Interleukin 4 acts on both high- and low-density murine B cell subpopulations to induce IgE and IgG1 synthesis in vitro

Murine B cells were activated for 24 h with either lipopolysaccharide (LPS) or polyribosyl-ribitol-phosphate (PRP), followed by addition of interleukin 4 (IL-4) and the immunoglobulin isotypes secreted into the supernatant quantitated. Without IL-4, both LPS and PRP induced mainly IgM and IgG3 and no IgE secretion. Addition of IL-4 to both LPS- and PRP-activated cells decreased the IgM and IgG3 secretion and induced a large IgG1 production. In contrast to IgG1, only LPS-activated cells secreted large amounts of IgE, demonstrating that the nature of the polyclonal B cell activator also plays an important role in the IL-4 induced IgE formation. The effect of LPS and IL-4 on high- and low-density sIgM+/sIgD+ cells was also investigated. LPS and IL-4 induced IgG1 and IgE secretion by both populations. Low-density B cells from mice infected with the parasite Nippostrongylus brasiliensis formed more IgE than low-density B cells from normal mice, presumably because these mice have more in vivo preactivated B cells committed to IgE formation. The data show that IL-4 can act on both small high-density resting B cells as well as on in vivo preactivated low-density B cells to induce IgG1 and IgE secretion.     

Suppression of IgE responses by hyperimmune serum in rats.

The capacity of various bacterial components to induce antibody formation in human lymphocyte cultures was studied in the present investigation. Antibody levels were determined by an enzyme-linked immunosorbent assay (ELISA). Lipopolysaccharide (LPS), bacterial cell walls (CW, isolated from Bacillus subtilis and Staphylococcus aureus Wood 46) and peptidoglycans (PG) appeared to stimulate IgM, IgG and IgA secretion, whereas lysozyme-solubilized PG and teichoic acids (TA) were ineffective. Also, umbilical cord blood lymphocytes produced IgM after stimulation with LPS, CW and PG. Coculture experiments with purified lymphocytes and monocytes indicated that B-cell differentiation was dependent on both T cells and monocytes, and that T-cell derived factors could derived factors could partially substitute for T cells.     

Pokeweed mitogen-induced immunoglobulin secretory responses of thymic B cells in myasthenia gravis: selective secretion of IgG versus IgM cannot be explained by helper functions of thymic T cells

The thymus, with its striking B cell infiltrates, is widely regarded as an important element in the pathogenesis of myasthenia gravis (MG) but its role remains to be elucidated. To gain further insight into the functional properties of MG thymic B cells, we studied the heavy chain isotype of immunoglobulin they produced in vitro in response to the T cell-dependent polyclonal activator pokeweed mitogen (PWM). MG thymic cells secreted prominent amounts of IgG but little IgM. In contrast, peripheral blood mononuclear cells (PBM) of the same subjects secreted similar amounts of IgG and IgM as did PBM of control subjects. In cell admixture experiments, MG thymic T cells, like PBM T cells, helped autologous PBM B cells produce IgM as well as IgG, although the overall magnitude of help for both isotypes appeared less than that of PBM T cells. Thus, in response to PWM, MG thymic B cells are largely committed to an IgG response and this likely reflects the intrinsic properties of these cells rather than the immunoregulatory properties of thymic T cells. This IgG isotype switch likely reflects in vivo activation events.     

Human interleukin-5 induces staphylococcal A Cowan 1 strain-activated human B cells to secrete IgM

