Calprotectin expression by gingival epithelial cells

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.

FIG. 1

27E10 reactivity in cultured immortalized and human gingival keratinocytes. Gingival keratinocytes were fixed and permeabilized before reaction with the antibody 27E10 or biotinylated 27E10 against the MRP8-MRP14 complex. (A) Human gingival keratinocytes (27E10 secondary antibody Cy3). (B) Immortalized gingival keratinocytes (Alexa 350-conjugated streptavidin). Nonspecific immunoglobulin G controls showed no detectable fluoresence. Magnification, ×400.

FIG. 2

Expression of calprotectin in gingival keratinocytes. (A) Calprotectin complex (MRP8-MRP14) was detected in keratinocyte cytosol by sandwich ELISA. Results are representative of four experiments. (B and C) MRP8 (B) and MRP14 (C) mRNA in keratinocytes detected by RNase protection assay.

FIG. 3

Constitutive expression of IL-8, MRP8, and MRP14 mRNA in immortalized keratinocytes detected by RNase protection assay. IL-8-, MRP8-, and MRP14-specific mRNA are represented by single protected fragments of 374, 282, and 333 bp, respectively. The internal control GAPDH included in each assay tube is represented by a band at 220 bp.

FIG. 4

Regulation of IL-8 mRNA expression in immortalized human gingival keratinocytes. (A) A representative storage phosphor scan demonstrates that, by using quantitative RNase protection assay, moderate IL-8 mRNA expression was detected in 8 μg of total RNA from immortalized keratinocytes under normal control conditions as seen at time zero. Following treatment of separate flasks of the same culture over 24 h with 50 ng of PMA per ml, IL-8 mRNA expression dramatically increases over a 6-h period, returning to nearly constitutive levels by 12 h. Test lanes, hours 0 to 24, show the protected probes after binding to IL-8 (374 bp) and GAPDH (220 bp) mRNA, respectively, within the sample, followed by RNase treatment. An increase in the band intensity of GAPDH at 2 to 6 h reflects an increase in the total amount of mRNA, as expected with PMA stimulation. This did not affect quantitation (see panel B, below), since the ratio of GAPDH to IL-8 was still proportionally constant. Control lanes (C1 and C2) show the two probes GAPDH (C1, 266 bp) and IL-8 (C2, 420 bp) to which no RNase has been added. The results are representative of three experiments. (B) Quantitation of IL-8 mRNA in immortalized keratinocytes following induction with PMA. From panel A the signal intensity determined for the IL-8 protected fragment was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation (n = 3).

FIG. 5

Regulation of MRP8 and MRP14 mRNA expression in immortalized keratinocytes. Quantitation of MRP8-specific (A) and MRP14-specific (B) mRNA in immortalized keratinocytes following induction with 50 ng of PMA per ml for 24 h. The signal intensity determined for the MRP8 and MRP14 protected fragments was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation (n = 3).

FIG. 6

Treatment of immortalized keratinocytes with LPS (0.1, 1, and 10 μg/ml) for 2 or 4 h. Immortalized keratinocytes were incubated with various concentrations of LPS (0.1, 1, and 10 μg/ml) for 2 h (black bars) or 4 h (crosshatched bars). Expression of mRNA for IL-8-, MRP8-, and MRP14-specific mRNA was determined by RNase protection assay. The signal intensity for the protected fragments was normalized to the abundance of the internal control GAPDH. The data shown are the mean ± the standard deviation (n = 3).

FIG. 7

Treatment of immortalized keratinocytes with IL-1β (0.1, 1, and 10 ng/ml) for 2 or 4 h. Immortalized keratinocytes were incubated with various concentrations of IL-1β (0.1, 1, and 10 ng/ml) for 2 h (black bars) or 4 h (crosshatched bars). Expression of mRNA for IL-8-, MRP8-, and MRP14-specific mRNA was determined by RNase protection assay. The signal intensity for the protected fragments was normalized to the abundance of the internal control GAPDH. The data shown are the mean ± the standard deviation (n = 3).