antibodies suggests that either FcRs or their associated inhibitory pathways change during B cell development.     

The modulation of membrane Ia on human B lymphocytes

Using flow cytometry techniques, changes in surface Ia (DR and DS) expression on human B lymphocytes were correlated with changes in the cell cycle following stimulation with anti-mu. The effect of interleukin (IL)-1, IL-2, B-cell growth factor (BCGF), and interferons on Ia expression on resting B cells was also examined. A population of resting B lymphocytes was cultured in vitro with 100 micrograms/ml of anti-mu and immunofluorescently stained for DR and DS at various times following stimulation. Detectable increases in DR and DS expression were found within 8 hr, and the major increases (twofold and fourfold) in DR and DS expression occurred over the next 48 hr. Using cell cycle inhibitors and propidium iodide staining, it was demonstrated that the enhanced DR and DS expression following anti-mu stimulation began during G0 to G1 transition and increased as the cells progressed through G1 phase. During S and G2/M phases, there were minimal further increases in surface Ia. Although prolonged exposure of B cells to anti-mu was required for cellular activation, cell size enlargement, and progression into S phase, a brief exposure to anti-mu, insufficient for cellular activation, markedly enhanced Ia expression. Thus anti-mu-stimulated resting human B lymphocytes rapidly increase their surface Ia expression. This increase occurs predominantly prior to entrance into S phase and can occur in the absence of significant cellular activation. Interferons have been reported to modulate surface Ia expression on a human lymphoid cell line and on monocytes and supernatants with BCGF activity to enhance surface Ia expression on murine B cells; however, neither alpha-interferon, gamma-interferon, IL-1, IL-2, nor BCGF modified surface DR expression on normal resting human B cells.     

The role of cytoplasmic free calcium concentration in B-cell tolerance

Calcium is an important factor in the immune response. Extracellular calcium is required for antibody production by B lymphocytes. Several investigators have demonstrated that crosslinking of receptors on B lymphocytes by anti-mu antibody induces an increase in intracellular calcium. There are few data on the role of intracellular calcium mobilization or calcium influx in tolerance induction in B cells. We studied changes in free intracellular calcium concentration ([Ca+2]i) induced by exposure of dinitrophenyl (DNP)-specific B cells to the tolerance-inducing conjugate DNP-murine IgG2a (DNP-MGG). Splenic B cells enriched for DNP-specific cells and DNP-specific continuous B-cell lines were used for the studies. Exposure of B cells to the tolerogen DNP-MGG, the antigen DNP-keyhole limpet hemocyanin (DNP-KLH), or the antigen DNP-Ficoll induced an increase in free [Ca+2]i which was due to both mobilization of Ca+2 from endoplasmic reticulum (ER) and influx of extracellular Ca+2. This increase was DNP specific since no significant change was seen with carriers alone and no change was seen in cells that were not DNP specific. The DNP-MGG and DNP-Ficoll induced the same amount of Ca+2 release from ER but the release induced by DNP-KLH was higher. When B cells, which were made tolerant by in vitro incubation with DNP-MGG, were incubated with antigens, a mobilization of Ca+2 from endoplasmic reticulum occurred that was the same as that of nontolerant B cells. Since Ca+2 mobilization is associated with Ig receptor-dependent early B-cell activation, it is likely that the tolerant B cell can still receive an activation signal through the Ig receptors.     

Ig-independent Ig beta expression on the surface of B lymphocytes after B cell receptor aggregation

In order for humoral immune responses to develop, B cells must be able to recognize, bind, and internalize Ags. These functions are performed by the BCR, which is also responsible for initiating and transducing activation signals necessary for B cell proliferation and differentiation. We have examined surface expression patterns of individual components of the BCR following anti-Ig- and Ag-induced aggregation. Specifically, the localization and expression levels of the Ag-binding component, surface Ig (sIg), and the Igbeta component of the Igalpha/Igbeta signaling unit were investigated to determine their individual participation in the internalization and signal transduction. Using primary murine B cells, we found that while >95% of the sIg is internalized following anti-Ig-induced aggregation, 20-30% of Igbeta remains on the surface. These results suggest that sIg and Igbeta may function independently following the initial stages of signal transduction.     

