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Biomedical subjects

A Muraguchi

Publications and source records attributed to A Muraguchi.

At least 73 records · Page 4Linked to original sources
PubMed1984

Inhibition of human B cell activation by diterpine forskolin: interference with B cell growth factor-induced G1 to S transition of the B cell cycle.

We have previously demonstrated that a series of sequential steps are involved in the induction of a resting human B cell to proliferate. In this system, initial activation signals were delivered by either in vivo or in vitro stimulation, and proliferative signals were delivered by a B cell growth factor (BCGF) that was derived from a human T-T cell hybridoma. The involvement of adenosine 3',5'-monophosphate (cAMP) in regulating the growth and differentiation of lymphocytes has been of considerable interest. Diterpine forskolin has been reported to be a unique adenylate cyclase activator in membranes from mammalian tissues that results in elevations of intracytoplasmic cAMP. We have examined the effect of this drug on the progression of human B cells through their activation cycle. It was found that forskolin causes a rapid and sustained increase of cytoplasmic cAMP in purified small and large B cells. In the in vitro costimulation of small resting B cells with anti-mu plus BCGF, forskolin inhibited the proliferative response of B cells in a dose-dependent manner. This forskolin-mediated suppression of B cell proliferation was found when the drug was added to the cultures as late as 36 hr after initial stimulation. Of note is the fact that the anti-mu-induced RNA synthesis as well as cell enlargement in resting B cells was not inhibited by the addition of forskolin, whereas BCGF-induced proliferative response of activated B cells was markedly inhibited by the drug. Thus, these data demonstrate that forskolin, an agent that elevates cytoplasmic cAMP levels, has relatively little effect on early events in the human B cell cycle (G0 to G1) transition, but selectively inhibits the progression of BCGF-induced G1 to S phase transition of B cells.

Adolescent↗
PubMed1984

Sequential requirements for cell cycle progression of resting human B cells after activation by anti-Ig.

We have previously demonstrated that in vivo activated normal human B cells reflected differential sensitivity on the basis of cell size to either an activation signal delivered by anti-mu or a proliferative signal delivered by a monoclonal B cell growth factor (BCGF) produced by a human T-T hybridoma. In this manuscript, we have shown that by using an in vitro co-culture system of stimulation with anti-mu and BCGF, purified small (resting) B cells could be induced by anti-mu stimulation to express functionally active BCGF acceptor sites concomitant with RNA synthesis and cell enlargement. We have demonstrated that purified small B cells could be activated to increase both cell size and RNA synthesis within 8 hr of anti-mu stimulation. DNA synthesis by anti-mu-stimulated B cells began at 36 to 44 hr from initial stimulation only when BCGF was added to the culture. In addition, large B cells that had been incubated with anti-mu for 24 to 48 hr manifested significant incorporation of [3H]thymidine if BCGF was added to cultures. These data strongly suggest that anti-mu may induce the G0 phase B cells into the G1 phase and that BCGF may then push at least a subset of these G1 phase-B cells into the S phase. This experimental system may provide a useful model of normal human B cell activation and proliferation and may allow a more precise delineation of each phase of this cascade in the B cell repertoire.

Adolescent↗
PubMed1984

Differential expression of cell activation markers after stimulation of resting human B lymphocytes.

The interaction of antigen with surface Ig receptors initiates a complex process by which resting B lymphocytes are activated. In this study, small resting B lymphocytes were isolated by countercurrent elutriation of tonsillar B lymphocytes and stimulated with a range of concentrations of anti-mu and B cell growth factor (BCGF). The subsequent expression of two glycoproteins known to be absent from resting B lymphocytes but present on activated cells were analyzed with the monoclonal antibodies 4F2 and 5E9. The antigen recognized by the 4F2 monoclonal was expressed early in the G1 phase of the cell cycle and correlated with blast transformation; this was demonstrated by stimulating resting B cells with a low concentration of anti-mu sufficient for cellular activation but not proliferation. The 5E9-defined antigen was not present unless the B cells were stimulated with a higher concentration of anti-mu or a BCGF was added to the low concentration of anti-mu. The addition of hydroxyurea to culture blocked the entrance of stimulated B cells into S phase but did not interfere with the expression of either 4F2 or 5E9. Thus, whereas the monoclonal antibody 4F2 recognized cells in early G1, 5E9 recognized cells in late G1. By double immunofluorescence staining with propidium iodide and either 4F2 or 5E9, all cells in S, G2, and M phases were demonstrated to be both 4F2- and 5E9-positive. The monoclonal antibodies 4F2 and 5E9 can be used to distinguish an activated from a resting human B lymphocyte and to delineate sequential steps in the activation process.

