INTRODUCTION & HYPOTHESIS
Our aim was to revisit the primary sterile epidermis lesion considering the alarmins constitutively expressed in the skin tissue and notably the IL-1 family members (IL-1fm) released by lesional keratinocytes.
At homeostasis, the non-lesional epidermis constitutes a barrier against various external aggressions.
When a lesion occurs, a dedicated inflammatory response is induced for the initiation of local protection and repair mechanisms, but this response should be adapted to the extent of the lesion and preserve the healthy surrounding skin tissue.
In order to model the epidermis lesion at cellular level, we first profiled the expression of IL-1fm in keratinocytes at mRNA and protein level and then assessed the effect of a keratinocyte extract on a panel of skin resident cells including fibroblasts, melanocytes, endothelial cells, leukocytes and keratinocytes themselves. IL-8 and IL-6 were used as consensus chemokine and pro-inflammatory cytokine read-out, respectively.
We then focused our work on the most responsive cell type (fibroblast) and on the cytokine that we demonstrated to be responsible for the major part of the observed effects (IL-1α).
MATERIALS & METHODS
Gene expression analysis was achieved using Affymetrix human genome strip array technology and the protein content in cell culture medium or cell extracts was analysed by ELISA.
For the analysis of IL-1fm in keratinocyte extract, cells were lysed in water using the same volume as the final volume of culture medium.
For the analysis of IL-6 and IL-8 release following recombinant IL-1fm or keratinocyte extract stimulation, the different cell types were incubated for 48 hours at similar density (except for leucocytes which were cultured at higher density in order to have a comparable culture format and protein quantity).
Keratinocyte extract on fibroblasts was also tested in presence of an anti-IL1R1 neutralizing polyclonal antibody.
Gene expression analysis were performed at 4h or 24 hours.
For titration experiments, IL-1α was tested at several concentrations, alone or in presence of increasing concentration IL-1Ra (at different ratios) and incubated on keratinocytes or fibroblasts for 48 hours.
1 – IL-1fm gene expression in keratinocytes
2 – IL-1fm protein expression in keratinocytes
Through the modeling of the epidermal lesion with a standardized keratinocyte extract, we have shown that fibroblasts are the most sensitive cell type among all other tested skin resident cells.
Stimulation of fibroblasts with keratinocyte extract induced a strong inflammatory response which we have demonstrated to be mediated by IL-1α.
Whereas fibroblasts are extremely sensitive to IL-1, they are overall insensitive to IL-1Ra, which is yet present in massive amounts in the keratinocyte extract.
On the contrary, IL-1Ra highly protects keratinocytes from IL-α stimulation.
Altogether these data indicate that fibroblasts may act as local sensors of primary epidermis lesion through keratinocyte IL-1α release.
In this context, IL-1Ra released together with IL-1α, should protect other skin cells from activation and limit the inflammatory response.