title: Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin.
abstract: One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.
authors.affiliation: Department of Microbiology, University of Iowa College of Medicine, Iowa.
authors.forename: J A
authors.initials: JA
authors.lastname: Wilder
authors.forename: R F
authors.initials: RF
authors.lastname: Ashman
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D001402:B-Lymphocytes / Q000502:physiology* / Q000648:ultrastructure; D002118:Calcium / Q000502:physiology; D015638:Cytochalasin D / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D009504:Neutrophils / Q000502:physiology / Q000648:ultrastructure; D010716:Phosphatidylinositols / Q000378:metabolism; D011108:Polymers; D011493:Protein Kinase C / Q000502:physiology; D011947:Receptors, Antigen, B-Cell / Q000502:physiology*
chemical_list: D000199:Actins; D010716:Phosphatidylinositols; D011108:Polymers; D011947:Receptors, Antigen, B-Cell; D015638:Cytochalasin D; D015759:Ionomycin; D000841:Anisomycin; D011493:Protein Kinase C; D002118:Calcium