Adolescent↗
PubMed1984

The effects of interleukin-1, interleukin-2, alpha-interferon, and gamma-interferon on human B lymphocytes.

The direct effects of interleukin-1 (IL-1), interleukin-2 (IL-2), alpha-interferon, and gamma-interferon on human B cell activation, proliferation, and differentiation have been examined and compared to the effects of B cell growth factor (BCGF) and B cell differentiation factor (BCDF). Human tonsillar B lymphocytes were separated according to cell size into two nonoverlapping and functionally distinct populations. The small fraction was incubated with IL-1, IL-2, alpha-interferon, or gamma-interferon, and subsequent DNA synthesis and immunoglobulin production were measured. None of the factors induced these cells to proliferate or to differentiate. Similarly, the large fraction of B cells was incubated with IL-1, IL-2, alpha-interferon, gamma-interferon, BCGF, or BCDF. BCGF augmented the proliferation, and BCDF augmented the differentiation of the large B cells. In comparison, IL-1, IL-2, alpha-interferon, or gamma-interferon had either a minor effect or no effect on large B cell proliferation and differentiation. Thus, this study suggests that IL-1, IL-2, alpha-interferon, and gamma-interferon are not important direct signals for mature B cell function when present in the absence of other signals.

Adolescent↗
PubMed1983

Selective suppression of an early step in human B cell activation by cyclosporin A.

The effect of cyclosporin A (CsA), a fungal metabolite with immunosuppressive properties, on the induction of human B cell proliferation and differentiation, has been described. CsA had a selective inhibitory effect on the activation phase of the cell cycle vs. the proliferation phase following preactivation of the cells. Cell enlargement and RNA synthesis of small resting B cells triggered by anti-mu were inhibited by addition of CsA (5-500 ng/ml). The inhibitory effect of CsA was found only when the drug was added within 24 h of initiation of culture. In marked contrast, once small B cells were activated by anti-mu, the resulting large, activated B cells could be induced to initiate DNA synthesis by incubation with B cell growth factor (BCGF), and addition of CsA (1-1,000 ng/ml) to the culture did not suppress this BCGF-induced B cell proliferation. Addition of CsA to cultures of B cells which had been preactivated with Staphylococcus aureus Cowan strain I (SAC) and were already proliferating did not suppress B cell differentiation factor (BCDF)-induced differentiation of these cells. Thus, these data indicate that CsA can be used as a pharmacologic tool to dissect out human B cell responses into two distinct steps: (a) the initial activation step induced by anti-Ig, which is characterized by cell enlargement, RNA synthesis, and expression of receptors for BCGF; and (b) the proliferative step induced by BCGF in these preactivated B cells that undergo DNA synthesis and can then go on to differentiate in the presence of BCDF. In this regard, CsA selectively suppresses an early step of human B cell activation and has little inhibitory effect on the subsequent factor-dependent proliferation and differentiation.

Adolescent↗
PubMed1983

Differential sensitivity of human B cell subsets to activation signals delivered by anti-mu antibody and proliferative signals delivered by a monoclonal B cell growth factor.

The present study demonstrates the minimal, optimal, and synergistic signals involved in the activation of normal human peripheral blood and tonsillar B cells to proliferation. Initial activation signals were delivered to B cells by low concentrations of anti-mu antibody which did not induce proliferation by themselves. However, marked synergy was seen when anti-mu antibody was added to cultures in the presence of monoclonal B cell growth factor (BCGF) obtained from a human T-T cell hybrid such that the B cells underwent substantial proliferation. This latter proliferation was seen without maturation into Ig-secreting cells, which indicates that the BCGF is not a differentiation signal but a signal that drives the cell up to but not beyond the proliferative phase. Of note was the fact that B cells reflected differential sensitivity on the basis of size to either the activation signal delivered by anti-mu antibody or the proliferative signal delivered by BCGF. BCGF directly stimulated the larger B cells in the normal tonsillar B cell repertoire to proliferate without the requirement for an in vitro activation signal, which indicates that the cells had already received some form of activation signal in vivo. Indeed, these cells expressed the 4F2 antigen found on activated but not resting lymphocytes. In contrast, the smaller tonsillar B lymphocytes did not express the 4F2 activation antigen and required activation by anti-mu antibody, which did not of itself induce proliferation, but which acted in synergy with BCGF for substantial proliferation of the B cells. These studies thus provide a useful model of human B cell activation, proliferation, and differentiation and allow a more precise delineation of each phase in this cascade.

