Tubuloreticular Inclusions and Paired Cisternae Induced in Human Lymphocytes Cultured with Staphylococcus Aureus Cowan 1

Tubuloreticular inclusions (TRI) and paired cisternae (PC) were induced in lymphocytes of normal individuals after incubation with Staphylococcus aureus Cowan 1. TRI were initially detected in lymphoid cells on day 2 (48-h culture). The frequency of TRI-positive cell sections on day 5 increased about twofold over those on days 2-4. On day 7, TRI were predominantly seen in lymphoplasmacytoid cells or plasmacytoid cells, with an incidence of up to 18% of sections. The regions in these cells were most extensive and anastomosed with the cisternae of adjacent well-developed rough endoplasmic reticulum (RER). TRI formation appears not to be essential for mitogen-induced B-cell differentiation to plasmacytoid cells, because poke-weed mitogen (PWM) failed to induce TRI. The diverse expressions of TRI induction between these two mitogens may be due to a difference in B-cell activation mechanisms.

Paired cisternae were observed in a great majority of mitotic cells at various stages. These were encountered most frequently on day 4. PC were also seen in the PWM-stimulated culture. Our observations suggest that PC formation may be related to new formation of RER as well as to reconstruction of the nuclear envelope.     

Tubuloreticular Inclusions and Paired Cisternae Induced in Human Lymphocytes Cultured with Staphylococcus Aureus Cowan 1

Tubuloreticular inclusions (TRI) and paired cisternae (PC) were induced in lymphocytes of normal individuals after incubation with Staphylococcus aureus Cowan 1. TRI were initially detected in lymphoid cells on day 2 (48-h culture). The frequency of TRI-positive cell sections on day 5 increased about twofold over those on days 2–4. On day 7, TRI were predominantly seen in lymphoplasmacytoid cells or plasmacytoid cells, with an incidence of up to 18% of sections. The regions in these cells were most extensive and anastomosed with the cisternae of adjacent well-developed rough endoplasmic reticulum (RER). TRI formation appears not to be essential for mitogen-induced B-cell differentiation to plasmacytoid cells, because poke-weed mitogen (PWM) failed to induce TRI. The diverse expressions of TRI induction between these two mitogens may be due to a difference in B-cell activation mechanisms.

Paired cisternae were observed in a great majority of mitotic cells at various stages. These were encountered most frequently on day 4. PC were also seen in the PWM-stimulated culture. Our observations suggest that PC formation may be related to new formation of RER as well as to reconstruction of the nuclear envelope.     

Mechanism of activation of human basophils by Staphylococcus aureus Cowan 1

We investigated the capacity of Staphylococcus aureus Cowan 1 and S. aureus Wood 46 to induce histamine release from human basophils in vitro. S. aureus Cowan 1 (10(5) to 10(7)/ml), which synthesizes protein A (Staph A), stimulated the release of histamine from basophils, whereas S. aureus Wood 46 (10(5) to 2 X 10(7)/ml), which does not synthesize Staph A, did not induce histamine secretion. Soluble Staph A (10(-3) to 10 micrograms/ml), but not staphylococcal enterotoxin A, induced histamine secretion from human basophils. Staph A binds through its classical site to the Fc region of human immunoglobulin G (IgG) and through its alternative site to the Fab portion of the different human immunoglobulins. Hyperiodination of Staph A, which destroys over 90% of the original Fc reactivity without altering the Fab-binding site, did not alter the ability of the protein to induce histamine release. The stimulating effect of Staph A was dose dependently inhibited by preincubation with human polyclonal IgG (0.3 to 100 micrograms/ml) and a human monoclonal IgM (0.3 to 100 micrograms/ml) which have F(ab')-Staph A reactivity. In contrast, rabbit IgG, which possesses only Fc-Staph A reactivity, and a Staph A-unreactive human monoclonal IgM did not inhibit Staph A activity. Similar results were obtained with intact S. aureus Cowan 1. Preincubation with either Staph A or anti-IgE (rabbit anti-Fc epsilon) resulted in complete desensitization to a subsequent challenge with the homologous stimulus. Staph A and anti-IgE induced partial cross-densensitization to the heterologous stimulus. Cells preincubated with anti-IgG (rabbit anti-Fc gamma) lost a small but significant part of their ability to release with Staph A but did not lose their response to anti-IgE. Basophils from which IgE had been dissociated by brief exposure to lactic acid no longer released histamine in response to anti-IgE and Staph A. When basophils from which IgE had been dissociated were incubated with human polyclonal IgE, they regained their ability to induce histamine in response to Staph A and anti-IgE. In contrast, two monoclonal IgEs which do not bind to Staph A did not restore the basophil responsiveness to Staph A. Furthermore, there was complete cross-desensitization between soluble Staph A and S. aureus Cowan 1, while cells desensitized to S. aureus Wood 46 released normally with Staph A and S. aureus Cowan 1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional maturity of cord blood B lymphocytes: Staphylococcus aureus Cowan 1 induces IgG secretion in the human neonate

