2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistant organic pollutant that causes profound suppression of the primary immunoglobulin M (IgM) response in animal models of T-independent humoral immunity. B cells, the antibody producing cell type, are directly sensitive to TCDD, but a mechanistic understanding for TCDD disruption of the primary IgM response is incomplete. B cell differentiation into antibody-secreting cells (ASC), called plasmacytic differentiation, is regulated by Activator Protein-1 (AP-1), B-cell CLL/Iymphoma (BCL-6), B lymphocyte induced maturation protein-1 (Blimp-1) Paired Box gene 5 (Pax5), and X-box Binding Protein-1 (XBP-1). It was hypothesized that TCDD disrupts the Toll-like receptor (TLR) ligand lipopolysacchride (LPS)-activated primary IgM response by altering expression of transcription factors known to regulate of plasmacytic differentiation. In vivo LPS administration induced a significant increase in the number of ASCs, which was dose-dependently impaired by TCDD (3, 10, or 30 μg/kg). Gene and protein expression analysis showed TCDD suppressed LPS-elicited increases in CD138 and IgM components. Blimp-1 and XBP-1 expression were dose-dependently impaired by TCDD.
Relationships between transcription factors regulating plasmacytic differentiation in purified B cells were assessed in vitro using LPS and combinations of TCDD (0.03, 0.3, 3 or 30 nM), then analyzed by flow cytometry 24, 48, and 72 h post-treatment. TCDD caused a concentration-related suppression of Blimp-1 and phosphorylated c-Jun expression, and elevated BCL-6 expression, relative to LPS + DMSO treated B cells. TCDD treatment caused a concomitant suppression of LPS-activated MHC Class II, CD69, CD80, and CD86 upregulation as early as 24 h post-treatment.
Kinases directly and indirectly regulate AP-1 and BCL-6. It was hypothesized that TCDD alters BCL-6 expression and AP-1 phosphorylation by changing AKT, ERK, and JNK phosphorylation. Multiparametric analysis of AKT, ERK, and INK phosphorylation activated by LPS, R848, or CpG DNA in the B cell lymphoma CH12.LX was used to assess the time- and concentration-dependent effects of TCDD (0.003, 0.03, or 0.3 nM). TCDD suppressed TLR-activated AKT, ERK, and JNK phosphorylation at 15, 30, and 60 min post-treatment. TCDD at 30 nM impaired R848-activated phosphorylation of AKT, ERK, and INK in primary B cells. These results suggest TCDD suppresses the primary IgM response by altering transcription factor and activation marker expression, potentially as a result of TCDD interference in TLR-activated kinase phosphorylation.