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.     

The effects of interleukin 1 on human B cell activation and proliferation

The precise role of B cell surface immunoglobulin (slg) in the activation of B cells is unclear at present. In particular, it is uncertain whether ligands interacting with the B cell slg suffice to induce proliferation, or simply induce a state of activation in which the B cell becomes responsive to growth factors made by accessory cells. We have examined the effects of two ligands, Staphylococcus aureus Cowan strain I (SAC) and antihuman mu chain (anti-mu), which interact with B cell slg on highly purified human peripheral blood and tonsillar B cells cultured at low cell concentrations. The effects on B cell proliferation of these ligands alone or in combination with highly purified interleukin 1 (IL 1) or a supernatant of a human T-T hybridoma containing a B cell growth factor (BCGF) were studied. SAC with its high cell wall content of protein A triggered maximal B cell proliferation which was not increased further by IL 1 or BCGF. High concentrations of soluble F(ab')2 fragments of goat anti-mu chain also induced significant B cell proliferation. Lower concentrations of anti-mu resulted in little or no B cell proliferation but activated the B cell to a state of responsiveness to both IL 1 and BCGF. IL 1 by itself had no effect on the proliferation of unstimulated B cells or on the proliferation of in vivo-activated B cells which responded to BCGF in vitro, but demonstrated clear synergy with low concentrations of anti-mu antibody. BCGF alone augmented the proliferation of unstimulated B cells, presumably by acting on B cells which had undergone some degree of activation in vivo. In addition, it showed marked synergy with anti-mu antibody, which resulted in proliferation similar in magnitude to that induced by SAC. This synergy was far greater than that seen between anti-mu antibody and IL 1, and the resulting proliferative response was only slightly increased by the presence of IL 1. We conclude that the importance of accessory cell factors for the initial rounds of B cell proliferation depends on the strength of the initial slg-mediated activation signal. When this is strong, the response is maximal and independent of accessory cells or accessory cell factors. When it is suboptimal, a moderate synergy is seen with IL 1 and a dramatic synergy with BCGF.     

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.     

Role of adherent cells in dextran sulfate-mediated enhancement of mitogen-induced B-cell proliferation

We have studied lipopolysaccharide (LPS)-induced proliferation in the rat and have found that the addition of the compound Dextran sulfate (DxS), which itself is not mitogenic, to LPS stimulated cultures results in significant enhancement of cell division. A "DxS-free" supernatant from DxS stimulated spleen cell cultures is able to substitute for DxS in stimulatory activity. This supernatant possesses interleukin 1 (IL-1) activity, however, the addition of purified recombinant IL-1 to LPS-stimulated cultures does not result in augmentation of proliferation. A DxS-free supernatant from DxS stimulated adherent cells is also able to substitute for DxS in stimulatory activity. The active molecule(s) present in the adherent cell-derived DxS-free culture supernatant appears to be distinct from classical IL-1.     

Control of B cell proliferation: arrest of B cells in late G1 underlies immunosuppression induced by plasma cell tumors

Immunosuppression in mice bearing plasma cell tumors (PC-mice) provides a model system for the study of negative B cell regulation. Our previous studies demonstrated that B cell proliferation is suppressed in these mice by a cascade of interactions involving macrophages and soluble factors. The present report pinpoints the G1 phase of the cell cycle as the stage of B cell proliferation inhibited in PC-mice. Modulation of surface immunoglobulin (sIg) with anti-mu, an early membrane activation event, occurred normally on B cells from the spleens of PC-mice. However, examination of the size profile and the expression of sIgD and sIgM on B cells from the spleens of PC-mice showed an accumulation of large-sized, low intensity sIgD+ cells, suggesting a block in B cell activation in the late G1 phase of the cell cycle. This was confirmed by experiments in vitro that demonstrated that although LPS-stimulated B cells from the spleens of PC-mice enlarged to a size characteristic of G1 phase, most did not additionally enlarge into S phase even after 3 days of culture, nor did they incorporate significant amounts of [3H]thymidine. Additional confirmation of a block in late G1 was obtained by using analysis of [3H]thymidine incorporation, cell size, and cell cycle after normal cells were cultured in supernatants from cloned PC lines containing the factor(s) that initiates the cascade of events leading to suppression of B cell proliferation. The relevance of these findings to PC-induced immunosuppression and to the regulation of normal B cell proliferation during the G1 phase of the cell cycle is discussed.     