Lymphocytes from newborn infants and adults were investigated with the polyclonal B cell activators Staphylococcus aureus Cowan 1, soluble protein A, and pokeweed mitogen (PWM). In cord blood lymphocytes (CBL), protein A and PWM induced only meagre IgM responses, whereas Cowan 1 bacteria induced synthesis of both IgM and IgG in quantities comparable to adult levels. We were also able to establish the neonatal origin of the Ig producing cells. Thus, cord blood lymphocytes seem to be functionally mature enough to produce any Ig class, provided they are given the right stimulus.     

Pokeweed mitogen and Staphylococcus aureus Cowan I induced immunoglobulin A synthesis by lymphocytes of IgA deficient blood donors.

In vitro IgA synthesis induced by pokeweed mitogen (PWM), Staphylococcus aureus Cowan I (STA), and combinations of STA and PWM (STA/PWM) was studied in lymphocytes of IgA deficient (sIgAd) blood donors. Cultures of T-depleted (non-T) cells with autologous or allogeneic control T, irradiated T (T), or T4 cells suggested abnormalities in non-T cell fractions in most (12/22) sIgAd donors. T cell abnormalities in themselves, detectable in six donors, did not appear to account for the failure of IgA synthesis. Repeat studies in 10 donors indicated fluctuations in in vitro IgA synthesis in four. IgA synthesis induced by STA/PWM combinations was observed in only one of eight sIgAd donors. Our findings suggest that in some donors defects leading to failure to produce IgA may not be constant and support the hypothesis of a maturation arrest in IgA+ B cells in sIgAd donors.     

The activation of T cells with Staphylococcus aureus Cowan 1 (SAC)

We reported our findings on the activation and functional capacity of human T cells stimulated by Staphylococcus aureus Cowan 1 (SAC). Serial determinations of surface markers on T cells stimulated by SAC showed that OKT4+ T cells remained relatively constant with the decrease of OKT8+ T cells and that Tac antigen was predominantly expressed on OKT4+ T cells. When the mixture of OKT4+-depleted T cells and OKT8+-depleted PBL was stimulated with SAC or PWM, PWM-stimulated OKT4+-depleted T cells suppressed immunoglobulin production by OKT8+-depleted PBL in a dose-dependent fashion, but SAC-stimulated OKT4+-depleted T cells did not show suppressive activity.     

Enhancement of the B-cell response to Staphylococcus aureus Cowan strain 1 by natural human gamma interferon.

The requirements for a primary, antigen-specific in vitro immunization of human peripheral lymphocytes using haemocyanin, a T-cell dependent antigen, have been studied. In order to obtain a specific response in vitro the peripheral lymphocytes had to be separated into B, T, accessory (A) and dendritic (D) cells. These cells were activated and reconstituted to give a population with a B:T ratio of 1:2. If the induction was supported by MHC-restricted, radioresistant T cells, this cell population could then be antigen-specifically activated using haemocyanin. The immunization had also to be supported by cytokines, such as B-cell growth and differentiation factors, interleukin-2 and gamma-interferon. A 5-day in vitro immunization using 2 micrograms haemocyanin/ml resulted in 200-300 cells secreting anti-haemocyanin-specific antibodies per 10(6) B cells.     

Immunoglobulin production of human lymphocytes stimulated by Staphylococcus aureus Cowan I and pokeweed mitogen: differential effects of recombinant interleukin-2.

