B cell maturation factor (BMF): a lymphokine or family of lymphokines promoting the maturation of B lymphocytes

Two in vitro B cell tumor lines have been used to characterize and partially purify a lymphokine, or family of lymphokines, from monoclonal helper T cell immune response supernatants. These lymphokines induce the pre-B-like 70Z/3 tumor cell to synthesize Ig L chains and express complete Ig molecules on its cell surface, and cause the mature B cell-like WEHI-279 tumor cell to increase its ratio of secretory to membrane mu production, begin high rate Ig secretion, and then die. Most of the activity responsible for these changes co-purifies during five different separation procedures, implying the existence of a discrete molecule or closely related class of molecules able to mediate all of these effects. The molecules active in these systems appear distinct from the other lymphokines IL 1, IL 2, G/M-CSA, TRF, IFN, BCGF, and the activity variously termed IL 3/BPA/PSF/HCGF/MCGF, etc. We call these B cell-differentiating molecules BMF, or B cell maturation factor(s). The BMF molecules are mildly acidic (pI 5 to 6 in various conditions), extremely hydrophobic, probably heterogeneously glycosylated glycoproteins, with an apparent m.w. of 50,000 to 55,000 by gel permeation chromatography and 16,000 by SDS-PAGE. BMF has been purified approximately 3000-fold by three sequential chromatographic steps, with the use of the B tumor line assay systems. BMF molecules thus purified also cause normal resting splenic B cells to mature to the state of active Ig secretion.     

Characterization of lymphokines mediating B cell growth and differentiation from monoclonal anti-CD3 antibody-stimulated T cells

Addition of anti-CD3 mAb 147 (IgG1), 446 (IgG1), or 454 (IgG2a) to cultures of T plus non-T cells can result in both B cell growth and differentiation. To determine whether lymphokines mediating these activities were similar to those described from conventional mitogen-induced T cell activation, normal peripheral blood T cells were stimulated with anti-CD3 mAb for 48 h. The supernatants were assayed for factors inducing B cell growth or differentiation (BCDF). A marked increase in Ig secretion was observed when either EBV-transformed B cell lines or normal B cells, pre-activated with Staphylococcus aureus Cowan I strain, were cultured in the presence of mAb 446 (anti-CD3) stimulated T cell supernatant whereas no significant increase in Ig secretion was noted with either mAb 454- or 147-induced T cell supernatant despite equivalent T cell proliferative responses to these antibodies. In contrast, IL-2 secretion was detectable in T cell supernatants from T cells stimulated with either mAb 454 or 147 but not 446. Factors promoting B cell proliferation were detected in all antibody-stimulated T cell supernatants but, contrary to BCDF, appear to act only on non-activated B cells. To determine whether these effector activities were due to distinct lymphokines, supernatants were pooled and concentrated by ammonium sulfate precipitation. Superose 12 permeation chromatography revealed BCDF activity with an apparent Mr of approximately 30,000 Da. The growth factor activity eluted over a wider and higher molecular weight range which overlapped the differentiation factor activity. Fractions containing BCDF activity were pooled, dialyzed, applied to a Mono Q anion-exchange column, and eluted with a linear NaCl gradient. The growth factor activity came off in a single-peak while BCDF was found divided into two major areas. The growth factor eluted at an ionic strength between the two BCDF activities. BCDF has an apparent isoelectric point (pI) of 6, in contrast to the reported pI 5 for IL-6 and more acidic than the documented basic pI of IFN-gamma. Lastly, peaks with BCDF activity were not active in assays for either IL-2 or IL-4. In addition, a rabbit anti-IL-6 heteroantiserum failed to inhibit the pI 6 BCDF, suggesting non-identity between IL-6 and anti-CD3 induced BCDF. Thus, anti-CD3 activated T cells generate both growth factor activity and BCDF as separate molecular entities distinct from IFN-gamma, IL-2, IL-4, and conventional IL-6.     