Surface IgM mediated regulation of RAG gene expression in E mu-N-myc B cell lines.

Transgenic mice carrying either the c-myc or N-myc oncogene deregulated by the immunoglobulin heavy chain enhancer element (E mu) develop both pre-B and B cell lymphomas (E mu-c-myc and E mu-N-myc lymphomas). We report here that B cell lines derived from these tumors, as well as a line derived from v-myc retroviral transformation, simultaneously express surface immunoglobulin (a hallmark of mature B cells) as well as a common subset of genes normally restricted to the pre-B stage of development-including the recombinase activating genes RAG-1 and RAG-2. Continued RAG-1 and RAG-2 expression in these lines is associated with VDJ recombinase activity detected with a VDJ recombination substrate. Cross-linking of the surface immunoglobulin on these lines with an anti-mu antibody leads to rapid, specific and reversible down-regulation of RAG-1 and RAG-2 gene expression. We also find that a small but significant percentage of normal surface immunoglobulin bearing bone marrow B cells express the RAG-1 gene. These findings are discussed in the context of their possible implications for the control of specific gene expression during the pre-B to B cell transition.     

Effects of diacetyl diamines on in vitro activation and proliferation of human B lymphocytes

N,N'-Diacetylputrescine (tetramethylenebisacetamide [TMBA]) and its six carbon analog, hexamethylenebisacetamide (HMBA), inhibited the proliferative response of human B lymphocytes to anti-mu and formalinized Cowan I strain Staphylococcal aureus (SAC) stimulation. In contrast, B cell growth factor-stimulated proliferation of human B cells was minimally inhibited by TMBA or HMBA. The antiproliferative effect of these diamine derivatives was specific for anti-mu (or SAC) activation of normal B cells, because the proliferation of PHA-stimulated human T cells and transformed human B cells was not affected by the presence of TMBA or HMBA. The inhibitory effect of diacetyl diamines on anti-mu (or SAC)-induced B cell activation was dose dependent and persisted after removal of the diamine derivatives from the culture media. These studies show that diacetylated derivatives of polyamines modulate human B cell activation in vitro by specific abrogation of anti-mu or SAC activation.     

Polyclonal activation of primed rat B cells

In recent years, murine and human virgin B lymphocytes have been used to examine the steps necessary for polyclonal activation. In these models mitogens are used in conjunction with lymphokines to determine which signals are responsible for regulating B-cell triggering, proliferation, and differentiation. While progress has been made in understanding these events as they occur in virgin B cells, very little evidence exists to suggest whether these models of activation also apply to the memory B-cell population. In this report we have described an antigen-specific, secondary in vitro immune response using cells isolated from lymph nodes draining the site of antigen injection. Unfractionated cells, B cells, and size-fractionated cells from dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH)-primed rats were challenged in vitro with DNP-KLH, lipopolysaccharide plus dextran sulfate (LPS/DxS), and T-cell factors. We have consistently found, under all these conditions, that antigen challenge of primed cells results in the production of DNP-specific IgG antibody while stimulation with LPS/DxS plus T-cell factors results only in the polyclonal activation of virgin B cells; no antigen-specific IgG secretion is seen. This suggests that acquisition of memory status is associated with a loss in responsiveness to LPS/DxS-induced differentiation.     

Cell cycle analysis of lymphocyte activation in normal and autoimmune strains of mice

