Tyrosine phosphorylation is an essential event in the stimulation of B lymphocytes by Staphylococcus aureus Cowan I.

Staphylococcus aureus Cowan I (SAC) is a potent mitogen for purified human B cells. By using Western blotting with antiphosphotyrosine antibodies, we demonstrated that the mitogenic effect of SAC is associated with rapid tyrosine phosphorylation of proteins of 45, 68, 75, 97, and 145 kDa. This tyrosine phosphorylation was detected within 30 s of the addition of SAC; it reached a maximum within 10 min, after which it declined gradually. In contrast to SAC, most soluble anti-IgM antibodies do not induce proliferation of isolated human B cells. As indicated by Western blotting, soluble anti-IgM antibodies induced a similar pattern of tyrosine phosphorylation, with the exception of the 68-kDa protein, which was the most heavily phosphorylated protein in SAC-treated cells. A similar but less intense 68-kDa band was also induced by mitogenic anti-IgM bound to beads. This suggested that tyrosine phosphorylation, especially of p68, may play an important role in B cell mitogenesis. To test this hypothesis, we determined the effect of specific tyrosine kinase inhibitors (tyrphostins) on SAC-induced tyrosine phosphorylation, oncogene expression, and B cell proliferation. The concentration dependencies of inhibition of these processes suggested that they were linked. Nonspecific toxic effects of the tyrphostins were ruled out by the demonstration that the tyrphostins did not alter cell viability and did not inhibit B cell proliferation induced by phorbol esters, which do not induce tyrosine phosphorylation. For maximal inhibition of SAC-induced cell proliferation, the tyrophostins needed to be added before or shortly after addition of SAC. Taken together, these data indicate that tyrosine phosphorylation is an obligatory early signal in B cell proliferation.     

The major rheumatoid factor cross-reactive idiotype is dominantly expressed by Staphylococcus aureus Cowan strain I-activated CD5+ control B cells.

Some seropositive (RF+) and seronegative (RF-) rheumatoid arthritis (RA) patients selectively express high concentrations of the major RF cross-reactive idiotype (RCRI) in their sera and generate high frequencies of RCRI+ pokeweed mitogen (PWM)-induced plasma cells from their peripheral blood mononuclear cells (PBM). To determine if normal individuals can express RCRI in vitro, B cells from controls were activated with Staphylococcus aureus Cowan strain I (SAC) bacteria to identify RCRI and RF production. In addition, we studied the relationship of RCRI expression with the subset of B cells bearing CD5. Control CD5+ B cells are responsible for RCRI expression following SAC activation. We also observed that RCRI is dominantly expressed by control SAC-induced B cells in frequencies comparable to that expressed by some RA and juvenile rheumatoid arthritis patients' PBM activated by PWM. Therefore, the frequency of RCRI+ B cells in control and arthritis patients' PBL may be similar, or the selection and/or regulation of RCRI+ B-cell expression in vitro and in vivo may be different in arthritis patients compared to normal individuals.     

Physico- and immunochemical properties of staphylococcal protein A extracellularly produced by a set of mutants from Staphylococcus aureus Cowan I.

Mutant strains of Staphylococcus aureus have been isloated using a simple cosedimentation technique. While the parental strain contains predominantly a cell-bound protein A, the mutant strains exclusively produce extracellular protein A. The mutant forms of protein A all have lower molecular weights than that of protein A from the parental strain. They showed the same antigenicity as the parental protein A and gave similar reactivity with immunoglobulin to the parental one except for one mutant. A conspicuous spur was observed between the parental protein A and that produced by the mutant against normal dog serum in the micro-Ouchterlony immunodiffusion test.     

