The actin cytoskeleton regulates exocytosis of all neutrophil granule subsets.
A comprehensive analysis of the role of the actin cytoskeleton in exocytosis of the four different neutrophil granule subsets had not been performed previously. Immunoblot analysis showed that, compared with plasma membrane, there was less actin associated with secretory vesicles (SV, 75%), gelatinase granules (GG, 40%), specific granules (SG, 10%), and azurophil granules (AG, 5%). Exocytosis of SV, SG, and AG was measured as increased plasma membrane expression of CD35, CD66b, and CD63, respectively, with flow cytometry, and GG exocytosis was measured as gelatinase release with an ELISA. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated exocytosis of SV, GG, and SG with an ED(50) of 15, 31, and 28 nM, respectively, with maximal response at 10(-7) M FMLP by 5 min, while no exocytosis of AG was detected. Disruption of the actin cytoskeleton by latrunculin A and cytochalasin D induced a decrease in FMLP-stimulated CD35 expression after an initial increase. Both drugs enhanced the rate and extent of FMLP-stimulated GG, SG, and AG exocytosis, while the EC(50) for FMLP was not altered. We conclude that the actin cytoskeleton controls access of neutrophil granules to the plasma membrane, thereby limiting the rate and extent of exocytosis of all granule subsets. Differential association of actin with the four granule subsets was not associated with graded exocytosis.
Jog
Neelakshi R
NR
Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA.
Rane
Madhavi J
MJ
Lominadze
George
G
Luerman
Gregory C
GC
Ward
Richard A
RA
McLeish
Kenneth R
KR
D000199:Actins / Q000378:metabolism*; D015703:Antigens, CD / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005089:Exocytosis / Q000187:drug effects; D005434:Flow Cytometry; D058851:GPI-Linked Proteins; D018093:Gelatinases / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D007781:Lactoferrin / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D017463:Receptors, Complement 3b / Q000378:metabolism; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism*; D060149:Tetraspanin 30; D053778:Thiazolidines / Q000494:pharmacology
D000199:Actins; D015703:Antigens, CD; D019086:Bridged Bicyclo Compounds, Heterocyclic; C554586:CD63 protein, human; C504120:CEACAM8 protein, human; D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; D010980:Platelet Membrane Glycoproteins; D017463:Receptors, Complement 3b; D060149:Tetraspanin 30; D053778:Thiazolidines; D015638:Cytochalasin D; D009240:N-Formylmethionine Leucyl-Phenylalanine; D007781:Lactoferrin; D018093:Gelatinases; C037067:latrunculin A