Early activation and cell cycle entry of resting B cells after Fab-anti-Ig treatment: role of receptor crosslinking

Comparison of the effect of goat anti-rabbit Ig (GARIg) and its monovalent fragment (Fab-GARIg) demonstrates that surface Ig (sIg) crosslinking is not necessary to effect G0 to G1 transition in rabbit peripheral blood B cells but is required for induction of DNA synthesis. Five micrograms per milliliter or more of GARIg is sufficient to induce DNA synthesis but up to 50 micrograms/ml of Fab-GARIg is not. However, the monovalent reagent induces microscopically observable cytoplasmic and nuclear changes (blast transformation) in a dose-dependent manner. These differ qualitatively and quantitatively from the morphological changes seen with comparable doses of GARIg; Fab anti-Ig produces "small blasts" whereas complete GARIg induces large blasts. The monovalent reagent, in a wide range of concentrations, is as effective as the complete antibody in modulating sIg from rabbit B cells. Fab-GARIg treatment modulates sIg in a biphasic manner. It clears the high-density sIg within 5 min, whereas the remaining low-density receptors disappear after 4 hr. Cytosolic protein kinase C levels decline equally after treatment with either Fab-GARIg or whole anti-Ig. RNA synthesis, as measured by [3H]uridine incorporation, increases for the first 12 hr in cells activated with either reagent. It declines to basal levels in Fab-GARIg stimulated cells, but a continuous increase occurs in cells stimulated with 5 and 50 micrograms/ml of complete antibody. Simultaneous addition of 50 micrograms/ml Fab-GARIg with 5 microgram/ml of GARIg causes greater RNA synthesis for 12 hr after stimulation than is caused by GARIg alone. After 12 hr the monovalent reagent has an inhibitory effect on RNA synthesis. Fluorescence-activated cell sorter analysis of acridine orange-stained cells shows that Fab anti-Ig-stimulated cells have higher RNA content than resting cells, but lower than GARIg-activated cells. These findings suggest that rabbit B cells can be activated from the G0 stage of cell cycle to G1 by monovalent anti-Ig reagents but further cell cycle progression requires maintenance signals provided by receptor crosslinking. The implications of these results for B cell activation signalling are discussed in the context of the floating receptor model.     

Anti-Ig-mediated proliferation of human B cells in the absence of protein kinase C

Cross-linking of surface Ig has been shown to stimulate phosphatidylinositol hydrolysis in murine B cells, leading to increases in [Ca2+]i and activation of protein kinase C (PKC). Preliminary evidence suggests that a similar activation mechanism occurs in human B cells. We wished to examine whether anti-Ig antibody-stimulated human B cell proliferation is as dependent upon the presence of PKC as is anti-Ig-mediated murine B cell proliferation. Using highly purified, small, dense peripheral-blood B lymphocytes from healthy adult donors, we confirmed that PMA, a direct activator of PKC, is a potent mitogen for human B cells that synergizes with anti-mu antibody. Furthermore, we demonstrated that PMA treatment abolishes detectable cellular stores of immunoreactive PKC. However, after such depletion of cellular PKC, anti-mu antibody is still capable of delivering a proliferative signal to human B cells. It is unlikely that this signal occurs solely on the basis of increases in [Ca2+]i, because the calcium ionophore A23187 does not induce a proliferative response in PMA-treated B cells similar in magnitude to that seen with anti-mu. Additionally, the finding that pretreatment of B cells with PMA ablates the ability of anti-Ig antibody to mobilize intracellular and extracellular calcium also suggests that the ability of PMA to enhance anti-Ig mediated stimulation does not depend on elevations of [Ca2+]i induced by anti-Ig. Together, these observations suggest that anti-Ig signaling of human B cells may occur via other pathways in addition to the phosphatidylinositol system of calcium influx and PKC activation.     

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.     

