CCL19 promotes TNF-alpha/IFN-gamma-induced production of cytokines by targeting CCR7/NF-κB signalling in HaCaT cells
Abstract
Introduction. Atopic dermatitis (AD) is the most common allergic skin disease. The dysfunction of keratinocytes is closely associated with AD progression. Nevertheless, the specific functions of CC chemokine ligand 19 (CCL19) and its receptor CC chemokine receptor 7 (CCR7) in human HaCaT keratinocytes are still unclear.
Material and methods. AD cell models in vitro were established by treating HaCaT cells with TNF-alpha (TNF-α, 10 ng/mL) and IFN-gamma (IFN-γ, 10 ng/mL). Cell viability was estimated by MTT assay. The protein levels of CCL19 and CCR7 were tested via Western blotting. The expression of CCL19 protein was knocked down by transfecting si-CCL19 into HaCaT cells. The contents of inflammatory factors i.e. thymus and activation-regulated chemokine (TARC), interleukin 6 (IL-6), and prostaglandin E2 were measured by ELISA, and the nitric oxide content was detected by Griess reagent. The protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) were tested via Western blotting.
Results. TNF-α and IFN-γ induced cytotoxicity and upregulated the expression of CCL19 and CCR7 in HaCaT cells. CCL19 knockdown alleviated cytokines-induced cytotoxicity and the release of TARC, IL-6, PGE2 and nitric oxide in TNF-α + IFN-γ-treated HaCaT cells. Furthermore, the protein levels of iNOS and COX-2 were also repressed by CCL19 knockdown. In addition, knockdown of CCL19 decreased CCR7 protein content and inhibited the phosphorylation of IκBα and p65, implying that knockdown of CCL19 inactivated CCR7/NF-κB signalling in HaCaT cells. Rescue assays validated that CCR7 overexpression reversed the effects of CCL19 silencing on the viability and levels of inflammatory factors in TNF-α + IFN-γ-induced HaCaT cells.
Conclusions. This study proves that CCL19 can promote TNF-α + IFN-γ-induced skin inflammatory responses by targeting CCR7/NF-κB pathway in HaCaT cells.
Keywords: atopic dermatitisCCL19CCR7inflammatory responsesTNF-αIFN-γ
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