The number of immunoglobulin-secreting cells (ISC) upon stimulation with pokeweed mitogen (PWM) or Staphylococcus aureus Cowan I (SAC) in recombinant interleukin-2 (rIL-2)-supplemented cultures of human peripheral blood mononuclear cells (PBMC) and co-cultures of B and T cells was studied. It has been shown that the addition of rIL-2 to culture can enhance or depress the number of ISC depending on the polyclonal B-cell activator used for culture stimulation. The SAC-induced response was enhanced in the presence of rIL-2, while the number of ISC in PWM-stimulated cultures was depressed. Moreover, in cultures stimulated simultaneously by both activators, the suppressive effect of rIL-2 prevailed, indicating that the reported direct effect of the lymphokine on SAC-activated B cells cannot overcome the suppressive activity of PWM-induced suppressor T cells. rIL-2 could not activate suppressor T cells in the absence of PWM, and has no effect on the number of helper or suppressor cells in the culture.     

Recombinant interleukin 2 induces immunoglobulin secretion in Staphylococcus aureus Cowan strain I activated human B-cells

Human B-cells, exhaustively depleted for T-cells, were activated with Staphylococcus aureus Cowan strain I (SAC) and responded to recombinant human interleukin 2 (rIL2) by secretion of immunoglobulin (Ig), as measured by a protein A hemolytic plaque assay. The rIL2, however, had to be present early, since addition later than 24 h after SAC-activation of the B-cells reduced the response to background levels. No clear dose response was observed and Ig-secreting cells (ISC) could be induced even with rIL2 at 0.5 U/ml. The monoclonal antibody anti-TAC prevented the rIL2-promoted induction of ISC. Ig production could be induced in SAC-activated cultures with supernatants of Xenopus laevis oocytes injected with sucrose-gradient-fractionated poly(A+) RNA derived from a stimulated human spleen cell culture. This activity coincided with the IL2 mRNA activity and was well separated from the interferon-gamma mRNA activity. Our results suggest that IL2 is not only a B-cell growth factor but also promotes the differentiation of activated human B-cells towards Ig secretion.     

Mechanisms of internalization of Staphylococcus aureus by cultured human osteoblasts

Staphylococcus aureus is an important bone pathogen, and evidence shows that this organism is internalized by chick osteoblasts. Here we report that S. aureus is internalized by human osteoblasts. Internalization was inhibited by monodansylcadaverine and cytochalasin D and to a lesser extent by ouabain, monensin, colchicine, and nocodazole. We propose that internalization occurs via a receptor-mediated pathway, requiring the participation of cytoskeletal elements, principally actin.     

Effect of Staphylococcus aureus Cowan i bacteria on the mitogenic response of human B-cell subsets.

We have made a detailed investigation to determine which of the B-cell subsets could be stimulated by Staphylococcus aureus Cowan I bacterium (SpA CoI). B-cell subsets were separated from peripheral blood and tonsil lymphocytes by means of rosette formation with E, EAIgG, anti-immunoglobulin (Ig) conjugated OE (OE-Pro A) or by separation on a bovine serum albumin (BSA) discontinuous density gradient. The cells responding to SpA CoI included E receptor negative (E-), C3 receptor positive (C3R+), and surface Ig positive (SIg+) B-cell subsets. Among these B-cell subsets, FcR-n cells were more responsive than FcR+ cells. These B-cell subsets responded alone to SpA CoI and significantly proliferated, although, they failed to respond alone to pokeweed mitogen (PWM) and Protein A of S. aureus (Protein A). Among the SIg+ B-cell subsets stimulated with SpA CoI, IgM+ and IgG+ B cells showed much less response. Both Protein A receptor positive (Pro A . R+) and negative (Pro A . R-) cells responded well to SpA CoI. Fractionation of B cells on a BSA gradient revealed that comparatively small sized and denser B-cell subsets responded well to SpA CoI. From these criteria, it is suggested that B cells responding to SpA CoI are capable of stimulating not only mature B cells, but can also stimulate immature B cells.     

Potentiation of natural killer cell activity of human lymphocytes in vitro: the participation of interferon in stimulation with Staphylococcus aureus Cowan I bacteria but not with protein A.