Recombinant interferon-alpha, -beta, and -gamma enhance the proliferative response of human B cells

Recombinant interferons (IFN-alpha, -beta, and -gamma) were examined for their effects on B cell activation. Relatively small IgM+ B cells from human blood samples were isolated by fluorescence-activated cell sorting and were used as target cells. Although the interferons themselves were nonmitogenic, each enhanced the proliferative response induced by a mitogenic anti-mu monoclonal antibody, with IFN-beta usually showing the greatest enhancement and IFN-gamma the least. Pretreatment with the interferons primed resting B cells to undergo enhanced DNA synthesis in response to the anti-mu antibody DA4. Conversely, anti-mu pretreatment, followed by IFN treatment, did not induce B cells to enter the S phase. Time-course analysis revealed that IFN could augment the anti-mu response even when added as late as the final 24 hr of a 3-day culture interval. Combinations of IFN-gamma plus IFN-alpha or -beta were synergistic in the anti-mu response, whereas the IFN-alpha plus IFN-beta combination was not. The data suggest that interferons produced by both lymphocytes (IFN-gamma) and nonlymphoid inflammatory cells (IFN-alpha and -beta) can enhance B cell growth via different mechanisms.     

Altered signal transduction secondary to surface IgM cross-linking on B-chronic lymphocytic leukemia cells. Differential activation of the phosphatidylinositol-specific phospholipase C

To further study the mechanisms by which surface Ig triggering activates the inositol phospholipid signaling pathway, we have used B cells from chronic lymphocytic leukemia patients which, as previously described, display two patterns of response upon sIg cross-linking: in one group this cross-linking induces an inositol phosphate release, an intracellular free Ca2+ concentration elevation and a subsequent cell proliferation; in a second group none of these events occur although there is an increased class II Ag expression following anti-mu stimulation as in the first group. We have been able to demonstrate that the phosphatidyl inositol specific phospholipase C (PI-PLC) can be activated in permeabilized B cells from the first group by direct stimulation, with GPT gamma S, of a guanine nucleotide binding (G) protein. In addition, since anti-mu + GTP gamma S stimulate an increased inositol phosphate production in these cells, this suggests that surface Ig cross-linking activates PI-PLC via a G protein. However, in cells from the second group no inositol phosphate is released after GTP gamma S stimulation although PI-PLC can be directly activated by high Ca2+ concentrations. This reflects in these cells, an interruption of the signaling cascade sIg/G protein/PI-PLC at the level of the G protein or at the G protein/PI-PLC coupling. In cells from both groups PMA treatment, which is known to alter phosphatidyl inositol metabolism in B cells, completely inhibits PI-PLC activation even by high Ca2+ concentrations. These studies show that the phosphatidyl inositol-dependent signaling cascade after surface Ig triggering can be altered at different levels in B cells.     

Human B cell responsiveness to B cell growth factor after activation by phorbol ester and monoclonal anti-mu antibody

The effect of phorbol ester on human B cell activation was examined. Picomolar to nanomolar concentrations of phorbol ester induced a high level of proliferation in small IgM-positive B cells isolated from peripheral blood by fluorescence-activated cell sorting. The addition of optimal doses of anti-mu antibody resulted in enhanced proliferation of phorbol ester-activated B cells. The addition of B cell growth factor (BCGF) to phorbol ester-activated B cells also resulted in a dose-dependent synergistic effect and maximal enhancement on day 3. BCGF activity could be absorbed with either phorbol ester- or anti-mu-activated B cells, but not with resting B cells, thus confirming the induction of functional BCGF receptor expression. Cell proliferation was not necessary for the induction of functional BCGF receptors. Phorbol ester was a more efficient inducer of BCGF receptor expression than was anti-mu antibody; gamma-interferon treatment had no effect. BCGF enhanced transferrin receptor expression by phorbol ester-activated B cells. The results suggest that phorbol ester-activated small B cells can be used to monitor BCGF activity, and this synergistic combination may be useful in establishing BCGF-dependent B cell clones in culture.     

