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Human neonates are immunologically immature, particularly in their humoral antibody responses to T cell-independent antigens, as exemplified by their increased susceptibility to infections with polysaccharide-encapsulated bacteria. To clarify the mechanism(s) underlying the unresponsiveness of neonates to polysaccharide antigens, we used an in vitro model with neonatal cord blood cells that has been shown to mimic surface Ig-dependent signaling in the adult by T cell-independent antigens. We studied the ability of cord blood human B cells to become activated after ligation of their surface Ig by unconjugated anti-Ig, dextran-conjugated anti-Ig, and Staphylococcus aureus Cowan A1, and compared their response with that of adult B cells. After the addition of nanogram concentrations of anti-Ig-dextran, neonatal cord blood B cells proliferated at levels comparable to that observed with adult B cells. The majority of cord blood B cells showed a marked rise in intracellular calcium, increased surface expression of human leukocyte antigen DR, and an increase in cell size. Direct activation of protein kinase C by phorbol esters in neonatal B cells led to cellular proliferation, and when combined with anti-Ig, a synergistic effect on proliferation was observed. These data suggest that the unresponsiveness of human neonates to polysaccharide antigens does not represent an inability of these antigens to induce early activation events in circulating B cells.

Stimulation of B lymphocytes through the Ig receptor initiates a cascade of biochemical changes, which can ultimately lead to either activation and growth, or cell-cycle arrest and cell death. One of the critical events that occurs in both cases is the activation of tyrosine kinases, and the resulting phosphorylation of a variety of proteins on tyrosine residues. In this report we identify one of the substrates of phosphorylation as the 85-kD subunit of the enzyme phosphatidylinositol-3 kinase (PI3K), and show that both anti-IgM and anti-IgD stimulation results in an increase in the anti-phosphotyrosine-precipitable PI3K activity. Furthermore, we show that the potent and specific inhibitor of PI3K, Wortmannin, can completely abrogate anti-Ig-mediated growth inhibition without affecting tyrosine kinase induction or protein kinase C (PKC) activation. Treatment of intact cells with Wortmannin results in an irreversible decrease in anti-Ig-induced PI3K activity, suggesting that the effect of Wortmannin on anti-Ig-mediated growth inhibition is caused by its inactivation of PI3K activity. Taken together, these data show that activation of PI3K is a critical component of the anti-Ig-initiated signaling cascade that leads to growth inhibition of human B lymphoma cells.

Surface immunoglobulin (sIg) cross-linking on B lymphocytes by high concentrations of anti-Ig antibody has been used to mimic antigen-stimulated B cell activation. In order to develop a system to study sIg-mediated T cell-independent B cell activation using low concentrations of anti-Ig antibody that more closely resemble the concentrations of antigen that are achieved under in vivo conditions, we conjugated monoclonal anti-human IgM antibody (anti-mu) to dextran (molecular weight 2 X 10(6)) thereby increasing its valency. This dextran conjugate (anti-mu-Dex) stimulated comparable levels of thymidine incorporation and B cell size increases as were seen with unconjugated anti-mu but at 100- to 1000-fold lower concentrations. Anti-mu-Dex also stimulated increases in intracellular ionized calcium ([Ca2+]i) in a higher percentage of cells, of greater magnitude and of longer duration than that stimulated by unconjugated anti-mu. Interestingly, there was no direct correlation between the increases in [Ca2+]i that were stimulated by anti-mu-Dex and its ability to stimulate B cell proliferation. The concentrations of anti-mu-Dex (10 micrograms/ml) that led to the highest increase in [Ca2+]i resulted in thymidine incorporation that was no greater than that of medium control, whereas 0.01 to 0.1 microgram/ml stimulated significant thymidine incorporation with 50% lower levels of stimulation of [Ca2+]i. These data demonstrate that anti-mu-Dex is a potent activator of human B lymphocytes, is effective even at ng/ml concentrations which over a 2-h time period do not induce detectable modulation of sIg, and its stimulation of B cells into G1 and S may not be directly related to its ability to stimulate increases in levels of [Ca2+]i.

