Signaling Through TLR4 Regulates B Cell Development

Objective B lymphocytes, like all blood cells, are derived from hematopoietic stem cells(HSCs). Mutiple stages of development between the HSC and newly produced surface IgMpos B lymphocytes have been defined, and progenitors must successfully navigate each of these in order to mature into functional B lymphocytes capable of participating in a productive immune response.[1]B cell development can be interrupted in many pathologic states, especially inflammation. Toll-like receptors are a family of proteins recognizing different pathogen-associated molecular patterns and form part of the first line of defense against pathogens. Once engaged by the TLRs, pathogen-associated molecular patterns trigger a signaling cascade resulting in specific gene expression profiles that stimulate the immune system to eliminate the invading pathogen.[2]TLRs can induce the production of inflammatory factors and these inflammatory factors can inhibit B cell development. Because of these two facts we intrests in the association between TLR signal and B cell development. As we know, TLR signal can seriously affect the function of mature B cells. We wonder whether TLR signal has similar effects on the developing B cells. In the present study, we detected the difference of B cells between C3H/He and C3H/HeJ mice, detected the expression level of TLR4 in every stage of B cells and detected the effects of TLR4 signal on developing B cells in vivo and in vitro.Methods To determine the expression pattern of TLR4 in subgroups of B cells, four subgroups of B cells were analysed by real-time quantitative RT-PCR; C57BL/6 mice were treated with a single i.p injection of 100ul PBS or LPS(1mg/kg) in 100ul PBS and detected the changes of B cells in BM at different time; BM cells were obtained from 7-8wk C3H/HeJ or normal C3H/He mice and analysed by flow cytometry; FACs purified B220lowIgM+IgD- immature B cells were cultured in 96-well flat-bottom plates with or without LPS and the expression of IgD, CD21, CD23 and CD86 were detected at 24h; FACs purified B220lowIgM+IgD- immature B cells were cultured in 96-well flat-bottom plates with LPS, anti-μor LPS plus anti-μ. After 24h culture, cells were analyzed by flow cytometry for differentiation and apoptosis.Results (1) Four subgroups of B cells in BM all expressed TLR4 and the expression level of TLR4 in mature B cells was higher than in pro, pre and immature B cells. Besides, the expression level of TLR4 in mature B cells from spleen was higher than four subgroups of B cells from BM; (2) After use of LPS, the count of total bone marrow cells was decreased in the first few days, then rose to an abnormal higher level in the second week and decreased to normal in the third week. The same changes were also observed in the counts of B cells, pre B cells and immature B cells in BM; (3)The percent of B cells in BM was obviously increased in C3H/HeJ mice. And the percents of pro B cells and pre B cells in BM were also increased in C3H/HeJ mice; (4) The rates of IgD, CD21, CD23 and CD86 positive B cells in groups treated by LPS were obviously higher than rates in control groups; (5) The percent of IgD+ mature B cells in LPS group was obviously higher than the percents in groups treated by anti-μ, LPS plus anti-μand control; Apoptosis assay showed that the rate of annexin-V+ apoptotic B cells in anti-μgroup was obviously higher than the rates in groups treated by LPS, LPS plus anti-μand control.Conclusions (1) The expression level of TLR4 is increased with the maturation of B cell; (2) In vivo use of LPS can change the composition of B cells in BM; (3) The compositions of B cells in C3H/HeJ and normal C3H/He mice are different; (4) LR4 signal can induce immature B cells to differentiate into mature B cells; (5) Anti-μcan inhibit the differentiation of immature B cells induced by LPS while LPS can reverse the apoptosis of immature B cells induced by anti-μ. These results reveal that Signaling through TLR4 can promote B cell development in vivo and in vitro.