Properties of anti-Lyb-2-mediated B-cell activation and the relationship between Lyb-2 molecules and receptors for B-cell stimulatory factor-1 on murine B lymphocytes

The murine B-cell differentiation antigen Lyb-2 has been shown to be involved in B-lymphocyte activation and has been postulated by some to be related to a receptor for B-cell stimulatory factor I (BSF-1) (H. Yakura et al., J. Immunol. 137, 1475, 1986). Here we have demonstrated that monoclonal antibody (mAB) to Lyb-2 resembles BSF-1 in its ability to activate small resting B cells and enhancement of surface Ia. Anti-Lyb-2 antibodies bound B cells with very high avidity and were able to induce mobilization of cytosolic-free calcium. Anti-Lyb-2 mAB differs from BSF-1 in that BSF-1 but not anti-Lyb-2 is able to synergize with anti-mu in induction of B-cell proliferation. The relation between Lyb-2 molecules and BSF-1 receptors was tested in assays that measure binding of anti-Lyb-2 or BSF-1 in B cells and were found not to compete with each other. It appears that the two B-cell agonists anti-Lyb-2 and BSF-1 may exert their effects on B cells through different cell surface moieties as well as different intracellular pathways.     

Physiology of adaptive immunity regulation via signaling pattern-recognition receptors

In the past fifty years, adaptive immune response has been studied from the standpoint of Burnet’s clonal selection theory. Much progress in understanding the mechanisms of specific cellular (T-cell) and antibody (B-cell) immune response has been made. However, it remained unclear why different pathogen types induce principally different types of immune response. Effective immune response in different cases may develop either by cellular or humoral type, and antibodies are produced on the basis of immunoglobulins of different classes. These facts could only be explained by specific regulation of differentiation of immunocompetent cells during the development of adaptive immune response to different pathogens. The discovery of the system of signaling pattern-recognition receptors (PRRs) in immunocompetent cells made it possible to understand these specific physiological mechanisms of regulation of T- and B-cell response to various pathogens. Upon interaction with pathogens, signaling PRRs activate the synthesis of various cytokines in the cells, which then regulate further activation of cells in different directions. Dendritic cells not only provide naive T cells with a processed antigen but also supply them with various cytokines inducing formation of type 1 or 2 T-helpers; as a result, adaptive immune response develops by the cellular or humoral type, respectively. Antigens of pathogens activate PRRs in B lymphocytes, which initiate the synthesis of various cytokines in cells. These are cytokines that determine predominant production by plasma cells of class A, M, G, or E antibodies depending on the pathogen type.     

Complex formation of CD23/surface immunoglobulin and CD23/CD81/MHC class II on an EBV-transformed human B cell line and inferable role of tetraspanin

CD23, a low-affinity IgE receptor, is a type II transmembrane protein having a C-type lectin domain and it associates noncovalently with MHC class II on B cells. The results of our immunoprecipitation analysis suggest that CD23 co-exists with at least two additional molecules, surface immunoglobulin (sIg) and CD81 (and/or CD9), on the cell surface of L-KT9 cells (an Epstein-Barr virus (EBV)-transformed human B cell line). When both CD23 and sIg molecules were stimulated simultaneously by the corresponding antibodies, a large increase in CD81 in the immunoprecipitation was observed as compared with the case of stimulation by only one antibody. Simultaneous stimulation by anti-CD23 and anti-Ig may mimic the situation of B cells stimulated by an antigen/IgE complex. In addition, a large increase in MHC class II in the immunoprecipitation was also observed by cross-linking of CD23 with anti-CD23 and its second antibody as compared with the case of stimulation by anti-CD23 alone. The cross-linking of CD23 with anti-CD23 and its antibody may mimic the situation of B cells stimulated by an IgE/antigen/IgE complex. Therefore, the complex formation among CD23, sIg, MHC class II, and CD81 on the cell surface of L-KT9 cells by the antigen/IgE or IgE/antigen/IgE complex is most likely to be closely related to B cell regulatory events by signaling through sIg or MHC class II. Tetraspanins such as CD81 and CD9 are thought to be involved in the formation and the preservation of various different membrane complexes consisting of several functional proteins.     

