interleukin-8 and loxoribine

Plasmacytoid dendritic cells (pDCs) are unique with respect to their capacity to produce unsurpassed amounts of IFN-alpha and coexpress TLR7 and TLR9, mediating IFN-alpha production. Although TLRs are critical receptors of innate immunity, little is known about the immunological effects of TLR7/TLR9 costimulation. We have analyzed the effects of TLR7/TLR9 costimulation on IFN-alpha production by leukocytes and pDCs. Our experiments revealed that both synthetic (resiquimod and loxoribine) and natural (ssRNA40) TLR7 ligands abrogate CpG-A- and CpG-C-oligodeoxynucleotide (ODN)-induced IFN-alpha production by human leukocytes. Because TLR7 ligands themselves represent important IFN-alpha inducers, we demonstrated that substimulatory TLR7 ligand concentrations significantly inhibited CpG-A-induced IFN-alpha. Delayed addition of TLR7 ligands still resulted in complete suppression of CpG-A-ODN-induced IFN-alpha production, suggesting that the inhibition is unlikely to be caused by a kinetic uptake advantage. Unlike for CpG-A and CpG-C, TLR7 ligands did not inhibit CpG-B-ODN-induced IFN-alpha production. Experiments with purified human pDCs demonstrated that the inhibitory effects of TLR7/TLR9 costimulation were mediated directly by pDCs. Suppression of IFN-alpha production was not related to increased cell death and was also detectable in enriched mouse pDCs. Analyses of pDCs suggested that the TLR7 signal regulates the outcome of TLR7 ligand/CpG-A-ODN costimulation and can either inhibit (IFN-alpha) or promote (IL-8/CD40) cytokine and surface marker expression. Our data reveal for the first time a strong inhibitory effect of TLR7 stimulation on IFN-alpha production induced by CpG-A- and CpG-C-ODNs. These findings provide novel insight into the effects of TLR7/TLR9 costimulation and may support the development of novel TLR9 inhibitors.

Topics: Animals; CD40 Antigens; Dendritic Cells; Guanosine; Humans; Imidazoles; Interferon-alpha; Interleukin-8; Ligands; Mice; Oligodeoxyribonucleotides; Signal Transduction; Toll-Like Receptor 7; Toll-Like Receptor 9

Toll-like receptors (TLRs) are important pattern recognition molecules that activate the nuclear factor (NF)-kappaB pathway leading to the production of antimicrobial immune mediators. As keratinocytes represent the first barrier against exogenous pathogens in human skin, we investigated their complete functional TLR1-10 expression profile. First, reverse transcription-polymerase chain reaction (PCR) analysis revealed a very similar pattern of TLR mRNA expression when comparing freshly isolated human epidermis and cultured primary human keratinocytes. Thus, further experiments were carried out with primary keratinocytes in comparison with the spontaneously immortalized human keratinocyte cell line HaCaT. The quantitative expression of TLR1-10 mRNA in real-time PCR of primary human keratinocytes and HaCaT cells was analysed. Both cell types constitutively expressed TLR2, TLR3, TLR5, and to a lesser extent TLR10. TLR4 was only found in HaCaT cells, TLR1 to a higher degree in primary keratinocytes. In line with this, LPS induced mRNA expression of CD14 and TLR4 only in HaCaT cells. After stimulation with various TLR ligands, the NF-kappaB-activated chemokine interleukin-8 (IL-8) was measured. In primary keratinocytes and HaCaT cells the TLR3 ligand poly (I:C) was the most potent stimulator of IL-8 secretion. The TLR ligands peptidoglycan, Pam3Cys and flagellin which bind to TLR2, TLR1/TLR2 heterodimer, and TLR5, respectively, also induced IL-8 secretion, whereas no IL-8 was induced by LPS, R-848, loxoribine and cytosine guanine dinucleotide-containing oligodeoxynucleotide. A corresponding pattern was found in the RelA NF-kappaB translocation assay after ligand stimulation of primary keratinocytes. These studies provide substantial evidence for a functional TLR expression and signalling profile of normal human keratinocytes contributing to the antimicrobial defence barrier of human skin.

Topics: Biological Transport; Cell Culture Techniques; Cell Line, Transformed; Dendritic Cells; Epidermis; Gene Expression; Guanosine; Humans; Imidazoles; Interleukin-8; Keratinocytes; Ligands; Lipopolysaccharides; Membrane Glycoproteins; NF-kappa B; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stimulation, Chemical; Toll-Like Receptor 1; Toll-Like Receptor 10; Toll-Like Receptor 2; Toll-Like Receptor 3; Toll-Like Receptor 4; Toll-Like Receptor 5; Toll-Like Receptors