Influence of human T lymphocytes identified by antibodies to dipeptidyl peptidase IV on differentiation of human B lymphocytes stimulated with Staphylococcus aureus Cowan I and pokeweed mitogen

The influence of human T lymphocytes expressing the enzyme dipeptidyl peptidase IV (DPP IV) was investigated with respect to human peripheral B-lymphocyte differentiation. B cells stimulated with pokeweed mitogen in the presence of DPP IV-positive T cells produced high amounts of immunoglobulin. Moderate amounts of immunoglobulin could be measured when B cells were cultured in the presence of DPP IV-negative T cells. DPP IV defines a T-cell subset partially overlapping the subsets characterized by the differentiation antigens Leu 3a (helper/inducer) and Leu 2a (suppressor/cytotoxic). DPP IV-positive T cells exert, in contrast to DPP IV-negative T cells, high interleukin-2 activity after stimulation with phytohemagglutinin and pokeweed mitogen. To further functionally characterize DPP IV-positive and DPP IV-negative T cells, the helper effects of Leu 3a-positive T-cell subsets, differing in DPP IV expression, were investigated in pokeweed mitogen- and Staphylococcus aureus-driven B-cell differentiation systems. After pokeweed mitogen stimulation, immunoglobulin production was markedly reduced when B cells were cultured in the presence of Leu 3a-positive T cells expressing DPP IV (DPP IV+/Leu 3a+). In contrast, high amounts of immunoglobulin were produced in cultures with Leu 3a-positive but DPP IV-negative T cells (DPP IV-/Leu 3a+). This difference in immunoglobulin production of B cells cultured with DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells could not be observed in Staphylococcus aureus-stimulated cultures. Here, both T-cell subsets supported terminal differentiation of B cells. We conclude that in the pokeweed mitogen-driven culture systems, DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells may differ in the production of growth and/or differentiation factors distinct from interleukin-2.     

Induction of monocytic suppression after stimulation of peripheral human mononuclear cells with staphylococcal protein A and Staphylococcus aureus

Staphylococcal protein-A (SPA) and Staphylococcus aureus are known to be polyclonal human B-cell activators. It was noted that they induced plaque-forming-cell (PFC) responses lower than those induced by pokeweed mitogen (PWM) and the possibility of early triggering of a suppressor cell was investigated in the present series of experiments. Peripheral mononuclear cells (MNC) were passed through Sephadex G-10 columns to eliminate monocytes. The PFC responses to SPA and S. aureus were thereby increased. PWM-driven PFC responses are suppressed by the simultaneous presence of SPA in a dose-related way, if present in the early phases of the cultures. MNC precultured with SPA or S. aureus have the ability to suppress the PFC response of autologous MNC to PWM. Interestingly this suppressor cell activity was radiation resistant and could not be abrogated by treatment with anti-T-cell monoclonal antibody plus complement. The above experiments clearly demonstrate that the observed low PFC responses of MNC after stimulation with SPA and S. aureus are due to the induction of suppressor cells by these stimulants. The suppressor cells are apparently of monocytic origin.     

Analysis of protein A encoded by a mutated gene of Staphylococcus aureus Cowan I

The protein A (spa) genes from Staphylococcus aureus Cowan I and a mutant strain of Cowan I called V-1 earlier suggested to produce a monovalent IgG-binding protein A have been cloned in Escherichia coli. The DNA sequences coding for the IgG-binding part of the spa genes from both strains have been determined and compared with each other and with a partial amino acid sequence of purified protein A from strain V-1. The nucleotide sequence of the spa gene from strain V-1 reveals an NH2-terminally located IgG-binding region homologous to region E first reported for strain 8325-4, region D and the major portion of region A. The amino acid sequence analysis of the purified protein A from this strain also shows the presence of regions E and D but only a minor part of region A. Reversed-phase high-performance liquid chromatography fractionation of purified protein A from strain V-1 revealed that the preparation was heterogeneous, containing mainly two peptides with different abilities to bind IgG molecules. A shuttle vector containing the cloned protein A gene from V-1 was constructed and transformed into different strains of S. aureus and the produced protein A was purified and analysed using sodium dodecyl sulfate/polyacrylamide gel electrophoresis.     