Studies on the role of human interleukin (IL)-5 in B cell growth and differentiation have yielded conflicting results. To clarify this issue, we studied the role of purified recombinant IL-5 on activated human B cells which were depleted of Tcells and adherent cells. Human IL-5 augments IgM secretion, but not IgG or IgA secretion of purified human B cells activated with staphylococcal A Cowan 1 strain (SAC). However, the period of B cell activation with SAC is critical for the B cell to respond to IL-5. After 24 h of SAC activation, human B cells are responsive to the IL-5 signal, but with longer periods of activation, IL-5 responsiveness diminishes. This may explain some of the previous conflicting results. The IgM enhancement was not seen when B cells were activated with pokeweed mitogen. In addition, human recombinant IL-4 synergized with IL-5 in augmenting IgM secretion by SAC-activated B cells, while IL-5 synergized with IL-2 to augment IgM, IgG and IgA secretion by SAC-activated B cells. As the purified IL-5 was derived from a COS-1 cell supernatant, and COS-1 cells secrete IL-6, we examined whether a polyclonal IL-6 antibody blocked the IgM-enhancing activity of IL-5. IL-6 antibody did not block the IL-5 enhancement of IgM secretion, but a monoclonal antibody to IL-5 inhibited the human IL-5 activity on human B cells. These results demonstrate that human IL-5 augments IgM secretion of SAC-activated human B-cells. In addition, this lymphokine synergizes with IL-4 and IL-2 in supporting Ig secretion.     

Antibody Secretion and DNA Synthesis Induced by Lipopolysaccharide in Enriched Human Lymphocyte Subpopulations

Lipopolysaccharide (LPS)-induced human lymphocyte activation was analysed with regard to DNA synthesis and antibody secretion. Antibody secretion was measured as plaque-forming cells (PFC) against fluorescein isothiocyanate (FITC)-coupled sheep erythrocytes (SRBC) or protein-A-coupled SRBC in the presence of developing antiserum against human IgG, IgA and IgM subclasses. By co-culturing lymphocytes with various concentrations of mitomycin, without impairing cell viability, inhibition of DNA synthesis and antibody secretion by LPS was correlated. Antibody secretion against FITC-SRBC or protein A-SRBC was not stimulated by LPS in B cells enriched by elimination of SRBC rosette-forming cells (E-RFC). In mixtures of enriched T and B cells the number of PFC was much lower than in unseparated lymphocytes, but the highest number of PFC was seen in the enriched E-RFC. However, DNA synthesis by LPS was induced in enriched B cells and not in enriched T cells. The highest DNA synthesis was seen for unseparated lymphocytes. Furthermore, autoradiography studies and experiments in which activated lymphocytes were separated revealed that LPS predominantly stimulated DNA synthesis in non-E-RFC, presumably B cells and only activated a small proportion of E-RFC. Finally, antibody secretion induced by LPS was unchanged following iron treatment but was inhibited by cells adherent to plastic.     

The construction and use of a human-mouse myeloma analogue suitable for the routine production of hybridomas secreting human monoclonal antibodies

A human-mouse myeloma analogue termed HMMA2.11TG/O was constructed by fusion of the mouse myeloma cell line P3x63Ag8.653, a mutant derivative of MOPC21, with bone marrow mononuclear cells from a patients with IgA myeloma. The HMMA2.11TG/O cell line is resistant to 6-thioguanine and ouabain and sensitive to HAT. The cell line secretes no detectable immunoglobulin and has a hybrid karyotype and cell surface phenotype. An average fusion efficiency for growth of hybridomas of 1/17,000 fused cells was obtained in fusions with human peripheral blood mononuclear cells (PBM), Pokeweed Mitogen (PWM) stimulated PBM, and Epstein-Barr Virus (EBV) transformed polyclonal B cell lines. Over 75% of hybrids secrete detectable immunoglobulin and the cloning efficiency of the hybrids at 1 cell/well averages 25%. Antibody secreting cloned hybridoma cell lines were obtained by fusion directly with PBM from an immunized volunteer and by fusion with in vitro, secondarily immunized, EBV transformed polyclonal cell lines. Five hybridomas secreting human monoclonal IgM anti-tetanus antibodies and 2 secreting human monoclonal IgG anti-tetanus antibodies were selected and cloned from 6 fusions performed specifically for anti-tetanus antibody. Immunoglobulin and antibody secretion by cloned hybrids has been stable for 5-10 months at present. Immunoglobulin and antibody secretion in routine cultures passaged every 3-4 days has been 8-42 micrograms/ml. This human-mouse myeloma analogue should prove useful for the routine production of human monoclonal antibodies.     