The possibility that injection of mice with an affinity-purified goat antibody to mouse IgD (GaM delta) that stimulates polyclonal IgG1 secretion might also stimulate differentiation of B cells into IgE-secreting cells was suggested by the observation that such treatment induces T cells from those mice to secrete a lymphokine, B cell stimulatory factor 1 (BSF-1), that can stimulate both IgG1 and IgE secretion in vitro. Studies described in this paper show that injection of BALB/c mice with 200 to 3200 micrograms of GaM delta greatly increased the quantity of splenic epsilon chain-encoding mRNA, the number of spleen cells with cytoplasmic IgE, and the concentration of serum IgE 7 days after injection. Serum IgE levels obtained in these mice were approximately 100 times baseline levels and were comparable with those found in mice infected with the nematode parasite Nippostrongylus brasiliensis, but were approximately 2000-fold less than the peak serum IgG1 levels induced by GaM delta injection. Both IgE and IgG1 secretion in GaM delta injected mice were T dependent (blocked by anti-L3T4 antibody). These observations are consistent with the hypothesis that BSF-1 may play a role in the in vivo stimulation of IgE secretion and provide an easy to apply model for the investigation of in vivo regulation of IgE responses.

The lymphokine B-cell stimulatory factor 1 (BSF-1) has been shown to greatly enhance the differentiation of lipopolysaccharide-activated B cells into IgG1- and IgE-secreting cells in vitro. To determine whether in vivo IgG1 and IgE antibody responses are BSF-1 dependent, the ability of a monoclonal rat IgG1 anti-BSF-1 antibody, 11B11, to affect polyclonal IgG1 and IgE production in mice infected with the nematode parasite Nippostrongylus brasiliensis or injected with a purified goat antibody to mouse IgD was studied. 11B11-containing ascites fluid or purified 11B11 strongly inhibited IgE production in both systems but did not affect IgG1 production, while control ascites or normal rat IgG1 had no IgE-inhibitory activity. These results indicate an important physiologic role for BSF-1 in the generation of IgE antibody responses and suggest means for limiting the production of antibodies responsible for allergic reactions without inhibiting protective antibody responses.

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.

Counterflow centrifugal elutriation (CCE), a technique which separates cells by size and density, was used to separate human peripheral blood mononuclear cells into fractions enriched for T lymphocytes, B lymphocytes, and monocytes. These morphologically and phenotypically distinct fractions were analyzed for their ability to respond in several functional assays. B-Cell-enriched fractions devoid of monocytes did not proliferate nor produce significant quantities of lymphokines in response to antigens. These B cells did proliferate to anti-IgM antibodies but not to anti-IgD antibodies. B-Cell fractions served as stimulators of the autologous mixed-lymphocyte reaction (AMLR). T-Lymphocyte fractions were unable to respond to antigen challenge, but both proliferation and lymphokine production could be restored by the addition of monocytes. Monocyte fractions produced PGE2, displayed chemotaxis, and functioned as stimulators in the AMLR. Thus, CCE appears to be a useful technique for reproducibly obtaining highly enriched subsets of human peripheral blood mononuclear cells with unique phenotypic and functional properties. These isolated populations can consequently be used to identify the independent and collaborative roles of the cells in immunological events.

