Comparison of actin changes and calcium metabolism in plastic- and fibronectin-adherent human neutrophils.
Human neutrophils adherent to a polystyrene plastic surface are vigorously activated, whereas those adherent to fibronectin manifest only a priming response. The basis of these metabolic differences was further characterized; polystyrene-adherent cells, which were shown to spread quickly upon adhesion, exhibited an increase of cytoskeleton-associated actin (F-actin) (measured by a nitrobenzoxadiazole-phallacidin fluorescent staining assay) and a decrease of monomeric G-actin concentration (measured by a DNase inhibition assay); in contrast, fibronectin-adherent cells exhibited little spreading and decreased their F-actin, after 1.5 min of adhesion, to 33.49 +/- 6.9% (mean +/- SD, n = 5) of initial levels found in suspended cells before plating. Actin depolymerization in fibronectin-adherent cells was confirmed by measuring G-actin, which sharply increased during the first minute of adhesion, rising from 0.065 +/- 0.007 to 0.20 +/- 0.035 microgram/microgram of protein (mean +/- SEM, p less than 0.05), and then remained elevated during 5 min of observation. In contrast, soluble fibronectin induced a decrease of G-actin in suspended cells. Cells pretreated with 1 microM cytochalasin D and allowed to adhere to a plastic surface did not spread, failed to generate O2-, and exhibited elevated concentrations of G-actin (0.1 to 0.2 microgram/microgram of protein) during the 5 min of observation. Actin changes, as well as respiratory burst, in adherent cells were shown to proceed through a pertussis toxin-insensitive pathway. Fluo-3 measurements of intracellular Ca2+ concentrations ([Ca2+]i) showed a fourfold and twofold [Ca2+]i increase in polystyrene- and fibronectin-adherent cells, respectively, after 2 min. The small rise in [Ca2+]i in fibronectin-adherent cells corresponds to a primed response of these cells to subsequent activation with FMLP. Ionomycin (1 microM) added to neutrophils just before adhesion on fibronectin induced full activation, i.e., O2- production and actin polymerization. The metabolic events controlling metabolic priming and actin depolymerization are as yet uncharacterized, but fibronectin receptor-linked responses beyond the mediation of cell adhesion have now been identified, suggesting complex metabolic functions of integrin receptors.
Ginis
I
I
William B. Castle Hematology Research Laboratory, Boston City Hospital, MA.
Zaner
K
K
Wang
J S
JS
Pavlotsky
N
N
Tauber
A I
AI
D000199:Actins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion; D003599:Cytoskeleton / Q000648:ultrastructure; D003600:Cytosol / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology* / Q000378:metabolism; D037342:Pertussis Toxin; D011137:Polystyrenes; D016897:Respiratory Burst / Q000187:drug effects; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000199:Actins; D005353:Fibronectins; D011137:Polystyrenes; D010566:Virulence Factors, Bordetella; D015759:Ionomycin; D037342:Pertussis Toxin; D002118:Calcium