Antibodies, Anti-Idiotypic↗
PubMed1983

Development of a human T-T cell hybridoma secreting B cell growth factor.

The success of long-term culture of normal human and murine B cells has been hampered by the limited availability of soluble factors capable of maintaining proliferation of activated B lymphocytes. Previous experiments using various culture-derived supernatants in a human system were unable to separate the activities of B cell growth factor (BCGF) and interleukin 2 (IL-2) by immunochemical means. Thus, purified factors with BCGF activity in the absence of IL-2 activity have not been available for study. In the present study, normal human peripheral blood T cells were fused with the hypoxanthine/aminopterin/thymidine-sensitive human T-leukemic cell line, CEM-6. Supernatants from the resulting hybrid cells were tested for the ability to maintain proliferation of normal human B cells in a recently described assay system for human BCGF. Hybrids demonstrating BCGF activity were cloned by limiting dilution. One hybrid clone, 2B11, continued to support proliferation of B cells in both long-term cultures and 6-d assays at a level significantly above that seen with conventionally produced growth factors. No IL-2 activity was found in the supernatant from hybrid 2B11. The hybridoma supernatant was fractionated by gel filtration, and maximum proliferation of B cells was supported by the 18-20,000 mol wt protein fraction. Thus, a human T-T cell hybridoma that has BCGF activity in the absence of any demonstrable IL-2 activity has been developed. Human T-T cell hybridomas secreting discrete immunoregulatory factors should prove to be powerful tools in dissecting the mechanisms of immunoregulation of human lymphocyte function.

B-Lymphocytes↗
PubMed1983

Soluble factors involved in B cell differentiation: identification of two distinct T cell-replacing factors (TRF).

Highly purified B lymphocytes cultured with affinity-purified goat anti-mouse IgM antibodies and B cell growth factor (BCGF) proliferate but fail to differentiate into immunoglobulin-synthesizing cells during 4-day cultures. Induction of immunoglobulin (Ig) synthesis depends on the presence of two additional T cell-derived factors (T cell-replacing factors [TRF]). One TRF, designated B15 1K 12-TRF, is found in the supernatant of B15 1K 12 T hybridoma cells. It is required relatively early in these cultures and appears to cause an increase in cell yield as well as Ig synthesis. The second factor, designated EL-TRF, is found in PMA-induced EL-4 supernatant. It appears to be different from both BCGF and IL 2 in that it can be separated from the former by isoelectric focusing and from the latter by phenyl-Sepharose chromatography. EL-TRF is still active if added on the last day of a 4-day culture. A model for the action of anti-IgM and these three T cell-derived factors in B cell activation and differentiation is proposed.

Animals↗
PubMed1983

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.

B-Lymphocytes↗
PubMed1982

The requirement for esterase activation in T cell replacing factor (TRF)-induced IgG production in a human B blastoid cell line.

Involvement of stimulus-activatable serine esterase in the process of TRF-induced IgG production in a human B blastoid cell line (CESS) was demonstrated. Addition of DFP or PMSF (1 X 10(-3) M to 1 X 10(-5) M) showed a dose-dependent inhibitory effect on TRF-induced IgG induction in CESS cells. Other organophosphorous inhibitors, such as p-NPEPP and PEPP, were also effective, but a nonphosphorylating analogue of DFP, DMP, was not inhibitory. Simultaneous addition of DFP with TRF showed the inhibitory effect, and the inhibition was irreversible. However, pretreatment of the cells with DFP did not abrogate TRF-induced IgG production. In synchronized cells, signals provided by TRF could be transduced only when cells were in the G1-phase, and DFP showed an inhibitory effect only when it was added to cells in the G1-phase with TRF. One amino acid derivative (AAME) partially inhibited TRF-induced IgG production.