Characteristics of bacterial lipopolysaccharide induction of interleukin 1 synthesis and secretion by human monocytes.

The objective of these studies was to characterize some aspects of interleukin 1 (IL-1) synthesis and secretion by human monocytes after stimulation with bacterial lipopolysaccharides (LPS). Various molecular species of LPS were incubated with adherent monocytes for 24 h. IL-1 activity in monocyte supernatants (secretion) and lysates (synthesis) was determined by stimulation of collagenase production in rabbit articular chondrocytes and augmentation of mitogen-induced proliferation of murine thymocytes. The presence of cytochalasin B enhanced LPS-induced IL-1 secretion without altering IL-1 synthesis. Monocytes preincubated in dexamethasone or hydrocortisone failed to exhibit any IL-1 activity in supernatants after LPS stimulation but the cell lysates still possessed 50% of control IL-1 activity. Studies with different LPS preparations indicated that the presence of diphosphoryl groups in lipid A enhanced the IL-1-inducing activities. Butanol-extracted LPS preparations, containing associated proteins, were not completely inhibited by 5 micrograms/ml polymyxin B in induction of IL-1 production at LPS concentrations of 10 or 100 ng/ml. These results indicate that the failure of polymyxin B to inhibit stimulation of IL-1 production by tests materials cannot be assumed to mean an absence of contaminating LPS.     

In vitro immunoglobulin production by mononuclear cells in rheumatoid arthritis

Since patients with rheumatoid arthritis (RA) exhibit serum hypergammaglobulinemia and autoantibody (rheumatoid factor) production, we compared elaboration and control of in vitro RA mononuclear cell (MNC), Ig assayed by enzyme-linked immunoassays or by hemolytic plaque formation, in 37 RA patients and 17 normal subjects. We found (1) RA spontaneous plaque-forming cells were significantly reduced (RA 344 vs normal 627 PFC/10(6) MNC, P less than 0.002); (2) RA spontaneous IgG and IgM (but not IgA) elaboration was significantly diminished (IgG RA 339, normal 776; IgM RA 255, normal 869 ng/ml, P less than 0.001; IgA RA 87, normal 124); (3) RA stimulated IgG and IgM production (but not IgA) was also decreased (IgG RA 2434, normal 3862, P less than 0.06; IgM RA, 1676, normal 3323, P less than 0.005; IgA RA 1859, normal 2315); (4) reduced RA Ig elaboration was not clearly due to altered numbers of T or non-T cells, age, medications, clinical features of disease, or response kinetics; (5) relative improvement of RA in vitro IgG, but not usually IgM, secretion followed removal of adherent cells, addition of indomethacin or addition of mitomycin C-treated T cells; (6) MNC from synovial fluids, but not bone marrows, exhibited spontaneous Ig production in excess of stimulated synovial fluid cellular or peripheral blood Ig elaboration. These observations indicate selective impairment of peripheral blood MNC IgG and, particularly, IgM secretion in RA. This defect appears to reflect accessory cell influences which differ from normal as well as the sequestration of primed or activated cells in the synovial fluid.     

Expression and regulation of human alveolar macrophage-derived interleukin-1 receptor antagonist.