Low responsiveness of human neonatal lymphocytes to a B-cell mitogen, Staphylococcus aureus Cowan I (SpA CoI)

Responses of neonatal and adult lymphocytes to various mitogens were studied. Lymphocytes from umbilical cord blood (UCB) responded well to both phytohemagglutinin and concanavalin A, and also to pokeweed mitogen and Staphylococcus aureus Protein A. The responses of UCB lymphocytes to these mitogens were not significantly lower than those of adult peripheral blood lymphocytes (PBL). In contrast, UCB lymphocytes showed only a minimal response to killed Staphylococcus aureus Cowan I (SpA CoI), a potent B-cell mitogen for human PBL, although the proportion of B cells in UCB was not less than that in PBL. The low level of response of lymphocytes from UCB to SpA CoI was not ascribed to differences in dose response or kinetics. Purified B cells from UCB were not stimulated by SpA CoI either, suggesting tht the low responsiveness was not due to the suppressive effect of T cells or macrophages, but to some intrinsic defect in B cells in UCB. These results suggest that the B cells in neonates may be more immature than the T cells.     

Induction and suppression of immunoglobulin synthesis in cultures of human lymphocytes: effects of pokeweed mitogen and Staphylococcus aureus Cowan I

The synthesis and secretion of immunoglobulins by human peripheral blood mononuclear cells in cultures stimulated with pokeweed mitogen or Staphylococcus aureus Cowan I were evaluated by enumeration of cells containing cytoplasmic immunoglobulins and cells actively secreting immunoglobulins, and by quantitation of immunoglobulins released into culture supernatants. The two mitogens caused comparable stimulation of immunoglobulin production; however, in contrast to pokeweed mitogen, S. aureus was active in cultures depleted of T lymphocytes, and its stimulatory effects were resistant to the influence of suppressor T cells generated by co-stimulation with concanavalin A or by preincubation without mitogenic stimulus. These results indicate distinct pathways of induction and suppression of immunoglobulin synthesis for these two polyclonal B cell activators, and suggest that stimulation by S. aureus is less thymus dependent than that induced by pokeweed mitogen.     

Chemiluminescence of human lymphocytes in reactions with Staphylococcus aureus peptidoglycan

The capacity of S. aureus peptidoglycan (PG) for inducing the luminol-dependent chemiluminescence of human lymphocytes has been studied. Lymphocytes taken from adult donors have been found to give dose-dependent reaction to S. aureus PG, while lymphocytes from newborn infants have been inert under the same conditions. Essential differences in the kinetics of response to PG (the maximum intensity of chemiluminescence occurs in 25-30 minutes) and to phytohemagglutinin (the maximum intensity is reached in 1 minute) were observed. These results are considered as the manifestation of specific sensitization to bacterial peptidoglycans, which may be rapidly detected by reactive chemiluminescence.     

Fibronectin binding protein A of Staphylococcus aureus can mediate human T lymphocyte adhesion and coactivation

The extracellular matrix protein fibronectin (FN) mediates the adhesion of bacteria as well as T lymphocytes. Mammalian cells express integrins alpha(4)beta(1) and alpha(5)beta(1) as the major FN-binding cell surface receptors. Bacteria such as Staphylococcus aureus, also express FN-binding receptors that are important for adherence to host tissue and initiation of infection. The S. aureus FN-binding protein, FnbpA, has been previously identified, and recombinant proteins that correspond to distinct functional regions of this protein have been made. Three recombinant truncated forms of FnbpA, rFnbpA(37-881), rFnbpA(37-605), and rFnbpA(620-881), were examined for effects on in vitro adhesion and coactivation of human T lymphocytes. These proteins, when coimmobilized with anti-CD3 mAb, activated T lymphocyte proliferation. The coactivation signal generated by the rFnbpA proteins required medium containing serum with FN. Furthermore, the costimulatory signal could be restored in FN-depleted serum when the rFnbpAs were preloaded with soluble FN. Monoclonal Ab blocking studies revealed that integrin alpha(5)beta(1) is the major receptor responsible for the rFnbpA costimulatory signal. Shear flow cell detachment assays confirmed that lymphocytes can bind to FN captured by the rFnbpA proteins. These results suggest that the S. aureus rFnbpA can interact with integrin alpha(5)beta(1) via an FN bridge to mediate adhesion and costimulatory signals to T lymphocytes.     

