ALBERT

Description: To investigate the relatedness of soluble and pelletable vesicular erythroid burst-promoting activity (BPA) present in lymphocyte- conditioned medium (LCM), we immunized rabbits with partially purified lymphocyte plasma membranes and tested the antisera for biological and immunologic crossreactivity with LCM and its component fractions. When preincubated with IgG purified from post-immune but not from preimmune serum, BPA expression by unseparated LCM, LCM-derived pellets, and supernatants was abolished in a dose-related fashion. As little as 0.001 mg/mL post-immune IgG reduced burst formation by 50%. Antimembrane IgG crossreacted on immunoblots with multiple components of both supernatants and pellets of LCM. Crossreactivity was also seen in LCM-derived supernatants that were subjected to ultracentrifugation. Soluble BPA was adsorbed from LCM supernatants incubated with antimembrane IgG-coated Staphylococcus aureus. Conversely, incubation of purified antimembrane IgG with intact circulating lymphocytes removed BPA-neutralizing effects from the antibody preparation. Antimembrane IgG incompletely suppressed erythroid colony-forming unit (CFU-E)-derived colony formation, an effect that could not be explained by alteration in erythropoietin sensitivity or action. There was no effect of the antibody preparation on erythroid differentiation of K562 cells or on CFU granulocyte/macrophage-derived colony growth, (CFU-G/M) by human or murine bone marrow. Taken together, our findings suggest that antibodies directed against lymphocyte plasma membranes react with both soluble and vesicular BPA, and that these physically separable erythroid growth factors may share antigenic determinants.

Description: We have investigated the role of contractile proteins of circulating mononuclear cells in generation of membrane-associated, erythroid growth regulatory molecules. Lymphocytes and monocytes were incubated under serum-free conditions without and with cytochalasin B, cytochalasin D, or colchicine, and effects on positive and negative erythropoietic activities were determined in cell membranes and in surface membrane vesicle-rich pellets and supernatants of dialyzed medium conditioned by the cells. In serum-free cultures of human bone marrow, plasma membranes and exfoliated membrane-derived vesicles from cytochalasin-treated lymphocytes lost their capacity to support the formation of erythroid bursts, while monocyte membrane-associated inhibitory activity was abolished by preincubation with cytochalasin. In contrast, membrane-associated activities of colchicine-treated cells were unaffected. Cytochalasin-induced alterations of membrane regulatory molecules were observed in a dose-dependent fashion over a wide range of concentrations (1 to 100 micrograms/mL) tested. However, the capacity of membrane vesicle-free supernatants of medium conditioned by lymphocytes or monocytes was unaffected by cytochalasins, regardless of drug concentration used. Lysates of cytochalasin B-treated cells inhibited the activity of deoxyribonuclease I to a greater degree than did lysates of untreated cells, suggesting that the relative amount of monomeric actin is increased in the cytoplasm of treated cells. Furthermore, results of experiments with D-glucose and with cytochalasin D suggest that cytochalasin effects are independent of alterations in glucose metabolism. The data indicate that expression of plasma membrane- associated regulators is sensitive to agents that block polymerization of actin. They raise the possibility that changes in distribution of actin between unpolymerized and filamentous pools may influence the organization and/or function of mononuclear cell surface-associated erythroid regulatory molecules.

Description: Previously we have demonstrated that, in contrast to various panspecific or multilineage hematopoietic growth factors, lymphocyte- derived erythroid burst-promoting activity (BPA) is lineage specific, stimulating BFU-E proliferation in serum-free culture by up to 600% of control values while failing to enhance nonerythroid colony formation. To further determine the cellular source(s) of this important erythropoietic growth regulator, we have separated normal nonadherent peripheral blood and splenic lymphocytes by nylon wool fractionation, SRBC rosetting, and panning with monoclonal antibodies (MoAbs). These unstimulated T- and B-lymphocyte-enriched populations were used as cell sources to produce conditioned media (CM) and to prepare plasma membranes (PM). When CM fractions or purified PM were assayed in serum- free human bone marrow culture, BPA was localized entirely to the B- lymphocyte-derived fractions. While CM or PM from unstimulated T lymphocytes failed to stimulate BFU-E proliferation, activation of T cells by either phytohemagglutinin-M (1%) or concanavalin A (Con A; 5 micrograms/mL) induced the expression of a factor on the PM and in the resultant CM that stimulated the formation of erythroid bursts. In addition to enhancing BFU-E proliferation, this T-cell factor stimulated the proliferation of CFU-GM and CFU-GEMM in serum-free culture. When compared biochemically (in terms of temperature stability, localization by ammonium sulfate fractionation, and sensitivity to dithiothreitol) or immunochemically (using antibodies specific for lymphocyte-derived BPA, GM-CSF, or interleukin-3 [IL-3]), as well as by lineage specificity, B- and activated T-lymphocyte- derived growth factors appeared to be distinct. The burst stimulatory activities expressed by recombinant human GM-CSF and IL-3 were immunologically distinct from that associated with octylglucoside extracts of plasma membranes from resting B lymphocytes. Our results suggest that the BFU-E-directed growth-promoting activity released from activated T lymphocytes is apparently due to GM-CSF, while both resting and mitogen-stimulated normal B lymphocytes express erythroid-specific BPA and neither GM-CSF nor IL-3.