The alveolar macrophage (AM) is the sentinel immune cell of the distal airspace of the lung. These mononuclear phagocytic cells represent the major host defense against inhaled environmental agents. When activated, the AM has the capacity to release reactive oxygen and arachidonic acid metabolites and produce a number of cytokines, such as interleukin-1 (IL-1). This latter cytokine has pleiotropic effects on a variety of cells and has been implicated as one of the preeminent mediators of acute inflammation. Recently, an IL-1 receptor antagonist (IRAP) has been isolated, purified, and cloned from peripheral blood monocytes (PBM) stimulated with either adherent IgG (adhIgG) lipopolysaccharide (LPS), or phorbol myristate acetate. IRAP acts as a true receptor antagonist without agonist activity. We postulated that the AM would be a significant cellular source of IRAP from the lung. To test this hypothesis, normal human AM were immediately isolated or stimulated in a dose-dependent fashion with either LPS or adhIgG. For comparison, PBM were also isolated and treated in a similar manner. PBM expressed steady-state IRAP mRNA by Northern blot analysis only in response to LPS or adhIgG. In contrast, AM were found to express significant levels of antigenic IRAP by Western blot analysis, immunostaining, and specific ELISA, and express steady-state levels of IRAP mRNA under unstimulated culture conditions. Moreover, LPS or adhIgG failed to induce AM-derived IRAP antigen generation over unstimulated control.(ABSTRACT TRUNCATED AT 250 WORDS)     

LPS augments human B-cell differentiation by direct stimulation of PWM-responsive B cells

Bacterial lipopolysaccharide (LPS) augments production of IgM and IgG by two- to seven-fold in cultures of peripheral blood lymphocytes (PBL) stimulated by pokeweed mitogen (PWM), but only if monocytes are rigorously depleted. When PBL were separated into adherent cell (AC), B-cell-enriched, and T-cell-enriched fractions, pulsed with LPS, and recombined in culture with PWM, increased generation of plasma cells was seen only in cultures containing LPS-treated B cells. This effect of LPS appears to be independent of soluble factors. Supernatants from LPS-stimulated B cells or AC did not consistently increase PWM responses when cultured with fresh B cells in the presence of polymyxin B. Furthermore, pulsing of B cells with purified interleukin 1 from two different commercial sources failed to augment PWM-induced differentiation. When B cells were depleted of surface IgD (sIgD)-bearing cells by panning, no effect on LPS-mediated augmentation of PWM-driven differentiation was seen. B cells were also fractionated by rosetting with mouse erythrocytes. Treatment of BMR+ cells with LPS did not induce them to respond to PWM, while treatment of BMR- cells with LPS augmented generation of plasma cells. These results indicate that LPS acts directly to augment differentiation of PWM-responsive B cells, rather than recruiting sIgD+, BMR+ cells to become PWM responsive.     

Macrophages Suppress Direct B-Cell Activation by Lipopolysaccharide

The influence of macrophages on polyclonal B-cell responses to lipopolysaccharide (LPS) and on a primary, specific immune response to a hapten-LPS conjugate was studied in mouse spleen and lymph node cells in serum-free and serum-supplemented cultures. Macrophages were found not to be necessary, and B cells were directly activated by LPS. In serum-free cultures of macrophage depleted spleen cells, (a) proliferation and antibody secretion occurred at normal levels or were enhanced when compared with normal spleen cells, (b) the responsiveness of limiting cell numbers to LPS was better than in normal spleen cell cultures, (c) LPS did not increase the number or activate residual macrophages, (d) the primary specific response to a hapten-LPS conjugate developed normally, (e) peripheral lymph node cells, which are known to contain a very low concentration of macrophages, from normal or congenitally athymic (nude) mice mounted excellent proliferative responses to LPS Furthermore, in cultures supplemented with fetal bovine serum, depletion of adherent cells resulted in enhancement of LPS-induced B-cell responses. Addition of peritoneal macrophages to adherent cell-depleted spleen cells produced suppression at all concentrations of both mitogen and adherent cells. Suppressive activity could also be demonstrated in supematants from adherent cell cultures stimulated by LPS     

Polyclonal activation of B lymphocytes by lipopolysaccharide requires macrophage-derived interleukin-1.

Lipopolysaccharide (LPS) is a potent murine polyclonal B-cell activator which induces cellular proliferation and IgM secretion. The precise role of activated macrophages in the induction of LPS-dependent, B-cell responses has been unclear. Although early reports concluded that the LPS effect occurs independently of other cell types, other studies have suggested that adherent macrophages exert either potentiating or inhibitory effects. In the present study, B-cell mitogenesis and IgM production were measured in primary spleen cell cultures after removing adherent cells by a variety of experimental procedures. B-cell activation by LPS was found to be strictly dependent on the presence of adherent macrophages. Antibody neutralization and cytokine reconstitution studies demonstrated that macrophage-derived interleukin- (IL-1) is a necessary co-factor for LPS-induced polyclonal activation.     