The spontaneous spleen cell proliferation and the proliferation induced by in vivo or in vitro stimulation with such polyclonal B cell activators (PBA) as LPS, poly rI.rC, and anti-mu were studied in normal and autoimmune mice. The various murine models of autoimmunity differ in the level of naturally occurring splenic cellular hyperactivity as well as in the ability of their spleen cells to be further stimulated in vitro by polyclonal stimulators. Both the NZB strain and the MRL/Ipr strain had markedly increased numbers and percentages of spontaneously proliferating spleen cells, whereas the BXSB strain did not. Nonautoimmune strains were found to have very small numbers of activated cells in the spleen. However, such normal strains could be induced in vivo to mimic the natural splenic hyperactivity observed in older NZB and MRL/Ipr autoimmune strains by the injection of polyclonal B lymphocyte stimulators. In contrast, old hyperactive NZB mice were not further induced to undergo proliferation by in vivo administration of such stimulators. Density-separated, T depleted, spleen cells of normal and autoimmune mice were stimulated in vitro with PBA in 48-hr cultures. Cells from old MRL/Ipr and NZB mice were abnormal in both the anti-mu response and the LPS response; BXSB mice had normal anti-mu responses. These studies suggest that there is no prerequisite for spontaneous splenic hyperactivity in the development of autoimmunity. In addition, different PBA stimulate separate subsets of B cells that differ in their state of activation in the various autoimmune strains. Finally, different B cell subsets appear to be abnormal in different types of autoimmune mice.     

Comparative activation requirements of human peripheral blood, spleen, and lymph node B cells

Human peripheral blood, spleen, and lymph node B cells were stimulated with Cowan I Staphylococcus aureus (SA) or F(ab')2 fragments of anti-mu antibody (anti-mu) and various lymphokines and were analyzed for proliferation and generation of Ig-secreting cells (ISC). SA alone but not anti-mu stimulated minimal proliferation of each population. Recombinant IL 2 (r-IL 2) effectively promoted proliferation of SA-stimulated blood and spleen B cells, but supported less vigorous responses of lymph node B cells. By contrast, r-IL 2 enhanced DNA synthesis of all anti-mu-stimulated B cells early in culture, but did not promote sustained proliferation of anti-mu-stimulated lymph node B cells and only promoted ongoing DNA synthesis of some anti-mu-activated blood (eight out of 17) and spleen (five out of 14) B cell preparations. Recombinant interferon-gamma (r-IFN-gamma) and a commercial preparation of B cell growth factor (BCGF) also augmented DNA synthesis of all three B cell populations stimulated with SA or anti-mu early in culture, but neither alone was able to sustain maximal proliferation. Markedly enhanced sustained proliferation of all three anti-mu- and SA-stimulated B cell populations was noted when cultures were supported by the combination of r-IL 2 and BCGF, or to a lesser extent by r-IL 2 and r-IFN-gamma. The generation of ISC from SA-stimulated blood or spleen but not lymph node B cells was effectively supported by r-IL 2 alone. Differentiation of lymph node B cells required the combination of r-IL 2 and BCGF. These studies emphasize the importance of both the activation stimulus and the origin of the B cells in determining the lymphokine requirements of human B cell responsiveness.     

Selective suppressive effects of glucocorticoids on the early events in the human B cell activation process

The effect of hydrocortisone on the induction of human B cell activation and proliferation has been described. Hydrocortisone prevents the anti-mu-induced cell enlargement of small tonsillar B cells, blocks expression of the activation markers 4F2 and 5E9 induced by anti-mu, inhibits RNA synthesis of small B cells stimulated by anti-mu with or without BCGF, and suppresses B cell proliferation in response to anti-mu and BCGF or to Staphylococcus aureus Cowan I. In contrast, hydrocortisone does not affect the proliferative response of in vitro or in vivo preactivated B cells. Therefore, hydrocortisone has a selective inhibitory effect on early events in the human B cell cycle that subsequently leads to inhibition of total RNA and DNA synthesis. Possible mechanisms of this action are discussed. These studies further define the nature of glucocorticoid-induced modulation of human B cell activation and proliferation.     

Dextran sulfate-mediated enhancement of mitogen-induced B-cell proliferation

We have studied lipopolysaccharide (LPS)-induced proliferation of rat lymphocytes and have found that a serum-free tissue culture medium supports significant cell division. When the compound dextran sulfate (DxS), which itself is not mitogenic, is added to LPS-stimulated cultures, significant augmentation of proliferation results. DxS will significantly enhance anti-immunoglobulin-induced proliferation as well. An intact T-cell compartment is not required for DxS-mediated enhancement of LPS-induced cell division, in that lymphocytes derived from athymic rats proliferate and respond to the influence of DxS to the same degree as euthymic derived lymphocytes. A "DxS-free" supernatant from DxS-stimulated spleen cell cultures is able to substitute for DxS in all stimulatory activity. This supernatant possesses interleukin 1 (IL-1) activity. However, purified recombinant IL-1 does not cause enhanced proliferation when added to LPS-stimulated cultures as is seen with DxS or the "DxS-free" supernatant.     