Separable helper factors support B cell proliferation and maturation to Ig secretion

The 24-hr culture supernatant of Con A-activated spleen cells (SN) contains helper factors that enable maturation to high-rate polyclonal Ig secretion and enhance proliferation in cultures of mouse B cells activated with the F(ab')2 fragment of class-specific rabbit antimouse IgM antibody (anti-Ig). When interleukin 2 (IL 2), also called T cell growth factor, is removed from SN by absorption with an IL 2-dependent cell line at either 4 degrees C or 37 degrees C, all the helper activity for anti-Ig-activated B cells is also removed. Partial removal of IL 2 results in partial removal of helper activity for B cells. However, the IL 2-depleted SN appears to contain another helper factor, TRF, that enables anti-Ig-activated B cell cultures to mature to high-rate Ig secretion. This TRF activity is revealed by adding purified human IL 2 or an IL 2-containing supernatant of a cloned, lectin-activated T cell hybridoma line (FS6-14.13) to Il 2-depleted SN, which restores the polyclonal antibody response to anti-Ig. The hybridoma supernatant by itself supports proliferation of anti-Ig-activated B cell cultures, as measured by an increase in cell number, but not maturation to Ig secretion. This proliferative response is likewise IL 2 dependent, although purified IL 2 with anti-Ig is not sufficient. These experiments define separable combinations of factors acting on anti-Ig-activated B cell cultures, one of which (SN) results in both proliferation and maturation to high-rate Ig secretion, whereas the other (hybridoma supernatant) results in proliferation only. IL 2 appears to be an essential component of both combinations, although the target cell for IL 2 action in this system remains to be determined.     

Factors affecting cell viability during bisection of bovine embryos

The aim of this study was to investigate the effects of temperature, cytochalasin B, sucrose, cell number and developmental stage of embryos on cell loss and cell lysis during embryo splitting. Day-7 morulae and blastocysts were bisected using a metal blade. In Experiment 1, splitting of embryos in control medium (PBS + 10% fetal calf serum) was compared with splitting in the presence of 7.5 mul/ml cytochalasin B. In Experiment 2, the control medium was compared with medium supplemented with 200 mM sucrose. In Experiment 3, the control medium was compared with medium supplemented with sucrose and cytochalasin B. Cell viability was measured by staining nuclei of embryos with Hoechst 33258 and propidium iodide. Cells with nuclei exhibiting pink fluorescence were considered lysed, while blue fluorescence was considered an indication of viable cells. Cells disaggregated during splitting were classified as extruded cells. An effect of the developmental stage was observed in the pooled data from the control groups of the 3 experiments, with a higher proportion of viable cells in bisected morulae compared with bisected blastocysts (77.6 vs 70.0%; P = 0.003). However, as there was no effect of cell number (P = 0.85), the influence of the developmental stage can be contributed to morphological changes rather than to increase of cells associated with this change. In Experiment 1, the cytochalasin B-treated embryos contained a higher percentage of viable cells than the control embryos after removal of the developmental stage effect (P < 0.01). In Experiment 2, no effect on sucrose could be observed. In Experiment 3, the combined use of sucrose and cytochalasin B tended to increase the proportion of cells surviving bisection, but this difference was not significant. In Experiment 1, there was a correlation between viable cells and temperature during splitting (r = 0.42, P = 0.05; temperature range 8.1 degrees C to 15.6 degrees C). No correlation was found in any other group in any of the experiments, nor in the pooled data from the control groups in the 3 experiments.     

Regulation of peripheral B cell maturation

Although it is clear that the final phases of B cell maturation occur after newly formed B cells exit the bone marrow, the mechanisms underpinning the maturation, selection, and long-term survival of immature peripheral B cells remain poorly understood. Here, we review recent advances in our understanding of how B cell receptor (BCR)-mediated signaling events integrate with additional environmental cues to promote the selection and differentiation of immature B cells into functionally distinct subpopulations of mature B cells. We pay particular attention to the role of the Baff cytokine family and the Notch receptor-ligand family and their unique roles in promoting B cell survival and differentiation into follicular and marginal zone B cells.     

The poised B cell: lymphokines induce an Ia-increase and antigen-presenting function in B cells

Individual murine B cells express a wide range of Ia densities on the plasma membrane. Here we demonstrate that a dramatic increase in B-cell Ia could be induced by overnight exposure to an uncharacterized lymphokine (LK). Membrane I-A and I-E molecules were both increased after LK treatment, whereas membrane IgM remained unchanged. Two subpopulations of B cells were identified, based on their requirements for expressing maximal Ia; one subpopulation required only LK, the other required both LK and T cells in the overnight culture. Functional changes accompanied the Ia increase. The functional capacity to present antigens to T cells was lacking in normal resting B cells, but was acquired following LK treatment. We suggest that the LK-treated B cell has achieved a new differentiation state, one of preparation for interaction with T cells. We term this state the "poised" B cell, and propose that B cells in the poised state may significantly contribute to T-cell activation as antigen-presenting cells. Moreover, poised B cells may themselves find an advantage over normal B cells in successfully acquiring T-cell help.     