Mitogenic Activation of Human Lymphocytes: a Protein A Plaque Assay Evaluation of Polyclonal B-Cell Activators

Using the protein A plaque assay, the capacity of various polyclonal B cell activators to induce differentiation in human B lymphocytes was investigated. Dextran sulphate and native Dextran were both virtually devoid of mitogenic properties, Lipopolysaccharide, however, was round to be a potent mitogen in human cells that, although giving rise to low DNA synthetic response, induced high numbers of immunoglobulin-synthesizing cells. Mean plaque-forming cell (PFC) numbers in healthy blood donors assayed on the optimal day (days 5–7) were 23, 493 IgM/10⁴ cells, 11, 288 IgG/10⁶ cells, and 2643 IgA/10⁶ cells. Values obtained in spleen cells, peaking at days 4–6, were slightly higher. Purified protein derivative (PPD) was equally or oven more-effective than lipopolysaccharide (LPS) in generating PFC of different subclasses in peripheral blood with mean of 29, 241 IgM/10⁶, 21, 269 IgG/10⁶, and 3681 IgA/10⁶. PPD furthermore induced a marked DNA synthetic response in human lymphocytes. These data suggest that LPS and PPD may both be used as functional markers in human cells when analysing patients with a suspected immunodeficiency state. It is suggested that cultures should be assayed using the protein A plaque assay, thereby being able not only to investigate the individual immunoglobulin classes but also to avoid the possible hazards involved in measuring antigen-specific responses in patients whose prior immunization to the antigen tested can never be totally excluded     

Immunoglobulin G subclasses secreted by human B cells in vitro in response to interleukin-2 and polyclonal activators

The IgG subclasses secreted by human B cells in vitro in response to IL-2 have been analysed. B cells were prepared from tonsil, blood and spleen, and cultured with recombinant IL-2 in the presence or absence of two polyclonal activators: Staphylococcus aureus Cowan 1 (SAC) and bacterial lipopolysaccharide (LPS). Secretion of all four subclasses and of IgM was stimulated by IL-2, but the relative amounts varied according to (i) the tissue source of the B cells, and (ii) which polyclonal activator was used. The amount of IgG1 tended to be higher and IgG2 tended to be lower when SAC was the polyclonal activator (compared to LPS). This difference was most marked for tonsil B cells, and it was found that SAC had a negative effect on secretion of IgM and IgG2 in these cultures, whilst synergizing with IL-2 to stimulate the production of IgG1, 3 and 4. When the degree of stimulation of different pairs of isotypes was analysed, several interesting positive correlations emerged. In tonsil B-cell cultures, stimulation of IgM and IgG2 was linked with each other, but not with IgG1, whilst in blood B-cell cultures all isotypes appeared to be stimulated co-ordinately. Stimulation of IgG1 and IgG3 were positively correlated in cultures of B cells from all tissues. The results emphasize that the effects of a single cytokine on immunoglobulin isotype production can be influenced by the source of the B cells, and by other signals delivered to the cells.     

Circulating and pokeweed mitogen-induced immunoglobulin-secreting cells in systemic lupus erythematosus.

