An in vitro line of the B cell tumor BCL1 can be activated by LPS to secrete IgM1.

An in vitro line of the B cell tumor BCL1 was developed. The cell line carried u-, S-, and A-chains on the cell surface as judged by analysis of surface iodinated proteins but did not secret Ig. ASfter stimulation with LPS, limpid A, or bacterial lipoprotein, 20 to 40% of the tumor cells matured to IgM secretors when detected in a plaque assay. Two other polyclonal B cell activators, namely dextransulphate and PPD, had at most a marginal stimulatory effect. The ability of the cells to become activated to IgM secretion as well as the expression of cell surface IgM and IgD makes the BCL1 unique among murine B cell tumors.

Activation to IgG secretion by lipopolysaccharide requires several proliferation cycles.

We have investigated the relationship between IgG secretion and cell proliferation after polyclonal activation of murine spleen cells with lipopolysaccharide (LPS). It was found that IgG secretion was optimal when cells proliferated extensively. Under those conditions, DNA synthesis commenced 8 to 12 hr after exposure to LPS. Increased proliferative activity was observed up to day 3, when the majority of the lymphoblasts were mitotically active. Two inhibitors of DNA synthesis, thymidine (TdR) and hydroxyurea (HU) caused a reduction in both the IgM and the IgG response, but the latter was more severely reduced. The inhibition was strongest when TdR and HU were added to cultures early after exposure to LPS, indicating that the cells developing to Ig secretion were continuously proliferating. 5-bromo-2'-deoxyuridine (BrdU) caused a general inhibition of IgM and IgG secretion at high concentrations, and a selective inhibition at low concentrations. The selective inhibition of IgG secretion, when measured on day 4, was also observed after a pulse of BrdU on days 1 and 2. The data suggest that development to IgG secretion is a complex process, which requires several proliferation cycles.

Surface Ig isotypes on cells responding to lipopolysaccharide by IgM and IgG secretion.

Using the fluorescence-activated cell sorter (FACS), we have investigated the Ig isotypes present on murine B cells, which can be polyclonally activated by lipopolysaccharide (LPS) in low cell density cultures. The LPS response was partly inhibited as a result of staining with anti-IgD and anti-IgM reagents, but not with anti-IgG reagents. The IgM+, IgD+, or IgG- fractionated cell populations gave both an IgM and an IgG response comparable to controls, whereas the response of the IgM-, IgD- cells was 5- to 20-fold lower. IgG- cells separated 1 day after LPS stimulation could still mount an IgM and IgG response indistinguishable from controls at the peak of the response. It is concluded that IgM+, IgD+, IgG- cells constitute the major LPS-sensitive cell population in the low cell density culture system and that IgG is not a necessary cell surface isotype for precursors of IgG-secreting cells.

Cell differentiation in the presence of cytochalasin B: studies on the "switch" to IgG secretion after polyclonal B cell activation.

Mouse spleen cells were cultured with lipopolysaccharide in conditions that activate both IgM and IgG secretion. Addition of cytochalasin B (CB), an inhibitor of cytokinesis, lead to a high degree of polynucleation, with little effect on Ig secretion. Using cytoplasmic staining with fluorochrome conjugated antisera, we determined the numbers of IgG-containing cells that also contained IgM in their cytoplasm. Such double staining cells were relatively more frequent at early times of the cultures, but at all times single producing cells were in the majority. Addition of CB over the period when the IgG producing cells first appear, lead to a marked increased frequency of double staining, polynucleated cells. This characteristic was stable over a period of at least 42 hr, suggesting that each double staining cell actively synthesized both isotypes. When CB was added after IgG production had started, little increase in the numbers of double staining cells were observed, although polynucleation remained extensive. These data confirm previous findings that the lineage of one cell can produce both IgM and IgG. Furthermore, the results suggest that cells in the process of switching from IgM to IgG go through an asymmetric division leading to one IgM-producing and one IgG-producing daughter cell.