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Alterations in cell protein phosphorylation in human neutrophils exposed to influenza A virus. A possible mechanism
S E Caldwell1, L F Cassidy, J S Abramson
1Department of Medicine, Wake Forest University Medical Center, Winston-Salem, NC 27103.
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)
Insights
Influenza A virus (DV) depresses polymorphonuclear leukocyte (PMN) functions by altering protein phosphorylation. DV-exposed PMNs show inhibited phosphorylation upon stimulation, suggesting a mechanism for immune cell dysfunction during infection.
Area of Science:
- Immunology
- Virology
- Cell Biology
Background:
- Polymorphonuclear leukocytes (PMN) are crucial immune cells whose functions can be impaired by certain influenza A virus (IAV) strains.
- Depressing virus (DV) strains of IAV, unlike non-DV strains, significantly reduce PMN chemotactic, secretory, and oxidative activities.
- The mechanism by which DV impairs PMN function is not fully understood but may involve interference with cellular activation pathways.
Purpose of the Study:
- To investigate the effect of DV and non-DV influenza A viruses on protein phosphorylation in PMNs.
- To determine if altered protein phosphorylation in DV-exposed PMNs contributes to the observed functional deficits.
- To explore the impact of DV on intracellular signaling pathways, including calcium levels and transmembrane potential, in PMNs.
Main Methods:
- PMNs were loaded with 32P and exposed to DV, non-DV, or buffer, followed by stimulation with FMLP or PMA.
- Cellular protein phosphorylation was analyzed using SDS-PAGE and autoradiography.
- Intracellular calcium levels, cAMP levels, and transmembrane potential were measured in response to viral exposure and stimulation.
Main Results:
- Both DV and non-DV induced phosphorylation of several PMN proteins.
- However, DV-exposed PMNs exhibited inhibited further phosphorylation upon subsequent stimulation with FMLP or PMA compared to controls.
- DV-treated PMNs showed increased intracellular calcium levels and altered transmembrane potential, while cAMP levels remained largely unchanged.
Conclusions:
- DV-induced depression of PMN functions is associated with an inhibition of cellular protein phosphorylation following activation stimuli.
- DV may interfere with PMN activation by altering protein kinase activity or downstream signaling pathways.
- These findings suggest a molecular mechanism for DV-induced immune suppression involving dysregulated protein phosphorylation in PMNs.