Peripheral blood mononuclear cells (PBM) obtained from patients with active untreated systemic lupus erythematosus (SLE) were evaluated both for the number of cells spontaneously secreting immunoglobulin (Ig) as well as for their capacity to generate immunoglobulin-secreting cells (ISC) in vitro in response to pokeweed mitogen (PWM). ISC were enumerated by a reverse haemolytic plaque assay designed to quantify the number of cells secreting IgG, IgM and IgA. PBM obtained from eight patients with active untreated SLE contained markedly increased numbers of ISC compared to age-, sex-, and race-matched normal control PBM. SLE PBM contained a mean of 13,805 +/- 3266 ISC per 10(6) cells, of which 74% secreted IgG, 10% IgM and 22% IgA, while normal PBM contained a mean of 779 +/- 143 ISC per 10(6) cells, with 57% secreting IgG, 25% IgM and 33% IgA. PBM obtained from SLE patients were also examined for their ability to generate ISC in vitro in response to PWM. SLE PBM were markedly deficient in their capacity to respond to PWM with the differentiation of ISC. This diminished responsiveness could not be ascribed to serum factors, the presence of increased numbers of cells with suppressive capacity or the absence of potentially responsive B cells. Rather, a deficiency of helper T cell activity appeared to be responsible. This was indicated by the observation that PWM responsiveness could be restored to SLE PBM by co-culturing them with purified mitomycin C-treated normal T cells.     

Human monoclonal antibodies reactive with cell surface antigens on human leukemia cell lines: many antibodies are (auto)antibodies

Primary Epstein Barr Virus (EBV) transformants from peripheral blood mononuclear cells (PBM) established in macrocultures were screened for the secretion of antibodies reactive with cell surface antigens on one or another of two indicator human leukemic cell lines and fused with the HMMA2.11TG/O human fusion partner. Human monoclonal antibodies (HuMAbs) were readily obtained. Relative oligoclonality of the primary EBV macrocultures was documented by the number of antibody secreting hybridomas (1-100%). The method permitted preselection for fusion of transformants producing antibodies of certain specificities and/or class. Fourteen HuMAbs, primarily of the IgM class, have been obtained. Those IgM HuMAbs obtained from patients with active diseases, e.g. Acute Lymphoblastic Leukemia (3 HuMAbs), and HIV infection (4 HuMAbs), were found to have a relatively broad spectrum of reactivity with cell lines of various hematopoietic lineages and normal cells, although several show selective reactivity with T cell lineage tumors or a selected population of cells. HuMAbs from normal donors of both the IgG and IgM class were obtained. The IgM HuMAbs from one volunteer reacted primarily with autologous and allogeneic macrophages (autologous PBM from the other patients were not available) as well as a diverse number of hematopoietic cell lines. From others, the IgG HuMAbs demonstrated a more restricted spectrum of reactivity, while the IgM HuMAbs reacted with both autologous and allogeneic normal cells. Thus, the B cell repertoire contains cells capable of secreting cell surface reactive antibodies and many of these antibodies express characteristics of autoantibodies. Those that did not react with autologous or allogeneic PBM may react with other autoantigens which have been expressed on the malignant cells used as screening targets or may represent true antitumor antibodies.     

Stimulation of Human B Lymphocyte Differentiation by the Neuropeptides Substance P and Neurokinin A

Substance P (SP) at a concentration of 10(-7) M significantly increased the number of IgG-producing cells induced by the polyclonal activator Staphylococcus aureus protein A (SpA) in 11 out of 22 cultures of enriched human blood B lymphocytes, in nine cultures SP did not significantly affect the SpA response and in three cultures IgG secretion was decreased in the presence of SP. Stimulation by SP was observed in cultures at days 6 and/or 8. In 3 out of 4 cell cultures depleted of monocytes SP did not affect the cell response to SpA stimulation. SP antagonists inhibited the enhancing effect of SP on B-cell antibody secretion induced by SpA. SP alone did not stimulate B lymphocytes. Neurokinin A (NKA) had similar effects as SP and enhanced the IgG secretion induced by SpA in 5 out of 9 experiments, in two experiments was inactive, and in one decreased the IgG secretion. The effect of SP and NKA on B lymphocytes suggest that the neuropeptides interact with the regulation of the immune response.     

