Cultured human melanocytes express functional toll-like receptors 2-4, 7 and 9.
BACKGROUND
Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs), which have been evolutionarily conserved in microbes. Human melanocytes are not simply pigment-producing cells but also have the phagocytic capacity and can produce pro-inflammatory mediators. However, the mechanisms of recognition of microbes by melanocytes have not yet been fully established.


OBJECTIVE
We investigated the TLRs 1-10 expression profile in human epidermal melanocytes and assessed their functions after triggering by their specific ligands.


METHODS
TLRs mRNA expression was determined by RT-PCR, and the TLR protein expression was measured by flow cytometry and immunofluorescence assays. After stimulation with various TLR ligands, the production of inflammatory cytokine IL-8 and IL-6 was measured by ELISA and the mRNA for chemokine CCL2, CCL3 and CCL5 was analyzed by real-time PCR. Phosphorylation of IkappaBalpha in TLR ligands-triggered melanocytes was determined by Western blot and the nucleus translocation of NF-kappaBp65 was analyzed by immunofluorescence.


RESULTS
Human melanocytes constitutively expressed TLRs 1-4, 6, 7 and 9 mRNA. Ample amounts of TLRs 2-4, 7 and 9 were confirmed at protein level. Stimulation of melanocytes with TLRs ligands resulted in the release of cytokines (IL-8 and IL-6) and the mRNA accumulation of chemokines (CCL2, CCL3 and CCL5). Triggering of TLRs in melanocytes resulted in the up-regulation of phosphorylated IkappaBalpha and in the nucleus translocation of NF-kappaBp65.


CONCLUSIONS
Present study indicates human melanocytes express a panel of functional TLRs. The ligation of TLRs can turn these cells into active players of the skin innate immunity.
Yu
Ning
N
Department of Dermatology, Huashan Hospital, Fudan University, Shanghai 200040, PR China.
Zhang
Shujie
S
Zuo
Fuguo
F
Kang
Kefei
K
Guan
Ming
M
Xiang
Leihong
L
D021581:Active Transport, Cell Nucleus; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics; D054405:Chemokine CCL3 / Q000235:genetics; D018946:Chemokine CCL5 / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007113:Immunity, Innate; D007231:Infant, Newborn; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008024:Ligands; D008297:Male; D008544:Melanocytes / Q000276:immunology*; D000072000:NF-KappaB Inhibitor alpha; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D051196:Toll-Like Receptor 3 / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D051199:Toll-Like Receptor 7 / Q000378:metabolism; D051217:Toll-Like Receptor 9 / Q000378:metabolism; D051193:Toll-Like Receptors / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000378:metabolism
C111835:CCL2 protein, human; C515429:CCL3 protein, human; C483024:CCL5 protein, human; C508606:CXCL8 protein, human; D018932:Chemokine CCL2; D054405:Chemokine CCL3; D018946:Chemokine CCL5; D020672:I-kappa B Proteins; C508600:IL6 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D008024:Ligands; C000606204:NFKBIA protein, human; C498143:RELA protein, human; D012333:RNA, Messenger; C495342:TLR2 protein, human; C495344:TLR3 protein, human; C495345:TLR4 protein, human; C495348:TLR7 protein, human; C495371:TLR9 protein, human; D051195:Toll-Like Receptor 2; D051196:Toll-Like Receptor 3; D051197:Toll-Like Receptor 4; D051199:Toll-Like Receptor 7; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha