CHARACTERIZATION OF THE MITOGENIC RESPONSE OF MURINE CD5(-LYMPHOCYTESTO LIPOPOLYSACCHARIDE() AND CONVENTIONAL B)

The nature of the response of conventional and CD5(+) B cells to stimu lation bl vitro with optimal mitogenic concentrations of LPS was exami ned to elucidate the contributions of these B cell subsets in polyclon al B lymphocyte responses. Stimulation of murine splenic lymphocytes w ith LPS resulted in an increase in total biomass, peaking at 72 h of c ulture. The viability of the cultures remained high (> 90%) until 48 h of culture. A combination of trypan blue and 7-aminoactinomycin D (7A AD) exclusion in conjunction with PE-anti-CD5 and FITC-anti-B220 enabl ed more detailed analysis of the cultures. The total number of convent ional B calls, viable and non-viable, increased until 48 h of culture and then decreased when stimulated with LPS, while CD5(+) B cells incr eased over the culture period. The numbers of conventional B cells in the control cultures decreased, but the CD5(+) B cell numbers remained stable. An examination of the modes of death of the B cell subsets us ing 7AAD showed that unstimulated conventional B cells were apoptotic rather than degenerate but, following stimulation with LPS, apoptotic and degenerate cells were found. Apoptotic and degenerate CD5(+) B cel ls were Found in both stimulated and unstimulated cultures, but the pe rcentage of these apoptotic and degenerate cells was increased signifi cantly only at 72 h and 96 h of culture in stimulated cultures compare d with 24 h onwards in the control cultures. Morphological analysis an d gel electrophoretic studies of extracted DNA reflected these finding s. it was also found that the increase in the number and percentage of non-viable cells in the cultures was not. equal to the decrease in th e number and percentage of viable cells. Activation of B cells was exa mined using expression of B7-1 (CD80) as a marker. When stimulated wit h LPS a greater proportion of conventional B cells expressed B7-1 afte r 24 h of culture than in the control cultures; however, only at 72 h and 96 h of culture was the proportion of CD5(+) B cells expressing B7 -1 significantly higher than in the control cultures. These results sh ow that conventional B cells are stimulated to proliferate and to beco me activated by LPS and that death is apoptotic rather than degenerate or necrotic. CD5(+) B cells were also shown to be stimulated by LPS; they became activated and death was delayed. The data suggest that in addition to the proliferative role, LPS acts to delay death and to act ivate conventional and CD5(+) B cells.