Leukosialin (CD43, sialophorin) redistribution in uropods of polarized neutrophils is induced by CD43 cross-linking by antibodies, by colchicine or by chemotactic peptides.
We investigated a possible association of leukosialin (CD43), the major surface sialoglycoprotein of leukocytes, with neutrophil cytoskeleton. We first analysed the solubility of CD43 in Triton X-100 and observed that CD43 of resting neutrophils was mostly soluble. The small proportion of CD43 molecules, which 'spontaneously' precipitated in Triton, appeared associated with F-actin, as demonstrated by the fact that this insolubility did not occur when cells were incubated with cytochalasin B or when F-actin was depolymerized with DNase I in the Triton precipitate. Cell stimulation with anti-CD43 mAb (MEM59) enhanced this CD43-cytoskeleton association. By immunofluorescence as well as by electron microscopy, we observed a redistribution of CD43 on the neutrophil membrane, initially in patches followed by caps, during anti-CD43 cross-linking at 37 degrees C. This capping did not occur at 4 degrees C and was inhibited by cytochalasin B and by a myosin disrupting drug butanedione monoxime, thus providing evidence that the actomyosin contracile sytem is involved in the capping and further suggesting an association of CD43 with the cytoskeleton. Some of the capped cells exhibited a front-tail polarization with CD43 caps located in the uropod at the rear of the cell. Surprisingly, colchicine and the chemotactic factor fNLPNTL which induce neutrophil polarization associated with cell motility, also resulted in a clustering of CD43 in the uropod, independently of a cross-linking of the molecule by mAbs. An intracellular redistribution of F-actin, mainly at the leading front and of myosin in the tail, was observed during CD43 clustering induced by colchicine and in cells polarized by anti-CD43 mAbs cross-linking. We conclude that neutrophil CD43 interacts with the cytoskeleton, either directly or indirectly, to redistribute in the cell uropod under antibodies stimulation or during cell polarization by colchicine, thus highly suggesting that CD43 may be involved in cell polarization.
Seveau
S
S
INSERM U90 Hôpital Necker, Paris, France.
Lopez
S
S
Lesavre
P
P
Guichard
J
J
Cramer
E M
EM
Halbwachs-Mecarelli
L
L
D000199:Actins / Q000378:metabolism; D000328:Adult; D000906:Antibodies; D015703:Antigens, CD / Q000378:metabolism*; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D016764:Cell Polarity; D002630:Chemotactic Factors / Q000494:pharmacology; D003078:Colchicine / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003599:Cytoskeleton / Q000276:immunology / Q000378:metabolism; D003902:Detergents; D006801:Humans; D066298:In Vitro Techniques; D051917:Leukosialin; D016253:Microscopy, Immunoelectron; D009218:Myosins / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D017830:Octoxynol; D009842:Oligopeptides / Q000494:pharmacology; D012795:Sialoglycoproteins / Q000378:metabolism*; D012995:Solubility
D000199:Actins; D000906:Antibodies; D015703:Antigens, CD; D002630:Chemotactic Factors; D003432:Cross-Linking Reagents; D003902:Detergents; D051917:Leukosialin; D009842:Oligopeptides; C465014:SPN protein, human; D012795:Sialoglycoproteins; C021642:F-chemotactic peptide; D017830:Octoxynol; D009218:Myosins; D003078:Colchicine