Background: Calprotectin is a major cytosolic protein of monocytes and granulocytes. It is increased in inflammatory tissues and is detected at high levels in the gingival crevicular fluid (GCF) of periodontitis patients. We previously reported that lipopolysaccharide of Porphyromonas gingivalis (P-LPS) and cytokines induced the release of calprotectin from monocytes isolated from human peripheral blood. The mechanisms of calprotectin expression and presence of its regulation factors in periodontal disease are unknown. On the other hand, P-LPS and cytokines are significant etiologic factors in the initiation and progression of periodontal diseases. In this study, we investigated the expression and production of calprotectin from human monocytes by examining the effects of lipopolysaccharide of P-LPS, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta).
Methods: Monocytes were isolated from the peripheral blood of healthy donors and cultured in the presence or absence of P-LPS, TNF-alpha, or IL-1beta. The expressions of calprotectin mRNAs (MRP8 and MRP14) were detected by Northern blotting. The contents of calprotectin in the cells and medium fractions were determined by enzyme-linked immunosorbent assay (ELISA). The DNA binding activity of C/EBPalpha, a transcription factor of MRP14, was assessed by electrophoretic mobility shift DNA-binding assay (EMSA).
Results: P-LPS, TNF-alpha, and IL-1beta induced MRP8/14 mRNAs and calprotectin production in monocytes. These factors also induced DNA CEBPalpha binding activity in monocytes. P-LPS increased MRP14 mRNA expression in monocytes to the maximum level, about two times the control level after 24 hours treatment, but did not enhance the basal level of MRP8. When the effects of TNF-alpha and IL-1beta on those mRNAs were investigated, both MRP8 and MRP14 significantly increased to about 2- and 2.5-fold the control level, respectively. Increases of MRP8/14 mRNA expression were followed by their protein production at about 2-fold the basal amount. DNA binding activity of C/EBPalpha was increased in P-LPS, TNF-alpha, and IL-1beta-treated monocytes.
Conclusions: These results demonstrate that P-LPS, TNF-alpha, and IL-1beta induce calprotectin production from human monocytes and that this production is associated with the activation of DNA C/EBPalpha binding complex.