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Calprotectin expression by gingival epithelial cells
1Department of Preventive Science, School of Dentistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Abstract:
Calprotectin, a heterodimer of MRP8 and MRP14 with antimicrobial properties, is found in the cytosol of neutrophils, monocytes, and human gingival keratinocytes. During inflammation of the oral mucosa, the expression of immunoreactive calprotectin appears upregulated. Given the possible cell sources, we sought to learn if epithelial cells upregulate calprotectin in response to proinflammmatory agents. First, human gingival keratinocytes were maintained in primary culture until senescence. At each passage, cells were harvested and analyzed for quantitative expression of MRP8 and MRP14 subunit mRNA by RNase protection assays and calprotectin complex by enzyme-linked immunosorbent assay. Calprotectin expression was constitutive in the primary gingival keratinocytes, but calprotectin-specific mRNA and protein tended to increase as the cells neared senescence. To test whether calprotectin expression was inducible, immortalized gingival keratinocyte cultures were treated for 2 to 4 h with lipopolysaccharide (LPS) or interleukin-1 beta (IL-1 beta). As a positive control for inducible expression, immortalized keratinocytes were incubated with phorbol myristate acetate (PMA) (50 ng/ml) for 24 h. Incubation with PMA stimulated increased expression of MRP8 and MRP14 mRNA within 2 h, peaking within 5 h. MRP8- and MRP14-specific mRNA expression by immortalized keratinocytes appeared to be unaffected by LPS or IL-1 beta. In contrast, LPS, IL-1 beta, and PMA each upregulated IL-8. These data show that calprotectin mRNA is expressed constitutively in cultured keratinocytes, while expression by immortalized cells appears to be independent of the exogenous proinflammatory agents LPS and IL-1 beta.
Insights
Calprotectin is constitutively expressed in gingival keratinocytes, increasing with senescence. Proinflammatory agents like LPS and IL-1 beta did not induce calprotectin expression in immortalized cells.
Area of Science:
- Oral immunology
- Epithelial cell biology
- Inflammation research
Background:
- Calprotectin, an antimicrobial protein complex of MRP8 and MRP14, is present in immune cells and oral epithelial cells.
- Upregulation of calprotectin is observed during oral mucosal inflammation.
- The role of epithelial cells in calprotectin expression during inflammation requires further investigation.
Purpose of the Study:
- To investigate whether human gingival keratinocytes upregulate calprotectin expression in response to pro-inflammatory agents.
- To determine if calprotectin expression is constitutive or inducible in oral epithelial cells.
Main Methods:
- Primary and immortalized human gingival keratinocytes were cultured.
- Quantitative expression of MRP8 and MRP14 mRNA was analyzed using RNase protection assays.
- Calprotectin complex levels were measured by enzyme-linked immunosorbent assay.
- Cells were treated with lipopolysaccharide (LPS), interleukin-1 beta (IL-1 beta), and phorbol myristate acetate (PMA).
Main Results:
- Calprotectin mRNA and protein expression were found to be constitutive in primary gingival keratinocytes.
- Expression of calprotectin subunits (MRP8 and MRP14) tended to increase as primary cells approached senescence.
- Immortalized keratinocytes showed increased MRP8 and MRP14 mRNA expression upon PMA stimulation but not in response to LPS or IL-1 beta.
- LPS, IL-1 beta, and PMA all upregulated interleukin-8 (IL-8) expression.
Conclusions:
- Calprotectin is constitutively expressed in cultured gingival keratinocytes.
- Calprotectin expression in immortalized keratinocytes appears independent of exogenous pro-inflammatory agents LPS and IL-1 beta.
- These findings suggest that while calprotectin is present in epithelial cells, its induction by common inflammatory mediators like LPS and IL-1 beta may be limited in this cell type.