Actin assembly is a crucial factor for superoxide anion generation from adherent human eosinophils.
BACKGROUND
Cellular adhesion is crucial for eosinophil effector functions.


OBJECTIVE
We sought to elucidate the role of the actin cytoskeleton in cellular adhesion and superoxide anion generation by human eosinophils.


METHODS
Eosinophils were stimulated with platelet-activating factor (PAF) or complement component 5a on human serum albumin-coated plates with or without an actin-polymerization inhibitor, cytochalasin B (CB), or cytochalasin D (CD). Superoxide anion generation was measured on the basis of reduction of absorbance associated with cytochrome c.2 Eosinophil adhesion was assessed on the basis of eosinophil protein X content in adherent cells. Transient stimulus-induced increase of intracellular calcium and translocation of protein kinase C (PKC) betaII, PKC delta, PKC zeta, and p47 phagocyte oxidase (a component of nicotinamide adenine dinucleotide phosphate oxidase) were also investigated.


RESULTS
CB, CD, or antibodies against CD18 (the beta2 chain of integrin, alphaMbeta2) inhibited stimulus-induced eosinophil superoxide anion generation. Stimulus-induced eosinophil adhesion was unaltered by CB, whereas it was significantly suppressed by CD or anti-CD18 antibodies. Transient PAF-induced intracellular calcium increase was also unaffected by CB or CD, but stimulus-induced eosinophil shape changes and translocation of PKCs and p47 phagocyte oxidase to the cell membrane region were completely inhibited by CB. PAF-induced eosinophil degranulation was inhibited by CB, CD, or anti-CD18 antibodies, whereas complement component 5-induced degranulation was not suppressed by CB.


CONCLUSIONS
By itself, beta2 integrin-dependent cellular adhesion is not sufficient for promoting eosinophil effector function. Adequate actin assembly is required for eosinophil adhesion and also for full superoxide anion generation in eosinophils.
Suzuki
Masato
M
Department of Pediatrics, Gunma University School of Medicine, Maebashi, Gunma, Japan.
Kato
Masahiko
M
Hanaka
Hiromi
H
Izumi
Takashi
T
Morikawa
Akihiro
A
D000199:Actins / Q000737:chemistry*; D018821:CD18 Antigens / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion; D015550:Cell Degranulation; D003571:Cytochalasin B / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D004804:Eosinophils / Q000502:physiology*; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases; D010750:Phosphoproteins / Q000378:metabolism; D010972:Platelet Activating Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
D000199:Actins; D018821:CD18 Antigens; D010750:Phosphoproteins; D010972:Platelet Activating Factor; D013481:Superoxides; D015638:Cytochalasin D; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D011493:Protein Kinase C; D002118:Calcium