Relative expression of toll-like receptor (TLR)-9 protein in human basophils vs. plasmacytoid dendritic cells (PDC): Effects of CpG-ODN on FceRIa mRNA levels

Abstract

Rationale To further characterize functional TLR9 expression in basophils, comparing the relative protein levels of this receptor to those found in PDC and to identify novel effects of immunomodulatory CpG oligodeoxynucleotide (CpG-ODN) ligands on the expression of activation-linked markers found on both cell types. Methods Basophils and PDC were purified from blood using negative and positive selection, respectively. Flow cytometry and Western Blot analysis were used in assessing TLR9 protein levels. Real-Time RT-PCR quantified changes in FcϵRIα mRNA expression. Results Basophils constitutively expressed protein for TLR9 at levels exceeding those found in PDC. This was demonstrated by intracellular staining, with net MFI values of 6 ± 1 in basophils (n=7) compared to 1 ± 1 in PDC (n=6). Treatment with IL-3 (18h) markedly up-regulated TLR9 in PDC (net MFI=8 ± 2), with levels approaching those detected in basophils (10 ± 2) also receiving this treatment (n=6). These findings were confirmed using immunoblotting, which detected a prominent band (∼120 kd) that was quantitatively greater in basophils than in PDC (n=4). While previously reporting that CpG-ODN activate NFκB and inhibit IgE-mediated IL-4 and IL-13 secretion in basophils, these same oligos had no effect on FcϵRIα mRNA expression in these cells. In contrast, FcϵRIα mRNA was unexpectedly detected in PDC and treatment with CpG-ODN (16-18h) markedly down-regulated by more than 25-fold (n=3) the mRNA levels for this subunit in this cell type. Conclusions These data suggest that basophils are important cell targets for CpG-DNA and therapeutic ODN. Reduction of FcϵRIα in PDC may provide an additional mechanism by which CpG-ODN promotes Th1-like immunity.