Recently, it has been reported that in SJA/9 mice infected with Nippostrongylus brasiliensis there are increased numbers of lymphoid cells positive for surface IgM and IgE (sIgM+ and sIgE+) even though they fail to secrete IgE, that both the sIgM and sIgE on these cells are intrinsic, and that there has been no deletion of genes for the Ig heavy chain constant region in these cells. These observations support a nondeletional model for Ig isotype switching. We have now reexamined the nature of sIgE on sIgE+ spleen and mesenteric lymph node cells of N. brasiliensis-infected SJA/9 mice, and the following observations lead us to believe that this sIgE is cytophilic rather than intrinsic: (i) Only approximately 50% of the N. brasiliensis-infected SJA/9 mice have detectable percentages of sIgE+ lymphoid cells. All mice with detectable sIgE+ lymphocytes have lymphocytes positive for intracytoplasmic IgE (cIgE+) and secrete IgE in vitro, while cIgE+ cells and IgE secretion are absent from N. brasiliensis-infected SJA/9 mice that lack sIgE+ cells. (ii) SJA/9 B lymphocytes have receptors for IgE: expression of these receptors is increased in N. brasiliensis-infected mice that have sIgE+ lymphocytes, but not in infected SJA/9 mice that lack sIgE+ lymphocytes. (iii) Treatment of sIgM+ sIgD+ sIgE+ cells for 1 min with dilute acid removes most sIgE but does not affect expression of sIgM or sIgD. (iv) The removal of mouse IgE from sIgE+ B cells facilitates the binding of exogenous rat IgE. (v) The small amount of sIgE that is reexpressed during a period of in vitro culture after acid treatment is blocked by inclusion of exogenous rat IgE in the culture medium. These observations show that most sIgM+ sIgE+ B cells in N. brasiliensis-infected SJA/9 mice do not express intrinsic sIgE; thus studies using these cells to determine mechanisms of Ig isotype switching are inconclusive.

Rapid separation of large numbers of human peripheral blood mononuclear cells into fractions enriched for B lymphocytes, T lymphocytes, or monocytes was accomplished by counterflow centrifugal elutriation (CCE). The first fraction contained 98% of the platelets. Ten additional fractions containing subpopulations of mononuclear cells were collected by sequential increases in the flow rate while maintaining a constant centrifuge speed. Analysis of the fractions using monoclonal antibodies revealed that fraction 2, which was free of esterase-positive monocytes, was highly enriched for B cells. T lymphocytes (OKT3+) were the predominant cell type found in fraction 4. No enrichment for T-lymphocyte-helper (OKT4+) or -suppressor (OKT8+) subpopulations was observed in the lymphocyte containing fractions. Three fractions (7-9), highly enriched for esterase-positive cells, were predominantly OKM1+ monocytes with no evidence of selective separation of monocyte subpopulations. Thus, cell fractions enriched for B cells, T cells, and monocytes could be obtained, by utilizing CCE, in large enough quantities to enable analysis of their functional properties. Of particular interest was the ability to separate small, resting B lymphocytes from monocytes.

In order to compare the actions of interferons on the various pathways leading to human B-cell activation, we examined the effects of alpha-interferon (INF-alpha), beta-interferon (INF-beta), and gamma-interferon (INF-gamma) on B-cell responses to mitogenic stimuli which differ in their mode of B-cell stimulation. Utilizing highly purified small, dense peripheral blood B-cells or splenic lymphocytes, we demonstrate that (i) INF-alpha, -beta, and -gamma enhance human B-cell proliferation induced by cross-linking of surface Ig with either SAC or anti-mu in a dose-dependent fashion, analogous to and with a magnitude equal to or greater than that seen with human B-cell growth factor; (ii) INF-alpha and -beta but not IFN-gamma inhibit phorbol myristate acetate-mediated B-cell mitogenesis, again in a dose-dependent manner; and (iii) IFN-gamma does not effect B-cell cytoplasmic calcium ([Ca2+]i) influx, either in the resting state or following stimulation with anti-mu, making it unlikely that IFN-gamma exerts its stimulatory effects on B-cell function through changes in [Ca2+]i. Taken together, these findings suggest that all three types of interferons may have important immunoregulatory roles in B-cell activity, and that their ability to enhance or suppress B-cell activation depends on the nature of the mitogenic stimulus.