In the previous paper we reported that human natural killer (NK) cell activity was augmented greatly by preincubation with Staphylococcus aureus Cowan I bacteria (SpA CoI) or its Protein A. We examined here whether the augmentation with these stimulants is ascribable to the direct activation of NK cells or mediated by some soluble factors produced by the stimulants. It was found that a significant amount of interferon (IFN) was produced by the SpA CoI-stimulation but not by the Protein A-stimulation, although the latter usually induced augmentation of NK-cell activity not less than SpA CoI-stimulation. IFN produced by SpA CoI was considered to belong to alpha-type IFN, because it was stable at pH 2.0 and could be neutralized effectively by anti-IFN alpha antibody. Kinetics of NK-cell activation by SpA CoI (but not by Protein A) were very similar to those by IFN alpha. Furthermore, augmentation of NK-cell activity with SpA CoI-stimulated supernatant was inhibited almost completely by diluted anti-IFN alpha antibody, whereas augmentation with Protein A-stimulated supernatant could not be abolished by the same treatment. It was, therefore, suggested that augmentation of NK-cell activity with SpA CoI might be ascribable in most part to the IFN induced, whereas Protein A can stimulated NK or T cells directly or soluble factors other than IFN might work as well.     

Induction of CD5 antigen on human CD5 B cells by stimulation with Staphylococcus aureus Cowan strain I

We have examined whether the CD5 phenotype could be inducedon human B cell surfaces by the polycional B cell stimulator,Staphyiococcus aureus Cowan strain I (SAC). Fresh tonsillarB cells were prepared by Percoll density gradient from E^–cells. The proportion of CD5⁺ B cells In the 50/60% and 60/70%interface high-density fractions varied between 1.2 and 10.2%depending on the tonsil preparations when they were placed onthe in vitro culture 12–60 h prior to flow cytometrlcanalysis. The expression of CD5 antigen obviously increasedin the presence of SAC (1:10⁵ v/v). The percentage of CD5⁺ Bcells varied from tonsil to tonsil, from 25.1 to 65.9% in aseries of experiments. The CD5⁺ B cells were found both amongCD23⁺CD25⁺CD71⁺ and CD23^–CD25^–CD71^– B cells.The level of CD5 expression was related to the cell size eniargement.The addition of anti-CD5 antibody in the culture blocked theCD5 induction by SAC without interfering with the expressionof other activation markers. A time-course study showed thatCD5 antigen appeared to be induced on the cell surface duringthe G₀ to G₁ phase transition in the cell cycle. When CD5⁺ andCD5^– B cells were separated by magnetic isolation, theCD5^– B cells showed DNA synthesis to the stimulation bySAC and expressed CD5 antigen on their cell surface. These resultssuggest that human CD5^– B cells can express the CD5 phenotypeby stimulationwith the polyclonal B cell stimulator, SAC.     

Tubuloreticular inclusions in skin biopsies from patients with HIV infection

Skin biopsies obtained from apparently normal skin from 15 HIV infected patients and 6 anti-HIV negative patients were examined by electron microscopy. Tubuloreticular inclusions (TRI) were detected within the cytoplasm of capillary endothelial cells in 5/5 AIDS patients and in 2/5 patients with AIDS related conditions. Biopsies from 5 asymptomatic HIV positive patients and the 6 control subjects were without ultrastructural alterations. The occurrence of TRI was related to low numbers of CD 4+ lymphocytes. 5/7 patients with TRI had elevated serum interferon activity, and in all of the patients without TRI, interferon activity was below detection level. The occurrence of TRI was not dependent on the presence of free p24 antigen in serum. It is concluded that the occurrence of TRI in entothelial cells of skin capillaries is associated with late stages of HIV infection and this may indicate a generalization of this change.     

Growth of Staphylococcus aureus Cowan I in the presence of subbactericidal concentrations of povidone-iodine

Staphylococcus aureus, strain Cowan I, is cultivated in the presence of povidone iodine (PVP.I) at the concentration of 0, 640, 960 micrograms/ml; growth curves points are platted (OD 640 mm) at 0, 1, 2.50, 3, 4.50, 7 and 24 hours. Growth curves in the presence of povidone alone as well as in the standard without antiseptic are similar and reproducible. A bacteriostatic effect is observed with 640 micrograms/ml PVP.I concentration compared to an incomplete bactericidal effect with 960 micrograms/ml concentration. Proteinee A detected by conditioned hemagglutination method is present in the centrifugation pellet of the standard and only in the supernatant of the test samples during the early phases of the growth. Production of alpha-ribitol-teichoic acid detected by counterimmunoelectrophoresis is not significantly different in the standard and in the presence of antiseptic. Biochemical characteristics studied by Api Staph gallery are identical for the control strain and the strain grown in the presence of PVP.I. Observed by scanning and transmission electronic microscope the bacterial cells do not present structural modification during growth with PVP.I.     

