Up Regulation Of Cc Chemokine Ligand 20 And Its Receptor Ccr6 In The Lesional Skin Of Early Scleroderma: An Implication In Scleroderma

ObjectiveTo investigate the expressions of CC chemokine CCL20 and its receptor CCR6 in early scleroderma skin lesions and study whether CCL20/CCR6 pair are involved in pathogenesis of scleroderma.Methods1.The protein and mRNA expression levels of CCL20 and CCR6 in early scleroderma skin lesions were investigated by IHC and Real-time RT-PCR, respectively.2. Primary scleroderma dermal fibroblasts (SDF) and normal adult dermal fibroblasts (NDF) were isolated by tissue explants, cultured in DMEM with 10% FBS and were indentified by morphology and ICC. Cell proliferation was mesured by MTT colorimetric assay and cells were treated with gradient concentrations of Th1 or Th2 cytokines. At indicated time points following cytokine exposure, conditioned media and cells were collected for CCL20 protein and mRNA quantification, respectively.3. Biological activity of CCL20 protein in the supernatants of SDF and NDF was evaluated by chemotaxis assays. We investigated whether CCL20 from NDF and SDF had a difference in chemotactic activity.Results1. CCL20 and CCR6 were significantly up-regulated in early scleroderma lesional vs normal skins and the locations of both were totally consistent.2. Dermal fibroblasts were successfully isolated and cultured in vitro steadily and rapidly. SDF and NDF have no significant difference in morphology and ICC. Under basal conditions, the levels of CCL20 protein and mRNA were very low both in NDF and SDF, but were significantly higher in NDF than in SDF. Th1 cytokines (TNF-αand IL-1β) markedly upregulated CCL20 mRNA and protein in a time and dose-dependent manner in both NDF and SDF; On the contrary, CCL20 mRNA and protein expression in SDF decreased with the increase of IL-4 concentrations and time. 3. PBMCs migrated toward recombinant CCL20 and culture supernants of NDF and SDF in a concentration-dependent manner. Conditioned media derived from NDF and SDF both displayed stronger chemotactic activity for PBMCs than recombinant CCL20, while supernant from SDF showed a even stronger chemotactic activity than that from NDF.4. Chemotatic response to recombinant CCL20 was almost completely blocked by human CCL20/MIP-3αantibodies. However, this chemotatic activity was only inhibited by 43.98±10.48% in supernants from SDF.Conclusion1.The interaction between CC chemokine CCL20 and its receptor CCR6 are involved in the pathogenesis of scleroderma and may serve an important role in the selective recruitment of inflammatory cells in early scleroderma leisional skins.2.SDF is in a persistently activated condition and can produce CCL20 in the presence or in the absence of exogenous proinflammatory stimuli.3.Except for CCL20, some other chemokines might also participate in the recruitment of MNCs into scleroderma leisional skins.