Rheumatoid Synovial Fluid Reconstitutes the B-Cell Defect in CBA/N Mice

Synovial fluid from patients with rheumatoid arthritis (RA-SF) contains a biological activity which can replace T cells for activation of antibody secretion in human blood lymphoid cells and which can also induce the selective differentiation of IgG2b-secreting cells in lipopolysaccharide (LPS)-pre-activated mouse spleen cells. The B-cell activity of this factor was studied in CBA/N mice which have an X-linked B-cell immunodeficiency which manifests itself as a defective humoral response to certain thymus-independent antigens (TI-2). RA-SF has now been shown to reconstitute partly the B-cell deficiency in CBA/N splenic B cells in vitro. Addition of RA-SF to LPS-pretreated cell cultures results in IgG2b secretion in CBA/N spleen cells as well. In contrast to cells from normal CBA mice, cells from CBA/N mice cannot respond to interleukin 4 (IL-4) after addition of LPS with production of IgG1 antibodies in vitro. However, the addition of RA-SF completely restores a normal IL-4-induced IgG1 response. No other biologically active factors have been shown to allow the production of IgG antibody producing cells in CBA/N splenic B cells. It is postulated that the xid immunodeficiency could be the result of a deficient production of a biological activity which is abundant in RA-SF.     

Neuroimmune modulation of lymphocyte function--I. Substance P enhances immunoglobulin synthesis in lipopolysaccharide activated murine splenic B cell cultures.

Murine B cells have been shown to possess substance P (SP) receptors, but their functional and biological significance remains unresolved. While previous studies have suggested that SP can induce B cells to secrete Ig, the effect could be indirect since mixed cultures were used. In order to assess directly the ability of SP to trigger normal B cells, we have studied the effects of this neuropeptide on purified splenic B cells in vitro. Although an activation, e.g. lipopolysaccharide (LPS), was required, the functionality of the B cell SP receptors was clearly shown by the ability of subnanomolar concentrations of this neuropeptide to augment antibody secretion in a dose-dependent fashion. Specifically, IgM and IgG levels, determined by an isotype-specific sandwich ELISA, were greatly enhanced at 10(-10) M SP by as much as 500 and 572% respectively, while IgA levels were only modestly affected. Even picomolar concentrations of SP could significantly increase IgM levels. This observed enhancement of Ig production was SP specific since B cells co-cultured in the presence of excess SP antagonist were reduced to basal LPS-stimulated Ig levels. Furthermore, this synergistic stimulation by SP and LPS upon normal B cells could not be attributed to SP-induced cell proliferation since stimulatory concentrations of SP were not mitogenic and at high concentrations could inhibit cell proliferation. Rather, it was observed that the increased IgM and IgG secretion was in part attributable to a greater number of B cells secreting antibodies as demonstrated with an ELISPOT assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of Immunoglobulin Secretion by Protein A from Staphylococcus aureus in Human Blood and Bone Marrow B Cells

Antibody secretion was measured in vitro as plaque-forming cells (PFC), using an indirect haemolysis-in-gel assay. Protein A from Staphylococcus aureus (SPA) induced 4900 +/- 1800 (mean +/- SE from 12 individuals) IgG+A+M PFC/10(6) blood lymphocytes. This was comparable to responses induced by Staph. aureus Cowan 1, lipopolysaccharide, pokeweed mitogen, and purified protein derivative of tuberculin. In bone marrow, SPA induced the highest number of PFC (36,500 +/- 100/10(6) of all the B-cell activators. SPA mainly induced IgG PFC and only a few IgA and IgM PFC. In blood, B cells enriched by sheep erythrocyte sedimentation gave a higher PFC response to SPA than unseparated blood lymphocytes, whereas almost no stimulation was seen in enriched T cells. In bone marrow, the highest number of PFC stimulated by SPA was often seen among the enriched T cells. After the removal of adherent and phagocytic cells, the number of PFC induced by SPA was reduced in blood and bone marrow cells. In enriched B cells from blood, the number of PFC stimulated by SPA increased in the presence of enriched T cells depleted of OKT8-positive cells and decreased when cultured with T cells depleted of OKT4-positive cells. It is concluded that macrophages/monocytes and T helper and suppressor cells play a role in the regulation of human B-cell antibody secretion induced by SPA.