Effect of metalloproteinase from Staphylococcus aureus on in vitro stimulation of human lymphocytes

Metalloproteinase (MP) produced by the majority of Staphylococcus aureus strains exerts, in a wide concentration range (0.1-100 micrograms/ml), no cytotoxic action on mononuclear leukocytes of human peripheral blood. The enzyme itself does not appreciably stimulate proliferation and differentiation of lymphocytes in culture, but affects the stimulation of both T and B lymphocytes by polyclonal activators. The action is dose-dependent. High doses of MP (100 micrograms/ml) lower the blastic transformation after stimulation with Con A, SpA, NDCM, S. aureus strain Wood 46 and with suboptimal doses of PWM. Optimal concentrations of the enzyme potentiate the stimulation of lymphocytes by PWM, PHA, S. aureus strains Cowan 1 and Wood 46, and by NDCM. The same potentiation effect was achieved whether the enzyme was added concurrently with the mitogen or 18 h later. This implies that the beginning of cell activation is not affected. A high MP concentration decreases the production of Ig in culture after stimulation with PWM whereas lower concentrations of MP enhance this production. Production of Ig after stimulation with NDCM and Wood 46 is decreased by MP. The possible action of exoproteinase from S. Aureus on the immune response during infection is discussed.     

Activation of human lymphocytes in vitro by membrane-damaging toxins from Staphylococcus aureus.

Purified staphylococcal alpha-, gamma-, and delta-toxin were shown to cause activation of lymphoid cells from adult human donors and of cord cells in vitro as measured by [14C]thymidine incorporation after 7 days of incubation. T cell-enriched and T cell-depleted lymphocyte suspensions were activated in a similar fashion. Beta-toxin, on the other hand, exerted no valid stimulation of the various lymphocyte preparations. The lymphocyte-activating properties of alpha- and gamma-toxin were shown to be independent of their hemolytic capacity. The results probably reflect unspecific mitogen effects, but a component of specific reactivity cannot be excluded. We suggest that the unspecific triggering of lymphocytes in vitro is caused by surface-active properties of the toxins.     

Human B cell activation and cell cycle progression: stimulation with anti-mu and Staphylococcus aureus Cowan strain I

The responses of resting human B lymphocytes to a variety of activation signals were studied. Human tonsillar B lymphocytes were separated according to size by countercurrent elutriation. The small B lymphocytes were then stimulated in vitro with various concentrations of anti-mu antibody in the presence or absence of B cell growth factor (BCGF) or with Staphylococcus aureus Cowan strain I (SAC). Cellular volume changes and RNA synthesis were measured over the first 24 h of stimulation and were similar with either 15 micrograms/ml of anti-mu, 100 micrograms/ml of anti-mu, or SAC. In the subsequent 24 h, however, substantial increases occurred in the amount of RNA synthesis and cell enlargement only in those cultures stimulated with 100 micrograms/ml of anti-mu or SAC, but not in the cultures stimulated with 15 micrograms/ml of anti-mu. The addition of BCGF to those cultures stimulated with 15 micrograms/ml of anti-mu did not alter the increases in cellular volume and RNA synthesis found 24 h after stimulation with anti-mu alone. However, over the subsequent 24 h, the presence of BCGF in culture enhanced both B cell volume changes and RNA synthesis, when compared to cultures stimulated with 15 micrograms/ml of anti-mu alone. In addition, BCGF enhanced DNA synthesis in cultures stimulated with low and high concentrations of anti-mu. DNA content changes following stimulation with anti-mu, anti-mu plus BCGF, and SAC were also measured using propidium iodide staining and flow cytometry. Optimal concentrations of anti-mu induced 20% of the resting B cells to enter S phase, while optimal concentrations of anti-mu plus BCGF or SAC induced approximately 40%. Finally, prestimulation of resting B cells for 24 h with a low concentration of anti-mu, sufficient for cell enlargement but not S phase progression, allowed for rapid entrance of the prestimulated B cells into S phase when a high concentration of anti-mu or SAC was added. These findings suggest the existence of a control point in the progression of human B cells through the cell cycle. This control point is located in the G1 phase of the cycle and is reached 24 to 36 h after a surface immunoglobulin-mediated stimulus.