Proinflammatory cytokines upregulate expression of calprotectin (L1 protein, MRP-8/MRP-14) in cultured human keratinocytes
Normal skin contains no epidermal calprotectin. In biopsies from various inflammatory skin disorders, however, this antimicrobial protein occurs in the cytoplasm of keratinocytes.Objectives
To exclude the possibility of epidermal uptake of calprotectin from granulocytes and macrophages in diseased skin, we investigated whether cytokine-stimulated human keratinocytes can express calprotectin in vitro.Methods
Keratinocytes from healthy individuals were cultured in serum-free keratinocyte medium. The cells were stimulated with different cytokines [interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-10 and IL-13], both separately and in various combinations. Cytoplasmic protein levels of calprotectin were measured by an enzyme-linked immunosorbent assay performed on fixed adherent keratinocytes, and mRNA expression was determined by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR).Results
Calprotectin was produced by cytokine-stimulated keratinocytes, especially in response to combinations of the proinflammatory cytokines, which showed an additive upregulatory effect. When expression of mRNA for the light (MRP-8) and heavy (MRP-14) calprotectin chain was determined by RT–PCR, their respective levels were shown to be increased four- to ninefold and three- to fivefold after 24 h of combined stimulation with IFN-γ and TNF-α. The time course of calprotectin production showed no significant elevation for the first 16 h but then increased and peaked after 36 h.Conclusions
Cultured human keratinocytes stimulated with proinflammatory cytokines produce calprotectin, suggesting that epidermal expression of this antimicrobial protein in diseased skin reflects compartmentalized synthesis rather than uptake from dermal inflammatory cells.