Toll-Like Receptor 9 Can Be Expressed at the Cell Surface of Distinct Populations of Tonsils and Human Peripheral Blood Mononuclear Cells

Abstract

Unmethlylated CpG dinucleotides induce a strong T-helper-1-like inflammatory response, presumably mediated by Toll-like receptor 9 (TLR9). However, the nature and cellular localization of TLR9 in primary human cells remain controversial. Here we demonstrate, using flow cytometry and immunofluorescence microscopy techniques, that TLR9 can be expressed at the cell surface. The primary human cell subsets that were positive for TLR9 expression were distinct depending on the tissues analyzed. Specifically, in human peripheral blood mononuclear cells (PBMC) the majority of cell surface TLR9؉ cells were confined to the major histocompatibility complex (MHC) class II ؉ CD19 ؊ populations that express CD11c and/or CD14, whereas in tonsils the same gated population contained primarily MHC class II ؉ CD19 ؉ cells. Cells positive for surface expression represented a minor fraction of the total cell populations examined, varying between 2 and 10%. In addition, we found that TLR9 expression at the surface of PBMC was up-regulated approximately fourfold following stimulation with the gram-negative bacterial cell wall component lipopolysaccharide, suggesting a potential modulatory role of TLR4 agonists on TLR9 expression. Taken together, these data validate human TLR9 expression at the surface of primary cells, in addition to the previously described intracellular localization. Further, our results suggest that human antigen-presenting cells comprise the major cell populations expressing cell surface TLR9.To discriminate between self and nonself antigens, the immune system has evolved a series of pattern recognition receptors to identify invading pathogens and initiate the host immune response. The newly identified Toll-like family of receptors function in this fashion to activate both the innate and adaptive arms of the immune response (21). Toll was originally identified in Drosophila as a receptor required for the establishment of dorso-ventral polarity (19). However, it was also found to play a critical role in immunity, as flies lacking this protein were highly susceptible to infection with Aspergillus fumigatus (27). The mammalian Toll-like receptors (TLRs) were cloned based on sequence homology to the Drosophila Toll protein (12,13,15,32,35,37). Activation of human Toll results in NF-B activation and up-regulation of B7-1, interleukin-1 (IL-1), IL-8, and IL-6 mRNAs, suggesting a role for TLR in bridging innate and adaptive immunities (32). To date, 10 TLRs in humans and 9 TLRs in mice have been identified. TLR9 has been reported to be the receptor for unmethylated CpG dinucleotides found within bacterial but not human DNA (20,24). Expression profiling revealed TLR9 mRNA or protein in B cells, plasmacytoid dendritic cells (DCs), and cells of the monocyte/macrophage lineage (6,20,22,24,25). Synthetic CpG oligodeoxynucleotides (ODN) were used for TLR9 stimulation or ligation and were found to mediate adjuvant activity (10, 30) resulting in the stimulation of T-helper-like-1 (Th1) cytokine production (23) and maturation of D...

Adjuvants modulating mucosal immune responses or directing systemic responses towards the mucosa

“…Hemmi et al [86] demonstrated that TLR-9 is required for the immune activation by CpG-dinucleotides. This receptor is highly expressed on plasmacytoid precursor DC, B cells, and cells of the monocytes/MØ lineage [53,104,239]. Whereas a lot of studies demonstrated an intracellular presence, more recent studies also show cell surface expression.…”

“…Whereas a lot of studies demonstrated an intracellular presence, more recent studies also show cell surface expression. Upon IFNγ treatment, both TLR9 mRNA expression and responsiveness to CpG DNA are upregulated in PBMC, whereas LPS upregulates cell surface expression [53].…”

TLR9 Is Required for the Gut-Associated Lymphoid Tissue Response following Oral Infection of Toxoplasma gondii

“…It is now appreciated that TLR9 can engage a wide range of microbial infections including bacteria (42,43), viral (44,45), and parasitic (8,9,15). TLR9 has been localized to several tissues and cell types, including the gastric mucosa, tonsils (29,46), IECs, Paneth cells in the gut mucosa, B cells, and several DC populations (2,15,39,(47)(48)(49).…”

IFN-γ Primes RAW264 Macrophages and Human Monocytes for Enhanced Oxidant Production in Response to CpG DNA via Metabolic Signaling: Roles of TLR9 and Myeloperoxidase Trafficking

“…These cells could be stained with both murine CpG DNA and human CpG DNA. CpG DNA may bind to TLR9 as well as other binding proteins, thereby staining intracellular and plasma membrane compartments (23)(24)(25). Hence, these immunostimulatory DNA molecules stain appropriate cells, suggesting that differential responses are not due to cell association.…”