Remodeling of the plasma membrane after stimulation of neutrophils with f-Met-Leu-Phe and dihydrocytochalasin B: identification of membrane subdomains containing NADPH oxidase activity

Abstract

Superoxide (O2-) production by neutrophils stimulated with chemotactic peptides [e.g., formylmethionyl-leucyl-phenylalanine (fMLP)] is transient but increases in rate and duration after pretreatment of the cells with dihydrocytochalasin B (dhCB), suggesting a possible role for the plasma membrane and membrane skeleton in the regulation of the O2- generating system. Analysis of plasma membrane isolated from these cells by isopycnic sucrose density gradient sedimentation showed that there were no significant variation in the distribution of plasma membrane markers between control and dhCB-treated cells, whereas a significant redistribution of plasma membrane markers was observed in dhCB + fMLP-treated cells. Instead of sedimenting to 31-35% sucrose, as in the former two groups, plasma membrane markers were broadly distributed over 25-50% sucrose in the dhCB + fMLP-treated cells. In addition, approximately 80% degranulation was achieved in these cells, whereas little granule release (< 5%) was observed in control and dhCB-treated cells. Analysis of the gradient fractions for membrane skeletal (actin and fodrin) and NADPH oxidase (cytochrome b and p47-phox) components in dhCB + fMLP-treated cells demonstrated that the distribution of fodrin, actin, cytochrome b, and p47-phox followed the broad distribution of plasma membrane markers, with an overall eightfold increase in membrane-associated actin. Despite the broad redistribution of plasma membrane markers, the distribution of O2- generating activity remained confined to a narrower peak at approximately 50% sucrose. These results demonstrate that a heterogeneous surface membrane of higher density with a differential distribution of proteins and O2- generating activity are created after dhCB + fMLP treatment; however, domain structure is conserved in the new membrane, with only a subfraction of the reorganized plasma membrane containing all of the components necessary for active O2- generation. Our results support a role for plasma membrane lateral organization and participation of the membrane skeleton in the regulation of the O2- generating system.

Essential Requirement of Cytosolic Phospholipase A2for Stimulation of NADPH Oxidase-associated Diaphorase Activity in Granulocyte-like Cells

“…Stimulated or resting permeabilized cells were centrifuged, sonicated in the presence of 5 mM EGTA, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl 2 , 10 mM PIPES (pH 7.4), 1 mM PMSF, and 100 M leupeptin, and membrane and cytosol fractions were separated. Similar results were obtained in the presence or absence of preincubation with 5 g/ml cytochalasin B (data not shown), which inhibits actin polymerization induced by GTP␥S or FMLP in permeabilized neutrophils (25)(26)(27)(28), indicating that the translocation of the oxidase cytosolic components is to the membrane, rather than a cytoskeleton-associated movement. Protein (150 g) from cell membrane were separated by electrophoresis on 12% SDS-polyacrylamide gels and blotted to nitrocellulose.…”

Compromised Host Defense on Pseudomonas aeruginosa Biofilms: Characterization of Neutrophil and Biofilm Interactions

“…Fig. 6A shows that when neutrophils are exposed to biofilms in the absence of serum, myeloperoxidase is released into the extracellular medium to a level that is ϳ20% of the maximal release observed when a known degranulating stimulus or secretagogue (36,37) is used, such as 1 M fMLF in the presence of 10 M cytochalasin B. This release was Ͼ5-fold greater than that observed for neutrophils adhering to plastic.…”

Decreased phosphorylation of protein kinase B and extracellular signal-regulated kinase in neutrophils from patients with myelodysplasia

“…10,11 Respiratory burst and additional neutrophil effector functions such as granule trafficking and phagocytosis are dependent on the organization of the actin cytoskeleton. [12][13][14][15] There are 2 forms of actin, a monomeric form (G-actin) and a filamentous form (Factin), that exist in equilibrium in the cell. On the stimulation of neutrophils with chemotactic factors, rapid conversion of G-actin to F-actin occurs.…”