Analysis of Signaling Pathways Involved in Peptidoglycan-Induced RANTES Production from Murine Langerhans Cells

Abstract

Background: Our previous study showed that percutaneous application of peptidoglycan from Staphylococcus aureus induced eosinophil infiltration in murine skin through production of CCL5/RANTES (regulated upon activation in normal T cells expressed and secreted) from epidermal Langerhans cells. Although it is well known that peptidoglycan is an agonist of Toll-like receptor (TLR)-2, it is unclear whether other TLR agonists are able to induce RANTES production from Langerhans cells. Methods: Langerhans cells were purified from murine epidermal cells by the panning method using anti-IA^d monoclonal antibody. RANTES production by Langerhans cells was investigated by RT-PCR and ELISA. Analysis of the signaling pathways responsible for RANTES production by Langerhans cells was performed by ELISA using N-acetyl-L-cysteine, SP600125, SB203580 and PD98059, which are specific inhibitors of NF-κB activation, JNK, p38 MAPK and ERK, respectively, and was finally confirmed by Western blot analysis. Results: Peptidoglycan, poly(I:C), lipopolysaccharide and CpG DNA, which signal through TLR-2, TLR-3, TLR-4 and TLR-9, respectively, were found to strongly induce RANTES production. Treatment with N-acetyl-L-cysteine inhibited all TLR agonist-induced RANTES production. However, treatment with SP600125 did not inhibit CpG DNA-induced RANTES production. Furthermore, treatment with SB203580 inhibited only peptidoglycan- and lipopolysaccharide-induced RANTES production and the inhibition was correlated with that of p38 MAPK phosphorylation. Conclusion: These results suggest that the signaling pathway of RANTES production from murine Langerhans cells induced by different TLR stimuli is not necessarily the same, and that inhibition of p38 MAPK may be a more specific therapeutic target for eosinophilic inflammation in patients with atopic dermatitis associated with S. aureus colonization.

Expression of vascular endothelial growth factor and other cytokines in atopic dermatitis, and correlation with clinical features

“…35 Moreover, findings suggest a role for staphylococcal superantigens in the production of RANTES during the development of atopic skin inflammation. 36 Some studies have suggested a proangiogenic role of IL-6, 4,37 but in our study, we did not find any correlations between IL-6 levels in AD versus healthy individuals.…”

“…Augmented production of RANTES has correlated with inflammation associated with AD . Moreover, findings suggest a role for staphylococcal superantigens in the production of RANTES during the development of atopic skin inflammation …”

Peptidoglycan Synergistically Augments Production of Allergic Mediators from Murine Mast Cells in Combination with Muramyldipeptide

“…Polymerase chain reaction (PCR) was performed using a GeneAmp PCR System 9700 (Perkin-Elmer, Norwalk, CT, USA) in a 25-µL reaction volume containing 1.5 µL of cDNA (corresponding to 16 ng of the original mRNA) as described previously, and the PCR products were separated on a 2% agarose gel containing ethidium bromide [7]. The primers used for amplification of β-actin, CXCL9/Mig, CXCL10/IP-10, CCL17/ TARC, CCL22/MDC, TLR2 and NOD2 have been described elsewhere [7,[10][11][12][13][14][15]. The culture supernatants of mast cells were also collected after incubation with PEG and/or MDP for 48 h, and their chemokine concentrations were determined using enzyme-linked immunosorbent assay (ELISA) kits for quantification of murine CXCL10 and CCL17 (R & D Systems).…”