Description: In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.

Description: An Argentinian family with hereditary elliptocytosis (HE) associated with a shortened beta-spectrin (Sp) chain was studied. As with most of the other shortened Sp beta-chains that have been described, this variant, called SpTandil, has impaired ability to participate in Sp dimer self-association, has lost its ability to become phosphorylated, and is associated with the presence of increased amounts of the alpha I 74-Kd fragment after partial tryptic digestion of Sp. The 3′ ends of the beta-Sp gene of affected patients were analyzed. cDNA was prepared by reverse transcription of peripheral blood mRNA and amplified by the polymerase chain reaction (PCR) using primers corresponding to sequences 400 bp apart on the cDNA, spanning the last three exons (X, Y, Z) of the beta-Sp gene. Agarose gel electrophoresis of the cDNA amplification showed the presence of one band, the size of which was apparently the same as the band amplified from mRNA of a normal control. cDNA from one HE patient was subcloned and sequenced. Several clones showed the presence of a 7-bp deletion at codon 2041 in exon X. Genomic DNA of all the affected members of the family were amplified by PCR using primers flanking the deletion and corresponding to sequences 128 bp apart on exon X. Analysis of the PCR products using electrophoresis on polyacrylamide gel showed the presence of 121- and 128-bp bands in all HE subjects, and an additional doublet migrating more slowly than the two bands, which corresponded to the presence of heteroduplexes. The mutation results in a shift of the normal reading frame and leads to a new amino acid sequence at the C-terminus of the mutant beta-Sp chain. A new in-frame stop codon is encountered downstream, leading to premature chain termination. The identification of the molecular defect in Sp beta Tandil provides information regarding the region of the beta-Sp chain that is important for both Sp dimer self-association and an indication of potential sites of phosphorylation of the chain.

Description: To investigate the relatedness of soluble and pelletable vesicular erythroid burst-promoting activity (BPA) present in lymphocyte- conditioned medium (LCM), we immunized rabbits with partially purified lymphocyte plasma membranes and tested the antisera for biological and immunologic crossreactivity with LCM and its component fractions. When preincubated with IgG purified from post-immune but not from preimmune serum, BPA expression by unseparated LCM, LCM-derived pellets, and supernatants was abolished in a dose-related fashion. As little as 0.001 mg/mL post-immune IgG reduced burst formation by 50%. Antimembrane IgG crossreacted on immunoblots with multiple components of both supernatants and pellets of LCM. Crossreactivity was also seen in LCM-derived supernatants that were subjected to ultracentrifugation. Soluble BPA was adsorbed from LCM supernatants incubated with antimembrane IgG-coated Staphylococcus aureus. Conversely, incubation of purified antimembrane IgG with intact circulating lymphocytes removed BPA-neutralizing effects from the antibody preparation. Antimembrane IgG incompletely suppressed erythroid colony-forming unit (CFU-E)-derived colony formation, an effect that could not be explained by alteration in erythropoietin sensitivity or action. There was no effect of the antibody preparation on erythroid differentiation of K562 cells or on CFU granulocyte/macrophage-derived colony growth, (CFU-G/M) by human or murine bone marrow. Taken together, our findings suggest that antibodies directed against lymphocyte plasma membranes react with both soluble and vesicular BPA, and that these physically separable erythroid growth factors may share antigenic determinants.