Adenosine Monophosphate↗
PubMed1982

Induction of proliferation and Ig production in human B leukemic cells by anti-immunoglobulins and T cell factors.

The proliferation and differentiation of human leukemic B cells (B-CLL cells) with anti-Ig and T cell-derived helper factors are described. Stimulation of B-CLL cells with anti-Ig and T helper factors could induce proliferation as well as differentiation into IgM- and IgG-producing cells. Neither anti-Ig nor T helper factors alone could induce any proliferation and/or differentiation of B-CLL cells. Not only whole molecules of anti-Ig but also F(ab')2 fragments could induce proliferation and differentiation of B-CLL cells in the presence of T helper factors, but monovalent Fab' fragments were not effective. Induction of both IgM and IgG with the same idiotype was confirmed by immunofluorescent and SDS-PAGE analysis. By employing an IL 2-dependent cytotoxic T cell line and a TRF-responsive B cell line, T cell factors were separated into a fraction with IL2 activity but no TRF activity and a fraction with TRF activity but no IL 2 activity by chromatofocusing. Anti-Ig and IL 2 fraction could induce proliferation of B-CLL cells, but TRF fraction was not effective for the induction of proliferation in anti-IG-stimulated cells. For IgM and IgG production, anti-Ig and both IL 2 and TRF fractions were required. Depletion of IL 2 fraction in the first 2 days' culture inhibited Ig production, whereas the absence of TRF fraction in the first 2 days did not show any inhibitory effect on Ig production.

Animals↗
PubMed1982

Proliferative responses of normal human B lymphocytes. Development of an assay system for human B cell growth factor (BCGF).

A simple and reproducible system for inducing and measuring proliferation of normal human peripheral blood B lymphocytes was developed and employed as an assay for BCGF activity contained in supernatants of cultured human mononuclear cells. SAC was used to activate human peripheral blood B cells to develop into blasts with low levels of proliferation, and a marked synergistic effect on the proliferation of these activated B cell blasts was demonstrated when various culture-derived supernatants containing exogenous growth factors were added to the system. Substantial BCGF activity was obtained from culture supernatants of co-cultures of pooled allogeneic mononuclear cells from two donors, who were also stimulated with PHA. Kinetic studies demonstrated that maximal BCGF activity was produced in cultures carried for 72 hr or longer. A linear relationship was observed between the logarithm of dilution of added factor and incorporation of 3H-thymidine in the responding SAC-stimulated B cell blasts, suggesting the applicability of this system in screening and quantitating BCGF activity in supernatants of various sources, such as T cell clones, T cell neoplasms, and functional human T-T hybridomas. Furthermore, the system could potentially be adopted to the long-term culture of proliferating normal human B cells.

B-Lymphocytes↗
PubMed1982

IgG induction in a human B cell line by red cell-mediated microinjection of the cytoplasm from T cell factor-stimulated B cells.

Generation of a cytoplasmic factor(s) that induced IgG secretion in nonstimulated cells was demonstrated in TRF-stimulated cells by using red cell-mediated microinjection. Injection of the cytoplasm from a TRF-stimulated B lymphoblastoid cell line (CESS) into nonstimulated cells induced an increase of IgG-producing cells. Injection of TRF itself did not induce an increase of IgG-producing cells. Active substance(s) in the cytoplasm were generated at 2 hr after TRF stimulation, and IgG-producing cells reached their maximum level at 40 hr after injection of the factor(s). DFP, but not actinomycin D, inhibited the generation of the cytoplasmic factor(s). The activity of the cytoplasmic factor(s) was not destroyed by RNase and not absorbed with anti-IgG. These results suggested that binding of TRF with its acceptors induced the generation of the cytoplasmic factor(s) involved in the transmission of TRF-mediated signals from membrane to nuclei.

Antibody Formation↗
PubMed1982

Induction of IgG production in a human monoclonal B lymphoblastoid cell line by a B cell-specific monoclonal antibody.