Effects of lymphokines and immune complexes on murine placental cell growth in vitro

Isolated murine placental cells obtained at Day 16 of allogeneic gestation (C3H x DBA/2J) were cultured for 3 days alone or in coculture with irradiated mouse splenocytes at the end of which 3H-thymidine was added for an additional 18-h culture to assess cell proliferation. Placental cell proliferation was significantly enhanced at spleen cell:placental cell ratios of 10:1 and 25:1 above that observed in the absence of added spleen cells. The stimulatory effect of irradiated allogeneic (C3H plus Balb/cJ) spleen cell cultures was significantly greater (approximately 2-fold) than that of isogeneic spleen cells (C3H alone). Conditioned medium from murine spleen cells cultured with concanavalin A (ConA) to induce lymphokine production had dose-dependent inhibitory effects on proliferation when added to placental cell cultures over a range of concentrations from 10 to 40% (vol:vol). Addition of pseudo "immune complexes" in the form of heat-aggregated human gamma globulin (AHGG) to culture medium failed to alter placental cell proliferation over a range of concentrations from 2 to 200 micrograms/ml either in the absence or presence of ConA-conditioned medium. In contrast to late-gestational stage placental cells, cell suspensions obtained from Days 8-9 murine ectoplacental cone (EPC) outgrowths, or from earlier stage placentas (Days 12-14) responded to low concentrations of conditioned medium from ConA-stimulated splenocytes with increased proliferation. The effect was less impressive on placental cells at gestational ages later than 12 days than on earlier stage preparations. On all placental cell suspensions tested, as well as EPC cells, a clear-cut inhibition of growth was observed at high doses of conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation of events mediating B cell proliferation and Ig production by using T cell membranes and lymphokines

The initiation by Th cells of B cell proliferation and differentiation to produce Ig involves both cell contact- and lymphokine-mediated signals. Plasma membrane-enriched fractions from stimulated, but not unstimulated, Th cells induced Ag nonspecific and MHC unrestricted proliferation of 60 to 70% of small dense B cells. Induction of stimulatory membrane activity was inhibited by cycloheximide, and the activity was eliminated by both protease and heat treatment of membranes. Membrane-stimulated B cells did not differentiate to secrete Ig; however, addition of a lymphokine-containing supernatant from activated Th cells or the combination of IL-4 and IL-5 resulted in substantial Ig production, predominantly of the IgM, IgG1, IgA, and IgE isotypes. The quantity and isotype distribution of the antibodies secreted were similar to those produced after B cell activation by the intact Th cells and Ag. Therefore, membranes from activated Th cells in combination with lymphokines normally secreted by such cells can replace intact Th cells and provide a defined system to identify molecular events important for B cell activation.     

Effect of oxytocin on neuroblastoma cell viability and growth

Oxytocin, released in response to different physiological stimuli, could play a key role in reducing stress reaction. It was suggested that it has protective effect against inflammation and consequences of oxidative stress. Mechanisms how oxytocin effects mediated in the brain tissue are unclear. In this study, oxytocin effect on cell growth and neuronal viability was examined. Human neuroblastoma (SH-SY5Y and SK-N-SH) and glioblastoma (U87MG) cells were exposed to different concentrations of oxytocin for 12-96 h. Potential protective effect of oxytocin treatment was investigated after exposing cells to oxidative stress using hydrogen peroxide (50 mM, 2 h) or 6-hydroxydopamine (25 μM, 24 h). Cell proliferation was measured by cell counting and cell viability was examined by MTT assay. Protein expression of selected neurotrophic factors was measured as an additional parameter. Oxytocin (1 μM) significantly increased cell number in all three cell types. Viability of SH-SY5Y cells was increased in the presence of oxytocin without significant effect of dose (0.01-1 μM). Cell death induced by hydrogen peroxide was not prevented by incubation with oxytocin. Oxytocin pretreatment blunted neurotoxin 6-OHDA reduction of cell viability in SH-SY5Y cells. Oxytocin (1 μM, 12 h) elevated amount of total proteins without increasing levels of brain-derived neurotrophic factor and neurotrophic growth factor. In conclusion, oxytocin increases growth and viability of neuroblastoma and glioblastoma cells without activation of neurotrophic factors. Oxytocin does not have protective effect in oxidative stress; however, it might be important for neuroprotection to dopaminergic neurons. Its proliferative effect might be important in native cell life, euplastic processes, and tumor progression.     

