Surface molecule loss and bleb formation by human germinal center B cells undergoing apoptosis: role of apoptotic blebs in monocyte chemotaxis.
Human tonsil germinal center (GC) B cells rapidly undergo apoptosis in culture. Annexin-V binding shows an early event in this process. In the present study, this method has been used to label apoptotic GC B cells and to analyze additional surface molecules. The expression of all of the molecules studied was reduced in apoptotic (annexin-V(+)) GC B cells, and the reduction was more marked for CD11a, CD21, CD22, CD49d, and CD54, molecules that participate in survival interaction for GC B cells. The analysis of CD54, one of the molecules that was more drastically reduced, showed that GC, but not mantle zone, B cells actively secrete CD54 to the culture supernatant (SN). The secreted CD54 was partly released from the GC B cells in a particulate form as demonstrated by centrifugation. Further experiments using filtration, fluorescence microscopy, electron microscopy, and flow cytometry analysis showed that GC B cells released to the culture SN a population of spherical membranous vesicles of about 0.18 micrometers in size, similar to the blebs described in other apoptosis systems. Bleb formation depended on active metabolism, Ca(2+), and, in part, on microfilament integrity. GC B-cell-derived blebs were clearly associated with apoptosis, as antiapoptotic stimuli prevented their formation. In addition, GC B-cell-derived blebs contained the adhesion molecules previously studied. Consequently, bleb formation might contribute to the surface molecule loss occurring in apoptotic GC B cells. Finally, a chemotaxis assay showed that GC B-cell blebs were chemotactic for human monocytes, suggesting that this mechanism might operate in vivo.
Servicio de Inmunología, Hospital Universitario Puerta del Mar, Cádiz; and Servicio de Inmunología, Hospital Universitario Marqués de Valdecilla, Santander, Spain.
C
C
Segundo
F
F
Medina
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C
Rodríguez
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Martínez-Palencia
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F
Leyva-Cobián
J A
JA
Brieva
D015703:Antigens, CD / Q000276:immunology*; D017209:Apoptosis / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology* / Q000473:pathology; D015815:Cell Adhesion Molecules / Q000276:immunology; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte / Q000276:immunology; D003594:Cytoplasmic Granules / Q000276:immunology; D006801:Humans; D009000:Monocytes / Q000276:immunology / Q000473:pathology
D015703:Antigens, CD; D015815:Cell Adhesion Molecules