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Updated: Aug 13, 2026
Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin
Published on: July 14, 2017
Proinflammatory cytokines upregulate expression of calprotectin (L1 protein, MRP-8/MRP-14) in cultured human
G Mørk1, H Schjerven, L Mangschau
1Department of Dermatology, Institute of Pathology, University of Oslo, Rikshospitalet, N-0027 Oslo, Norway.
Background:
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)-gamma, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, 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-gamma and TNF-alpha. 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.