Evaluation of IgM,IgG, IgA,IgD, and IgE secretion by human peripheral blood lymphocytes in cultures stimulated with pokeweed mitogen and Staphylococcus aureus Cowan I

Sheep erythrocytes coated with staphylococcal protein A were used as target cells in a reverse hemolytic plaque assay. Monospecific antisera to human immunoglobulins G, M, A, D, and E were used to detect each of these classes in cultures of human peripheral blood mononuclear cells stimulated with the polyclonal B-cell activators pokeweed mitogen and Staphylococcus aureus (Cowan I). Both mitogens induced substantial increases in the numbers of cells actively secreting immunoglobulins; in time-course experiments, the peak response was seen on Day 5. The numbers of cell secreting IgM, IgE, and IgD were usually higher in cultures stimulated with S. aureus than with pokeweed mitogen.     

Expression of a cross-reactive idiotype on rheumatoid factor in patients with Sjogren's syndrome

Primary Sjogren's syndrome (SS) is a systemic autoimmune disorder characterized by lymphocytic infiltration of salivary and lacrimal glands. These patients have evidence of marked B cell hyperactivity, including the production of autoantibodies such as rheumatoid factor (RF) and an increased frequency of non-Hodgkin's lymphoma. We now demonstrate that RF from 12/15 SS patients contains a cross-reactive idiotype (CRI) on their kappa light chain defined by a monoclonal antibody (MoAb 17-109) and immunoblotting. This CRI was associated with immunoglobulin (Ig) A-RF, and to a lesser extent with IgM-RF molecules on the basis of direct binding studies. With the use of immunoperoxidase techniques to stain frozen tissue sections, B cells containing cytoplasmic Ig reactive with MoAb 17-109 were detected in the salivary gland biopsies from 11/12 SS patients at high frequencies, and in the blood from the same patients at much lower frequencies. One patient with pre-existant SS developed non-Hodgkin's lymphoma with tumor cells and RF paraprotein reactive with MoAb 17-109. Evaluation of serial biopsies over a 4-yr period showed a progressive increase in the proportion of B cells bearing the CRI. In contrast, synovial membrane biopsies from RA patients lacking sicca symptoms did not contain B cells expressing the CRI. Because previous studies have demonstrated that MoAb 17-109 detects a CRI on RF paraproteins from patients with lymphoma, B cells bearing this CRI may have increased frequency of neoplastic transformation. SS patients provide an opportunity to study the expression of this CRI and to understand the transition of B cell clones from autoimmune proliferation to neoplastic transformation.     

Response of human B cells to Staphylococcus aureus Cowan I: T-independent proliferation and T-dependent differentiation to immunoglobulin secretion involve subsets separable by rosetting with mouse erythrocytes

We separated T-depleted mononuclear cells into subsets by rosetting with mouse erythrocytes and studied proliferation and differentiation responses to Staphylococcus aureus Cowan I (SAC), pokeweed mitogen (PWM), and a combination of the two polyclonal activators. All of the T cell-independent proliferation of unfractionated B cells in response to SAC was attributable to mouse erythrocyte rosette-forming cells (BMR+). BMR- cells were not stimulated to proliferate by SAC in the presence or absence of T cells, but did proliferate to PWM plus irradiated T cells. Co-stimulation of BMR+ cells with SAC and PWM in the presence of autologous T cells did not lead to immunoglobulin secretion. The B cells stimulated to divide by SAC apparently do not become responsive to B cell differentiation factors and are distinct from those that undergo T cell-dependent differentiation.     

In vitro stimulation of human lymphocytes by alpha, beta and delta toxins and toxoids of Staphylococcus aureus.

Alpha, beta and delta toxins of Staphylococcus aureus stimulate human peripheral blood lymphocytes to blastic transformation and formation of IgM, IgG and IgA. The toxins are efficient at concentrations that are not toxic for the cells in culture. A dose of a toxin suitable for stimulation is 100 ng/ml but a stimulation can be observed also at 10 ng/ml, in the case of Ig formation even at a concentration of 1 ng/ml. Toxoids are approximately as effective to elicit blastic transformation as the toxins themselves, their efficiency to stimulate Ig formation being somewhat lower but significant. Alpha and delta toxins and toxoids at the appropriate concentration appear to act as medium-strength polyclonal activators of lymphocytes. Beta toxin and its toxoid are weak polyclonal activators.     

