Endogenous fibronectin of blood polymorphonuclear leukocytes: immunochemical characterization and subcellular localization.
Fibronectin, a large dimeric glycoprotein synthesized and secreted by several cell types, mediates cell adherence to surfaces. In infections and inflammatory responses, blood polymorphonuclear leukocytes (PMNs) adhere to cells and matrix proteins during extravasation and accumulation at inflammatory sites. The presence of fibronectin in blood PMNs has been poorly studied, and the characteristics and subcellular localization of this endogenous adhesive molecule are practically unknown. By immunofluorescence flow cytometry, purified rabbit antibodies and a monoclonal antibody to plasma fibronectin reacted with isolated blood PMNs, only after permeabilization of the cells. By Western blot analysis, the antibodies recognized, under reducing conditions, a protein with an apparent molecular mass of 230 kDa in the cell lysate. Eleven monoclonal antibodies to common frame fibronectin epitopes, including the RGD-containing cell-binding domain, also reacted with PMN fibronectin by Western blotting. In contrast, two antibodies to ED-A, the alternatively spliced region characteristic of "cellular" fibronectin, were unreactive, but recognized platelet fibronectin. On average, 1 million PMNs contained 6.8 ng +/- 1.4 (SD) of fibronectin, as measured by sandwich ELISA. Immunogold labeling and electron microscopy studies indicated localization of most fibronectin in PMN granules. Moreover, double-immunofluorescence and digital image analysis demonstrated colocalization of fibronectin with lactoferrin, a marker of specific (secondary) granules. The results indicate that blood PMNs contain approximately 8000 molecules per cell of intact ED-A-negative fibronectin localized mainly in their specific granules.
Salcedo
R
R
Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden.
Segura
C
C
Szekely
L
L
de Mesquita
R
R
Biberfeld
P
P
Patarroyo
M
M
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules / Q000276:immunology / Q000302:isolation & purification*; D002451:Cell Compartmentation; D000939:Epitopes; D005353:Fibronectins / Q000276:immunology / Q000302:isolation & purification*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D009504:Neutrophils / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D000939:Epitopes; D005353:Fibronectins