title: Alterations in cell protein phosphorylation in human neutrophils exposed to influenza A virus. A possible mechanism for depressed cellular end-stage functions.
abstract: When polymorphonuclear leukocytes (PMN) are exposed to most harvests of influenza A virus (depressing virus, DV) for 20 min, chemotactic, secretory, and oxidative functions are depressed upon subsequent exposure to soluble or particulate stimuli. Other harvests of influenza A virus (non-DV) do not alter these activities. The DV-induced changes in multiple functions suggest the virus may interfere with steps involved in PMN activation. Because some of these steps may be regulated by protein phosphorylation, we examined the effect of non-DV and DV on cellular protein phosphorylation. PMN loaded with 32P-labeled inorganic orthophosphate were exposed to non-DV, DV, or buffer for 30 min; cells were then treated with buffer, FMLP (10(-6) M), or PMA (100 ng/ml) for 30 s. Samples were sonicated and centrifuged; cytosolic and particulate fractions were analyzed by SDS-PAGE and autoradiography. Exposure of PMN to either non-DV or DV caused phosphorylation of several cell proteins. However, when DV-treated PMN were then stimulated with FMLP or PMA, further phosphorylation was inhibited compared to non-DV- or buffer-treated cells. This suggests that DV-induced depression of PMN end-stage functions may be due to changes in cell protein phosphorylation. DV could interfere with phosphorylation of PMN proteins by altering protein kinase activity. We therefore examined the influence of non-DV and DV on some parameters that could affect kinase function. PMN intracellular [Ca2+] was monitored by using the fluorescent Ca2+ indicator, Indo 1, and cAMP levels were measured by RIA. PMN treated with DV alone or DV plus FMLP had higher intracellular [CA2+] than PMN similarly treated with non-DV or buffer. Exposure of PMN to non-DV, DV, or buffer caused minimal changes in cAMP levels, and similar increases occurred in cAMP levels upon FMLP stimulation. To determine whether DV interferes with transmembrane signaling, the effect of influenza virus on PMN transmembrane potential was studied by using a fluorescent cyanine dye. Transmembrane potential changes were greater in PMN exposed to DV than to non-DV or buffer; however, subsequent stimulation with FMLP caused equivalent changes in transmembrane potential. Our data show that protein phosphorylation in PMN is induced by DV and non-DV infection; upon subsequent stimulation with FMLP or PMA, there is inhibited cellular phosphorylation only in PMN previously exposed to DV.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Medicine, Wake Forest University Medical Center, Winston-Salem, NC 27103.
authors.forename: S E
authors.initials: SE
authors.lastname: Caldwell
authors.forename: L F
authors.initials: LF
authors.lastname: Cassidy
authors.forename: J S
authors.initials: JS
authors.lastname: Abramson
mesh_terms: D002634:Chemotaxis, Leukocyte; D000242:Cyclic AMP / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D006801:Humans; D009980:Influenza A virus / Q000502:physiology*; D007424:Intracellular Fluid / Q000378:metabolism; D008564:Membrane Potentials; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism* / Q000382:microbiology / Q000502:physiology; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation
chemical_list: D010750:Phosphoproteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000242:Cyclic AMP