Clonal analysis of the synergistic mitogenic effect of lipopolysaccharide and dextran sulphate on B cell activation, growth, differentiation into Ig-secreting cells

Abstract

Splenic B cells of BALB/c mice were stimulated in vitro either with lipopolysaccharide (LPS), dextran sulphate (DxS), or with both LPS and DxS. The absolute frequency of B cells that differentiate into clones of immunoglobulin (Ig)-secreting cells upon activation with these mitogens was determined by means of limiting dilution analysis. Determination of the number of Ig-secreting cells in DxS-stimulated cultures with the protein A plaque assay proved to be difficult because of the anti-complementary activity of DxS. Therefore, we assayed the number of Ig-secreting cells with a reverse ELISA-plaque assay. This assay is complement-independent and is at least as sensitive as the protein A plaque assay. The results showed that LPS, DxS, the combination of LPS and DxS stimulate 1 in 27, less than 1 in 500 and 1 in 15 nucleated spleen cells of BALB/c mice to proliferation and differentiation into a clone of Ig-secreting cells, respectively, indicating that these mitogens have a synergistic effect on B cells at the precursor cell level. Analysis of the clone size of the activated B cells showed that the combination of both mitogens also caused a larger clone size. Thus, the synergistic effect of LPS and DxS that was previously observed in mass cultures is due to two separate effects. Quantitatively most important, however, is that more precursors are activated by the combination of the two mitogens.