The data are representative of three independent experiments. ALD-DNA and LPS synergize for inducing IgG production in naive B cells After demonstrating that ALD-DNA enhanced LPS-induced survival, proliferation and the up-regulation of CD86 expression, we next analyzed the effect of ALD-DNA around the antibody production by LPS-activated B cells. proliferation, plasma cell generation, and IL-10 production. (A) CFSE-labeled na?ve B cells were stimulated with ssDNA (0 g/mL, 10 g/mL, 50 g/mL, or 100 g/ml) in the presence or absence of 100 ng/ml LPS for 72 hours. The frequency of proliferating (B220+CFSE-low) B cells was determined by performing circulation cytometry analysis. Data, pooled from three impartial experiments, are shown as bar graphs (means SEM, n?=?5). **DNA, and ***DNA. (B) CFSE-labeled na?ve B cells were stimulated with ssDNA (10 g/mL or 50 g/ml) in the presence or absence of 100 ng/ml LPS for 72 h. Cells were analyzed by circulation cytometry for CD138 surface expression. Representative dot plots of three impartial experiments show the percentages of CD138+ plasma cells generated under different culture conditions. (C) Na?ve B cells were cultured in media containing ssDNA (50 g/ml) with or without LPS (100 ng/ml) for 72 h, and cell culture supernatants were collected for analysis of IL-10 by ELISA. Data, pooled from three impartial experiments, are shown as bar graphs (mean SEM, n?=?4).*and have been identified in humans with SLE . In SLE patients and murine lupus, excessive apoptosis with a defect in clearance of apoptotic cells is usually implicated as one source of extracellular DNA C. Furthermore, DNA-containing immune complexes (ICs) in serum of SLE patients were shown to activate plasmacytoid dendritic cells to overproduce type I IFN and the serum type I IFN levels correlated with both SLE disease activity and severity , . A direct correlation was established between endogenous DNA and autoantibody production in studies with transgenic AM14 B cells specific for autologous IgG2a (rheumatoid factor, RF). ICs made up of IgG2a mAbs specific for DNA or chromatins can directly activate autoreactive AM14 RF+ B cells to proliferate in a T-cell impartial (TI) manner by dual engagement of the B cell receptor (BCR) and intracellular Toll-like receptor (TLR) 9. DNA component in antigen is usually a critical factor for these immunostimulatory ICs to activate autoreactive AM14 RF+ B cells . TLR9 was first shown to uniquely recognize unmethylated CpG motif rich in microbial DNA and transmit mitogenic signals to B cells, although it was subsequently shown that TLR9 might also mediate mammalian DNA acknowledgement. It has been proposed that this endosomal localization of nucleic acid-sensing TLRs may be an evolutionary strategy to safeguard them from access to self nucleic acids , . Thus, DNA made up of ICs are actively involved in anti-nucleic acid and RF autoantibody production, and in the maintenance and exacerbation of autoimmunity . B cells play an important role in protective immunity by generating large amounts of antibodies against invading pathogens. B cells are also responsible for the development and pathogenesis of both systemic and organ-specific autoimmune diseases, as highlighted by the clinical efficacy of Peptide YY(3-36), PYY, human B-cell depletion therapies , . B cells sense antigens through antigen-specific BCRs and innate pattern acknowledgement receptors (PRRs) such as TLRs. In general, the antibody response against thymus dependent protein antigens Peptide YY(3-36), PYY, human (TD-Ags) requires the antigen-specific CD4+ T helper cells, which provide help for antigen specific B-cell activation via CD40-CD40L interactions and by cytokines in the germinal centers (GCs). Here, activated B cells proliferate and undergo class switch recombination (CSR), affinity maturation, and differentiate into memory B cells or high affinity antibody-secreting plasma cells. The TI antibody response can be elicited by microbial products in the absence of helper T cells. Both Peptide YY(3-36), PYY, human LPS (TLR4 Rabbit polyclonal to PELI1 ligand) and unmethylated CpG DNA (TLR9 ligand) can trigger polyclonal activation of na?ve mouse B cells and induce proliferation and differentiation into short-lived plasma cells . However, human na?ve B cells express low to undetectable Peptide YY(3-36), PYY, human levels of TLRs, and therefore require Peptide YY(3-36), PYY, human prior stimulation via BCR to respond to TLR ligands (microbial products) irrespective of the nature of T helper cells present . In contrast to na?ve B cells, human memory B cells have higher constitutively expressed TLRs and can respond directly to TLR stimulation to induce B cell proliferation and differentiation into.
The data are representative of three independent experiments