Cultured human melanocytes express functional toll-like receptors 2-4, 7 and 9
Ning Yu1, Shujie Zhang, Fuguo Zuo
1Department of Dermatology, Huashan Hospital, Fudan University, Shanghai 200040, PR China.
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.
Conclusion:
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.
Insights
Human melanocytes express functional Toll-like receptors (TLRs) 1-4, 6, 7, and 9. TLR activation triggers inflammatory responses, indicating melanocytes are key players in skin innate immunity.
Area of Science:
- Immunology
- Dermatology
- Cell Biology
Background:
- Toll-like receptors (TLRs) recognize conserved microbial patterns.
- Human melanocytes possess phagocytic and pro-inflammatory capabilities.
- Mechanisms of microbial recognition by melanocytes remain incompletely understood.
Purpose of the Study:
- To profile the expression of Toll-like receptors (TLRs) 1-10 in human epidermal melanocytes.
- To assess melanocyte function following TLR activation by specific ligands.
Main Methods:
- Quantitative reverse transcription PCR (RT-PCR) for TLR mRNA expression.
- Flow cytometry and immunofluorescence for TLR protein detection.
- ELISA for cytokine production (IL-8, IL-6) and real-time PCR for chemokine mRNA (CCL2, CCL3, CCL5).
- Western blot for IkappaBalpha phosphorylation and immunofluorescence for NF-kappaBp65 translocation.
Main Results:
- Human melanocytes constitutively express TLRs 1-4, 6, 7, and 9 at mRNA and protein levels.
- TLR ligand stimulation induced release of IL-8 and IL-6 cytokines.
- TLR activation led to increased mRNA for chemokines CCL2, CCL3, and CCL5.
- TLR triggering resulted in IkappaBalpha phosphorylation and NF-kappaBp65 nuclear translocation.
Conclusions:
- Human melanocytes express a functional repertoire of Toll-like receptors.
- TLR ligation activates melanocytes, positioning them as active participants in skin innate immunity.