Extracellular and intracellular factors affecting nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles

Nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles were examined. Oocyte-cumulus complexes were collected from small (1-2 mm in diameter), medium (3-6 in diameter) and large (7-8 mm in diameter) follicles and cultured in a modified tissue culture medium 199 for 44 h. Nuclear maturation was evaluated after orcein staining, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay. Oocyte diameter, cumulus morphology, steroid hormones and glutathione in the follicular fluid (FF), were also examined. Significantly higher proportions of oocytes collected from large and medium follicles reached metaphase II than did oocytes from small follicles. Oocytes from small follicles also had a smaller size. GSH content was significantly higher (p < 0.05) in oocytes from large (14.24 +/- 2.1 pmol/oocyte) and medium (13.69 +/- 1.5 pmol/oocyte) follicles than in oocytes from small (9.44 +/- 1.28 pmol/oocyte) follicles just after collection. After maturation, oocytes from medium follicles had a higher GSH concentration than oocytes from small follicles. It was found that between 49.7 +/- 5.18 nM and 52.25 +/- 0.78 nM GSH was present in FF but there was no statistical difference between follicle sizes. A significantly higher (p < 0.001) estradiol level was present in FF from large follicles (299.2 +/- 68.6 ng/ml) than from medium (40.0 +/- 6.4 ng/ml) and small (41.2 +/- 3.7 ng/ml) follicles. Progesterone concentrations in FF from large (281.6 +/- 45.9 ng/ml) and medium (267.5 +/- 38.6 ng/ml) follicles were significantly higher than that (174.7 +/- 22.0 ng/ml) from small follicles. These results indicate that the oocyte's ability to accumulate intracellular GSH during maturation, and extracellular steroid hormones and cumulus cells, affect the competence of porcine oocytes to undergo nuclear and cytoplasmic maturation.     

Production of lymphokines affecting tumor cells by T-T hybridomas

T-Cell hybridomas were constructed by fusing BW5147, an AKR lymphoma, with concanavalin A-stimulated murine splenic lymphocytes. The hybrids which were formed were studied for their ability to produce a lymphokine which inhibits tumor cell migration (TMIF) as well as macrophage migration (MIF) using in vitro assays. Clones were identified which affected tumor cell motility without exerting similar effects on murine macrophages, although the opposite effect was not observed. Although noncoordinate production of these factors cannot be unequivocally established, these results demonstrate that clones can be constructed that preferentially secrete TMIF. In these experiments, we also tested the supernatants for another lymphokine effect on tumor cells; namely, the ability to inhibit tumor cell binding to endothelial monolayers. A number of clones were identified that lacked TMIF activity, but could inhibit the tumor cell-endothelial interaction, suggesting the possibility that these effects may be due to separate mediators.     

Neuronal-derived factors regulating glial cell proliferation and maturation

We purified to homogeneity two active factors, named astroglial growth factors (AGFs: AGF2 and AGF1), from bovine brain after two and three chromatographic steps, respectively. We found that AGFs have a strong affinity for heparin. Therefore, heparin affinity chromatography was used to purify rapidly and efficiently these growth factors. The purified AGF1 is an acidic protein (pI: 5.5) with an apparent molecular weight of about 17,500 daltons; the AGF2 is a basic protein (pI: 9.5) of 18,500 daltons. The comparison of the physico-chemical properties, the aminoacid composition and the amino-terminal sequence of the AGFs with that of other growth factors isolated from the brain and affecting the proliferation of other cell types has indicated that AGF1 and AGF2 are identical to the acidic and basic fibroblast growth factor (FGF), respectively. Both factors stimulate the proliferation as well as the morphological and biochemical maturation of the astroglial cells. Both factors enhance also the multiplication of oligodendroglial cells. Polyclonal and monoclonal antibodies against AGFs have been prepared and used for immunocytochemical localization of these molecules in the rat brain and cerebellum. AGFs are found exclusively in neuronal cells.     

Engineering an APRIL-specific B cell maturation antigen

B cell maturation antigen (BCMA) is a tumor necrosis factor receptor family member whose physiological role remains unclear. BCMA has been implicated as a receptor for both a proliferation-inducing ligand (APRIL) and B cell-activating factor (BAFF), tumor necrosis factor ligands that bind to multiple tumor necrosis factor receptor and have been reported to play a role in autoimmune disease and cancer. The results presented herein provide a dual perspective analysis of BCMA binding to both APRIL and BAFF. First, we characterized the binding affinity of monomeric BCMA for its ligands; BAFF binding affinity (IC50 = 8 +/- 5 microm) is about 1000-fold reduced compared with the high affinity interaction of APRIL (IC50 = 11 +/- 3 nm). Second, shotgun alanine scanning of BCMA was used to map critical residues for either APRIL or BAFF binding. In addition to a previously described "DXL" motif (Gordon, N. C., Pan, B., Hymowitz, S. G., Yin, J., Kelley, R. F., Cochran, A. G., Yan, M., Dixit, V. M., Fairbrother, W. J., and Starovasnik, M. A. (2003) Biochemistry 42, 5977-5983), the alanine scanning results predicted four amino acid positions in BCMA (Tyr13, Ile22, Gln25, and Arg27) that could impart ligand specificity. Substitution of Tyr13 was tolerated for BAFF binding but not APRIL binding. Arg27 was required for high affinity binding to APRIL, whereas substitutions of this residue had minimal effect on affinity for BAFF. Further phage display experiments suggested the single mutations of I22K, Q25D, and R27Y as providing the greatest difference in APRIL versus BAFF binding affinity. Incorporation of the Q25D and R27Y substitutions into BCMA produced a dual specificity variant, since it has comparable binding affinity for both APRIL and BAFF, IC50 = 350 and 700 nm, respectively. Binding of the I22K mutant of monomeric BCMA to BAFF was undetectable (IC50 > 100 microm), but affinity for binding to APRIL was similar to wild-type BCMA. Based on these results, a BCMA-Fc fusion with the single I22K mutation was produced that binds APRIL, IC50 = 12 nm, and has no measurable affinity for BAFF. These results suggest that APRIL is the preferred ligand for BCMA and show that specificity can be further modified through amino acid substitutions.     

Role of DNA synthesis in secretion of immunoglobulin from murine B cells stimulated by T cell derived lymphokines

We have investigated whether cell division is required for induction of Ig secretion from three types of B cells, which represent distinct activation states: normal splenic B cells, anti-Ig-treated B cells, and a monoclonal murine B cell tumor, BCL1. Polyclonal Ig secretion was stimulated in vitro by LPS or by lymphokines produced by EL-4 cells (EL-4 SN), which includes B cell growth factor II (BCGF II). LPS and EL-4 SN were mitogenic for all three cell populations and stimulated substantial IgM secretion from both B cells and anti-Ig blasts. Aphidicolin, a reversible inhibitor of DNA synthesis, abolished IgM secretion from B cells and anti-Ig blasts induced by either mitogen, indicating that Ig-secreting cells in these cultures are part of a cycling population. BCL1 tumor cells respond to BCGF II (but not to interleukin 2 or B cell stimulatory factor 1) with IgM secretion and cell division, allowing a direct assessment of the influence of BCGF II-stimulated cell division on secretion of IgM. Secretion by these cells during the first 24 hr of culture was not substantially affected by aphidicolin, but secretion at 48 or 72 hr was markedly inhibited. Culture of BCL1 cells for 48 hr with aphidicolin alone had no effect on cell viability or on subsequent responsiveness if the drug was removed, eliminating non-specific toxicity as an explanation of the drug's effect. Addition of aphidicolin during the last 24 hr of culture to either normal B cells or BCL1 cells was much less effective at inhibiting IgM secretion. These results indicate that the cells that secrete IgM in response to BCGF II also synthesize DNA when exposed to this factor. Thus, induction of high-rate Ig secretion from murine B cells by some stimuli, including BCGF II, may require at least one round of cell division.     

Staphylococcus aureus S-6: factors affecting its growth, enterotoxin B production and exoprotein formation

The growth of Staphylococcus aureus S-6, enterotoxin production and exoprotein formation were always higher in NZ-amine A medium compared with brain heart infusion medium. The formation of exoproteins, including enterotoxin B, per bacterial cell in static culture was influenced by the addition of glucose. Lactate and amino acids were used by Staph. aureus S-6 in media without additional glucose. When both media were supplemented with glucose, lactic and acetic acids were produced. Different electrophoretic patterns for exoprotein formation were obtained when the organism was grown in shaken or static culture.     

Staphylococcus aureus S-6: factors affecting its growth, enterotoxin B production and exoprotein formation

The growth of Staphylococcus aureus S-6, enterotoxin production and exoprotein formation were always higher in NZ-amine A medium compared with brain heart infusion medium. The formation of exoproteins, including enterotoxin B, per bacterial cell in static culture was influenced by the addition of glucose.
Lactate and amino acids were used by Staph. aureus S-6 in media without additional glucose. When both media were supplemented with glucose, lactic and acetic acids were produced. Different electrophoretic patterns for exoprotein formation were obtained when the organism was grown in shaken or static culture.