Keratinocyte-derived cytokines after photodynamic therapy and their paracrine induction of matrix metalloproteinases in fibroblasts

Abstract

Recent studies have shown that collagen-degrading matrix metalloproteinase (MMP)-1 and MMP-3 are produced by fibroblasts in response to photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) and are considered to be involved in the antisclerotic effects of ALA-PDT observed in the treatment of localized scleroderma. As the primary target of topical PDT is epidermal keratinocytes, we studied the indirect participation of keratinocytes in the production of MMPs and collagen by dermal fibroblasts. Keratinocytes were treated with sublethal doses of ALA (100 micromol L(-1)) and red light. The conditioned media were collected 24 h after PDT and primary human fibroblasts were exposed to these media for 6-48 h. Further, a coculture model, keratinocytes seeded on to collagen type IV-coated transwells in the upper chamber and fibroblasts in the lower chamber, was used to study paracrine effects of keratinocytes after PDT. Keratinocyte supernatants after PDT showed a significant, up to 10-fold increase of interleukin (IL)-1alpha and a 2.5-fold increase of tumour necrosis factor-alpha as determined by enzyme-linked immunosorbent assay, while IL-6, MMP-1 and MMP-3 were not altered significantly. Fibroblasts treated with keratinocyte-conditioned media after PDT showed an induction of MMP-1 and MMP-3 protein levels up to threefold in both models used, suggesting that ALA-PDT modulates MMP-1 and MMP-3 production via indirect mechanisms. Collagen type I mRNA expression by fibroblasts was not altered significantly in either model. The addition of an IL-1 receptor antagonist to the keratinocyte-conditioned media completely inhibited the induction of MMP-1 and MMP-3 in stimulated fibroblasts, suggesting that IL-1 is mainly responsible for the observed paracrine effects. We present evidence that PDT can trigger MMP production in dermal fibroblasts not only directly as has been already shown, but also by an indirect paracrine loop mediated by soluble factors released by epidermal keratinocytes.