Description: Previously we have demonstrated that, in contrast to various panspecific or multilineage hematopoietic growth factors, lymphocyte- derived erythroid burst-promoting activity (BPA) is lineage specific, stimulating BFU-E proliferation in serum-free culture by up to 600% of control values while failing to enhance nonerythroid colony formation. To further determine the cellular source(s) of this important erythropoietic growth regulator, we have separated normal nonadherent peripheral blood and splenic lymphocytes by nylon wool fractionation, SRBC rosetting, and panning with monoclonal antibodies (MoAbs). These unstimulated T- and B-lymphocyte-enriched populations were used as cell sources to produce conditioned media (CM) and to prepare plasma membranes (PM). When CM fractions or purified PM were assayed in serum- free human bone marrow culture, BPA was localized entirely to the B- lymphocyte-derived fractions. While CM or PM from unstimulated T lymphocytes failed to stimulate BFU-E proliferation, activation of T cells by either phytohemagglutinin-M (1%) or concanavalin A (Con A; 5 micrograms/mL) induced the expression of a factor on the PM and in the resultant CM that stimulated the formation of erythroid bursts. In addition to enhancing BFU-E proliferation, this T-cell factor stimulated the proliferation of CFU-GM and CFU-GEMM in serum-free culture. When compared biochemically (in terms of temperature stability, localization by ammonium sulfate fractionation, and sensitivity to dithiothreitol) or immunochemically (using antibodies specific for lymphocyte-derived BPA, GM-CSF, or interleukin-3 [IL-3]), as well as by lineage specificity, B- and activated T-lymphocyte- derived growth factors appeared to be distinct. The burst stimulatory activities expressed by recombinant human GM-CSF and IL-3 were immunologically distinct from that associated with octylglucoside extracts of plasma membranes from resting B lymphocytes. Our results suggest that the BFU-E-directed growth-promoting activity released from activated T lymphocytes is apparently due to GM-CSF, while both resting and mitogen-stimulated normal B lymphocytes express erythroid-specific BPA and neither GM-CSF nor IL-3.

Description: An Argentinian family with hereditary elliptocytosis (HE) associated with a shortened beta-spectrin (Sp) chain was studied. As with most of the other shortened Sp beta-chains that have been described, this variant, called SpTandil, has impaired ability to participate in Sp dimer self-association, has lost its ability to become phosphorylated, and is associated with the presence of increased amounts of the alpha I 74-Kd fragment after partial tryptic digestion of Sp. The 3′ ends of the beta-Sp gene of affected patients were analyzed. cDNA was prepared by reverse transcription of peripheral blood mRNA and amplified by the polymerase chain reaction (PCR) using primers corresponding to sequences 400 bp apart on the cDNA, spanning the last three exons (X, Y, Z) of the beta-Sp gene. Agarose gel electrophoresis of the cDNA amplification showed the presence of one band, the size of which was apparently the same as the band amplified from mRNA of a normal control. cDNA from one HE patient was subcloned and sequenced. Several clones showed the presence of a 7-bp deletion at codon 2041 in exon X. Genomic DNA of all the affected members of the family were amplified by PCR using primers flanking the deletion and corresponding to sequences 128 bp apart on exon X. Analysis of the PCR products using electrophoresis on polyacrylamide gel showed the presence of 121- and 128-bp bands in all HE subjects, and an additional doublet migrating more slowly than the two bands, which corresponded to the presence of heteroduplexes. The mutation results in a shift of the normal reading frame and leads to a new amino acid sequence at the C-terminus of the mutant beta-Sp chain. A new in-frame stop codon is encountered downstream, leading to premature chain termination. The identification of the molecular defect in Sp beta Tandil provides information regarding the region of the beta-Sp chain that is important for both Sp dimer self-association and an indication of potential sites of phosphorylation of the chain.

Description: We have investigated the role of contractile proteins of circulating mononuclear cells in generation of membrane-associated, erythroid growth regulatory molecules. Lymphocytes and monocytes were incubated under serum-free conditions without and with cytochalasin B, cytochalasin D, or colchicine, and effects on positive and negative erythropoietic activities were determined in cell membranes and in surface membrane vesicle-rich pellets and supernatants of dialyzed medium conditioned by the cells. In serum-free cultures of human bone marrow, plasma membranes and exfoliated membrane-derived vesicles from cytochalasin-treated lymphocytes lost their capacity to support the formation of erythroid bursts, while monocyte membrane-associated inhibitory activity was abolished by preincubation with cytochalasin. In contrast, membrane-associated activities of colchicine-treated cells were unaffected. Cytochalasin-induced alterations of membrane regulatory molecules were observed in a dose-dependent fashion over a wide range of concentrations (1 to 100 micrograms/mL) tested. However, the capacity of membrane vesicle-free supernatants of medium conditioned by lymphocytes or monocytes was unaffected by cytochalasins, regardless of drug concentration used. Lysates of cytochalasin B-treated cells inhibited the activity of deoxyribonuclease I to a greater degree than did lysates of untreated cells, suggesting that the relative amount of monomeric actin is increased in the cytoplasm of treated cells. Furthermore, results of experiments with D-glucose and with cytochalasin D suggest that cytochalasin effects are independent of alterations in glucose metabolism. The data indicate that expression of plasma membrane- associated regulators is sensitive to agents that block polymerization of actin. They raise the possibility that changes in distribution of actin between unpolymerized and filamentous pools may influence the organization and/or function of mononuclear cell surface-associated erythroid regulatory molecules.

Description: In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.