Induction of CD40 expression and enhancement of monoclonal antibody production on murine B cell hybridomas by cross-linking of IgG receptors

The 55-6 murine B cell hybridoma line not constitutively expressing CD40 was treated with increasing amounts of intact anti-mouse surface immunoglobulin G antibody (anti-mIgG) either not preincubated or preincubated for 48 h with lipopolysaccharide (LPS). In vitro, cross-linking of surface immunoglobulin G (sIgG) with the whole molecule of anti-IgG antibodies induced the expression of CD69, CD40, and CD19 surface antigens on 55-6 cells. The effect of sIgG ligation was dose-dependent, and preincubation with LPS enhanced their responsiveness to anti-mIgG stimulation. The expression of these surface molecules reached the maximum value during the first part of the cell cycle, corresponding to the position of the G1 peak of the DNA distribution. Stimulation of cells with anti-mIgG did not induce changes either in the number of viable cells or in the fraction of cells undergoing proliferation (mitosis). However, preincubation of 55-6 cells with LPS for 48 h before stimulation with anti-mIgG increased both the maximum specific growth rate (micromax) and the percentage of cells in the G2/M phase, in comparison with non-preincubated cells. Moreover, on cells preincubated with LPS prior to anti-mIgG treatment, specific IgG2a production rate was enhanced significantly compared to that obtained in control cultures. The correlation between the antibody production rate and the amount of IgG that is detectable on the cell surface was analyzed by flow cytometry. A good correlation between secreted and surface IgG was observed, and the results of cell cycle analyses demonstrated that the 55-6 hybridoma cell line has a substantially higher sIgG content in G1 phase.     

Activation of human B cells and inhibition of their terminal differentiation by monoclonal anti-mu antibodies

Anti-mu antibody preparations have been found to exert both positive and negative effects on B cell activation and differentiation. To explore these paradoxical influences of IgM cross-linkage on human B cells, three gamma 1 kappa murine monoclonal antibodies specific for human mu-chains (DA4.4, AB6.4, 145.8) were examined for their comparative effects on activation of B cells and inhibition of terminal plasma cell differentiation. All three antibodies appeared equally efficient in immunoprecipitation of surface IgM molecules; however, fluorescence-activated cell sorter analysis revealed that the DA4.4 and AB6.4 antibodies saturated the B cell surface IgM at slightly lower concentrations than did the 145.8 antibody. When the affinity-purified antibodies were added in varying concentrations to cultures of small resting B cells, all three antibodies induced B cell enlargement and DNA synthesis, but with varying degrees of efficiency (DA4.4 greater than AB6.4 much greater than 145.8). In striking contrast, large B cells isolated either by FACS or density gradient separation were unresponsive. The anti-mu-induced proliferative response of small B cells required relatively high B cell densities, but not T cells or the Fc portion of the antibody molecules. The maximal proliferative response was obtained during the third day of culture, and the response curve suggested that anti-mu induced only one round of B cell replication. All three antibodies were capable of completely inhibiting T cell factor-induced differentiation of large B cells into IgM plasma cells; both F(ab')2 fragments and intact anti-mu antibodies were effective in final concentrations as low as 1 microgram/ml. Significant suppression of IgG and IgA plasma cell differentiation was also achieved, but required higher concentrations of the anti-mu antibodies. For each antibody, there was a close correlation between the efficiency of inducing small B cell proliferation and of inhibiting large B cell differentiation into plasma cells. The results show that the B cell response to cross-linkage of cell surface IgM varies according to the differentiation stage. We postulate that the mature resting B cell represents the only stage in the life history of the B cell during which surface Ig cross-linkage leads to a positive signal, negative signals being the rule at other stages in B cell replication and differentiation.