Detection of calprotectin in primary and immortalised gingival keratinocytes

Objective: Calprotectin, a heterodimeric S100 calcium-binding protein consisting of MRP8 and MRP14 subunits, is present in the cytosol of neutrophils, monocytes and gingival keratinocytes. In periodontitis calprotectin is upregulated and can be detected in higher concentrations in gingival crevicular fluid. The mechanisms how calprotectin influences periodontal inflammation are unknown. Methods: To investigate the induction of calprotectin and the direction of the protein-secretion by gingival keratinocytes, primary and immortalized gingival keratinocytes were seeded on TranswellCol inserts and stimulated with an IL-1alpha/beta cytokine mixture. Experiments were repeated 2 times, each in duplicates. Apical and basolateral cell supernatant and cell lysates were harvested separately at different points and tested for calprotectin protein by ELISA.

Results: After a period of 8 h in primary keratinocytes a mean value of 19 ng/ml calprotectin can be detected in the apical cell supernatant and 9 ng/ml in total cell lysate of non-stimulated cells. In stimulated cells 31 ng/ml (38.8 % increase) in apical cell supernatant and 44 ng/ml (79.5% increase) in total cell lysate were measured. In immortalised gingival keratinocytes non-stimulated cells showed a mean value of 17 ng/ml in the apical cell supernatant and 14 ng/ml in total cell lysate. In stimulated cells 37 ng/ml (54.1 % increase) Calprotectin was detected in apical cell supernatant and 30 ng/ml (53.3 % increase) in total cell lysate.

Conclusion: Our data show, that calprotectin is induced in primary as well as in immortalised gingival keratinocytes by the proinflammatory cytokines IL-1alpha/beta. The secretion of calprotectin seems to be directed into the apical compartment.