The A domain of fibronectin-binding protein B of Staphylococcus aureus contains a novel fibronectin binding site

The fibronectin-binding proteins FnBPA and FnBPB are multifunctional adhesins than can also bind to fibrinogen and elastin. In this study, the N2N3 subdomains of region A of FnBPB were shown to bind fibrinogen with a similar affinity to those of FnBPA (2 μM). The binding site for FnBPB in fibrinogen was localized to the C-terminus of the γ-chain. Like clumping factor A, region A of FnBPB bound to the γ-chain of fibrinogen in a Ca(2+)-inhibitable manner. The deletion of 17 residues from the C-terminus of domain N3 and the substitution of two residues in equivalent positions for crucial residues for fibrinogen binding in clumping factor A and FnBPA eliminated fibrinogen binding by FnBPB. This indicates that FnBPB binds fibrinogen by the dock-lock-latch mechanism. In contrast, the A domain of FnBPB bound fibronectin with K(D) = 2.5 μM despite lacking any of the known fibronectin-binding tandem repeats. A truncate lacking the C-terminal 17 residues (latching peptide) bound fibronectin with the same affinity, suggesting that the FnBPB A domain binds fibronectin by a novel mechanism. The substitution of the two residues required for fibrinogen binding also resulted in a loss of fibronectin binding. This, combined with the observation that purified subdomain N3 bound fibronectin with a measurable, but reduced, K(D) of 20 μM, indicates that the type I modules of fibronectin bind to both the N2 and N3 subdomains. The fibronectin-binding ability of the FnBPB A domain was also functional when the protein was expressed on and anchored to the surface of staphylococcal cells, showing that it is not an artifact of recombinant protein expression.     

Role of proteases in fermentation of the fused protein staphylococcal protein A-human insulin-like growth factor I in Staphylococcus aureus

Summary Fermentation of the fused protein staphylococcal protein A-human insulin-like growth factor I (SpA-IGF-I) was investigated. The fused protein was expressed in Staphylococcus aureus and secreted to the culture medium. The production of SpA-IGF-I was growth related and started after a short lag phase. Examination of the product quality by SDS-PAGE after IgG affinity purification showed that the product was partially degraded. Exoproteases appeared later than SpA-IGF-I and were responsible for some, but probably not all, of the degradation of the product. It was possible to influence the exoprotease activity and thereby the product degradation by varying the cultivation conditions.     

Human helper T cell factor(s). IV. Demonstration of a human late-acting B cell differentiation factor acting on Staphylococcus aureus Cowan I-stimulated B cells

At least two distinct B cell stimulatory factors (BSF) were found to be involved in the differentiation of Staphylococcus aureus Cowan I (SAC)-stimulated human B cells to IgG-producing cells. A factor tentatively called B cell differentiation factor I (BCDF I) was found in one fraction, and a second factor, BCDF II was found in another fraction. The BCDF I fraction alone induces IgG-production in SAC-stimulated B cells, but the BCDF II fraction does not. The BCDF II fraction enhances IgG production in SAC-stimulated B cells in the presence of the BCDF I fraction. Studies concerning the time-course of the action of the BCDF II fraction revealed that it contains a late-acting differentiation factor that acts on B cells most effectively when it is added to the SAC-stimulated B cell culture after the addition of BCDF I fraction; it induces IgG plaque-forming cells within 1 day. The pI value of a late-acting BCDF was in the range of 5 to 6; this pI range is different from that of BCDF I but similar to that of BCDF II, which was shown in our previous studies to be able to induce IgG production in Epstein Barr Virus-transformed B lymphoblastoid cell lines. In addition, the m.w. of a late-acting BCDF were about 35,000 and 20,000, which are the same as those of BCDF II, and thus its identity with BCDF II was suggested.