Multiple elevations of cytosolic-free Ca2+ in human neutrophils: initiation by adherence receptors of the integrin family.
Multiple spontaneous transient elevations of cytosolic-free calcium ([Ca2+]i) are observed in single human neutrophils during adherence. The interrelation between adherence and spontaneous [Ca2+]i transients was analyzed by simultaneous monitoring of [Ca2+]i and cell morphology. Fluorescent images of fura 2-loaded neutrophils attached to albumin-coated glass were recorded with a high sensitivity CCD camera while [Ca2+]i was assessed with a dual excitation microfluorimetry. The majority of the initially round cells studied showed changes in shape which started either before or at the same time as the onset of the [Ca2+]i transients. These data suggested that a rise in [Ca2+]i is not a prerequisite for shape change. This conclusion was confirmed by observation of movement and spreading in cells whose [Ca2+]i transients were abolished by chelation of extracellular Ca2+. Instead, our data suggest that spreading or adhesion itself initiates the [Ca2+]i activity. In keeping with this hypothesis, cytochalasin B, which prevents both cell movement and adhesion, completely inhibited generation of [Ca2+]i transients. To determine if the movement alone or adhesion alone is responsible for [Ca2+]i activity, we treated cells with antibodies against the beta chain (CD18, beta 2) or the alpha subunit (CD11b, alpha m) of the dominant leukocyte integrin (CR3). Antibody-treated cells showed normal extension of pseudopods but impaired ability to adhere. Inhibition of adhesion in this way inhibited [Ca2+]i activity. Taken together these results suggest that following sequence of events after contact of neutrophils with surfaces: (a) cell movement and shape change lead to enhanced contact of integrins with the surface; and (b) integrins-mediated adhesion generates multiple [Ca2+]i transients. The [Ca2+]i transients may then control exocytic events associated with movement and may provide a link between adherence and activation or priming of neutrophils to other stimuli.
Jaconi
M E
ME
Division of Infectious Diseases, Geneva University Hospital, Switzerland.
Theler
J M
JM
Schlegel
W
W
Appel
R D
RD
Wright
S D
SD
Lew
P D
PD
D000911:Antibodies, Monoclonal; D002118:Calcium / Q000097:blood*; D002448:Cell Adhesion / Q000187:drug effects; D002462:Cell Membrane / Q000502:physiology; D003571:Cytochalasin B / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D016257:Fura-2; D006801:Humans; D066298:In Vitro Techniques; D016023:Integrins / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D012984:Software
D000911:Antibodies, Monoclonal; D016023:Integrins; D003571:Cytochalasin B; D004533:Egtazic Acid; D002118:Calcium; D016257:Fura-2