IgG production was induced in a human B blastoid cell line, CESS, by stimulation with a human B cell-specific monoclonal antibody (IIIA4). The number of IgG-producing cells reached its maximal level within 48 hr after incubation with IIIA4. IgG-producing cells induced by an optimal concentration of the antibody was about one-half as many as those induced with TRF, but higher concentrations of the antibody were rather inhibitory. Simultaneous addition of an optimal concentration of the antibody with TRF partially inhibited TRF-induced IgG production in CESS cells, but higher concentrations of the antibody did not block TRF-induced IgG production. TRF activity was completely absorbed with IIIA4-coated CESS cells as well as nontreated CESS cells. These results suggested that the membrane antigen recognized by IIIA4 was not the acceptor molecule for TRF. IIIA4 did not show any inhibitory effect on PWM-induced proliferation of normal B cells, but completely inhibited PWM-induced Ig production of normal B cells.

Absorption↗
PubMed1982

B cell growth factor and T cell growth factor produced by mitogen-stimulated normal human peripheral blood T lymphocytes are distinct molecules.

Using a recently described assay for inducing and measuring the proliferation of normal human B cells in the absence of differentiation, we have demonstrated that B cell growth factor (BCGF) activity can be obtained from the culture of human peripheral blood mononuclear cells in the presence of mitogens. Using Sephacryl S-200 gel filtration, BCGF activity was demonstrated in the 20 to 30K m.w. fractions of mitogen-stimulated peripheral blood mononuclear cells. However, this fraction also showed substantial T cell growth factor (TCGF) activity. Despite this overlapping of m.w., BCGF activity could clearly be separated from TCGF activity by selective absorption of factor-containing supernatants with the interleukin 2 activity by using interleukin 2-dependent cells without any diminution of BCGF activity, strongly suggesting that these two factors may be distinct molecular entities.

B-Lymphocytes↗
PubMed1981

T cell-replacing factor- (TRF) induced IgG secretion in a human B blastoid cell line and demonstration of acceptors for TRF.

IgG-secretion was induced in a human B blastoid cell line, CESS, by the addition of partially purified T cell-derived helper factor(s) (TRF), which had been obtained from PHA-stimulated human T cells. The number of IgG-producing cells in CESS cells reached its maximal level (10% of total cells) within 48 hr after the addition of TRF. TRF did not affect the proliferation of CESS cells and the block of cell proliferation with hydroxyurea did not inhibit the increase of IgG-producing cells, showing that TRF induced IgG-production in CESS cells without any requirement of cell division. TRF activity was completely removed by CESS cells but TCGF-activity in the same preparation was not absorbed with CESS cells. On the other hand, TCGF-dependent human killer cells absorbed TCGF activity but not TRF activity in the same preparation. The binding of 125I-labeled factor(s) on CESS cells was also demonstrated. These results showed the presence of acceptors for TRF on the surface of CESS cells and this cell line will provide useful means for the chemical characterization of acceptors and for the study of the mechanisms of the signal transmission through acceptors.

Absorption↗
PubMed1980

In vitro immune response of human peripheral lymphocytes. VII. Effect of anti-mu and anti-delta antibodies on B colony formation and detection of abnormal B cells in patients with juvenile rheumatoid arthritis.

Effect of anti-mu and anti-delta antibodies on PHA- and protein A-induced B colony formation was studied. Anti-mu antibody at any concentrations tested did not show inhibitory or enhancing effect on colony formation. On the other hand, anti-delta antibody enhanced both PHA- and protein A-induced colony formation. Optimum concentration of anti-delta antibody for maximum enhancement was 0.1 microgram/ml. and F(ab')2 fragment of anti-delta antibody also showed comparable enhancing effect. Simultaneous addition of IgD with anti-delta antibody abrogated anti-delta-induced enhancement, and anti-delta antibody did not show any facilitation of colony formation in IgM+ IgD- cell population. In marked contrast with normal B cells, anti-mu antibody showed a remarkable enhancing effect on protein A-induced colony formation of B cells from JRA patients. F(ab')2 fragment of anti-mu antibody also showed comparable enhancing effect. Anti-mu antibody did not show any enhancement of colony formation of B cells from several other autoimmune diseases. The result indicated the presence of abnormal B cells in JRA patients.

Animals↗