Activation of human B cells: alternate options for initial triggering and effects of nonmitogenic concentrations of anti-IgM antibodies on resting and activated cells

The monoclonal antibody 1F5, which is reactive with the CD20 (Bp 35) pan-B cell antigen, was shown to activate resting human peripheral blood B cells into the middle to late G1 phase of the cell cycle. However, in contrast to F(ab')2 fragments of polyclonal anti-mu, 1F5 synergized only weakly with B cell growth factor (BCGF) for DNA synthesis in these cells. We provide evidence that the CD20 molecule and surface immunoglobulin represent two alternative activation pathways in resting B cells. We also show that anti-immunoglobulins, during co-stimulation with BCGF, may play an important role in G1 as well as for the initial cell triggering. Thus, anti-mu in nonmitogenic concentrations was shown to provoke distinct effects on 1F5-pretreated G1 cells, as monitored by increases in cellular volumes as well as in cytoplasmic Ca2+ levels. Moreover, anti-mu could increase c-myc mRNA levels in 1F5-primed cells, implying that c-myc expression can be regulated in G1 as well as during the initial G0 to G1 transition. Partially purified human BCGF neither induced G1 entry in resting peripheral blood cells nor primed the cells for DNA synthesis. The finding that BCGF did not influence c-myc mRNA levels in resting or in activated B cells suggests that its mitogenic action does not involve the c-myc function.     

Differential expression of a surface antigen recognized by a monoclonal antibody, J11d, on unprimed and primed B cells

Functional studies of both polyclonal and antigen-specific responses have suggested that murine B cells differ in the expression of an antigen recognized by a rat anti-mouse monoclonal antibody, called J11d. Using both positive and negative selection, we now demonstrate that the J11d marker is differentially displayed on B lymphocytes responding to LPS vs anti-mu, as well as on unprimed vs specific antigen-primed B cells. Thus, cytotoxic elimination of cells expressing high levels of J11d (J11d-hi) reduced LPS-driven B cell proliferation by 60 to 80% but had no effect on anti-mu stimulated B cell growth. Interestingly, equal numbers of positively selected J11d-hi B cells responded similarly to LPS and anti-mu plus B cell growth factors, a result that suggests that the response to anti-mu of the J11d-lo B cells is normally masked by the majority J11-d-hi cells. In further studies, the primary PFC response of normal murine spleen cells to fluorescein (FL)-coupled TI antigens or to LPS in vitro was reduced dramatically by cytotoxic J11d antibody treatment. In contrast, the anti-FL PFC response of spleen cells from mice primed 1 wk previously with FL-Ficoll was not affected by J11d antibody treatment, whereas the response of these FL-primed B cells to TNP (to which the mice were not primed) was greatly reduced by J11d + complement treatment. Our data indicate that antigen-experienced (activated) B cells are primarily found in the J11d-lo B cell subset and that unprimed (resting) B cells are found in the J11d-hi population, although both populations of murine B cells can respond to anti-mu. These studies also provide further evidence for B cell heterogeneity.     

Flow cytometric analysis of opposite effects of a monokine on proliferation and differentiation of murine B lymphocytes.

A 35,000 mw factor able to replace macrophages (FRM) in the induction of the in vitro antibody response to sheep erythrocytes has been isolated from the supernatant of murine resident peritoneal macrophage cultures. Purified FRM, when added at the outset of cultures, induced B cells to generate an antigen-specific antibody response. The signals provided by FRM in the process of B cell activation were analyzed using a polyclonal model. Cell cycle analysis by multiparameter flow cytometry after acridine orange staining showed that FRM, on its own, did not trigger the transition of B cells from the G0 to the G1 stage of the cell cycle. In addition, FRM affected neither the basal intracellular free calcium level ([Ca2+]i) nor the rapid increase in [Ca2+]i induced by crosslinking of membrane immunoglobulin (mIgM) with anti-mu antibodies. In parallel with its positive effect on B cell differentiation, FRM markedly reduced both proliferation and cell cycle progression of B cells stimulated with anti-mu plus interleukin 4 (IL-4). Indeed, the addition of FRM to such cultures led to a preferential accumulation of cells in the early G1 compartment of the cell cycle and to a decreased frequency of cells in all other phases including G1B, S and G2/M.