S100 and cytokine expression in caries

Abstract

The molecular immune response of the pulpal tissue during chronic carious infection is poorly characterized. Our objective was to examine the expression of potential molecular mediators of pulpal inflammation, correlate their levels with disease severity, and determine the cellular localization of key molecules. Results indicated that there was significantly increased transcriptional activity in carious compared to healthy pulp, and the increase correlated positively with disease severity. Semiquantitative reverse transcriptase PCR analysis in 10 carious and 10 healthy pulpal tissue samples of the S100 family members S100A8, S100A9, S100A10, S100A12, and S100A13; the cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-8, IL-6, and epithelial cell-derived neutrophil attractant 78 (ENA-78); and the structural protein collagen-1alpha indicated that all genes tested, with the exception of S100A10, were more abundantly expressed in carious teeth. In addition, we found that the closer the carious lesion front was to the pulpal chamber the higher the expression was for all genes except S100A10. Multiple-regression analysis identified a significant positive correlation between the expression levels of S100A8 and IL-1beta, ENA-78, and IL-6 and between collagen-1alpha and S100A8, TNF-alpha, IL-1beta, IL-8, IL-6, and ENA-78. Immunohistochemical studies in carious pulpal tissue indicated that S100A8 and the S100A8/S100A9 complex were predominantly expressed by infiltrating neutrophils. Gene expression analyses in immune system cells supported these findings and indicated that bacterial activation of neutrophils caused upregulation of S100A8, S100A9, and S100A13. This study highlights the complex nature of the molecular immune response that occurs during carious infection.

FIG. 1.

(a) Average amount of RNA isolated from 10 diseased and 10 healthy teeth. An asterisk indicates statistically significant difference. Error bars for one standard deviation from the means are shown. (b) Scatter plot displaying the relationship between the amount of RNA isolated and the distance from the microbial front for the 10 carious teeth analyzed (P < 0.003; equation of line was y = 0.1848x + 0.9689; R² = 0.6896). The solid horizontal line represents the mean total RNA isolated from healthy dental tissue, and dashed lines indicate one standard deviation from the mean.

FIG. 2.

(a) Semiquantitative RT-PCR analysis showing differential gene expression analysis in carious and healthy dental tissue samples. The cycle number at which PCRs were analyzed is shown in parenthesis. (b) Densitometric analysis of gel images. Volume densities of amplified products were normalized against GAPDH control values, and average values for each group were calculated and expressed as a percentage of the highest normalized individual volume density obtained. Error bars for one standard deviation from the means are shown. All genes, except S100A10, showed statistically higher expression in carious teeth (P < 0.01).

FIG. 3.

Analysis of gene expression levels for S100 family members (a) and cytokines and collagen-1α (b) in teeth with deep and shallow carious lesions. Carious teeth with microbial fronts within 2 mm of the pulp chamber were classified as having deep lesions, and carious teeth with microbial fronts further than 2 mm from the pulp chamber were classified as having shallow lesions. Averaged normalized values for each classification group were calculated and plotted. Error bars for one standard deviation from the means are shown. ENA-78 exhibited a statistically significant difference in expression levels between deep and shallow carious lesions (P < 0.05).

FIG. 4.

Representative scatter plots of GAPDH-normalized gene expression in carious teeth for ENA-78 and IL-6 (R² = 0.849) (a) as well as for collagen-1α and IL-1β (R² = 0.816) (b).

FIG. 5.

(a) Carious (i, iii, v, and vii) and healthy (ii, iv, vi, and viii) teeth stained with H&E (i and ii), anti-S100A8 (iii and iv), anti-S100A8/S100A9 (v and vi), and anti-neutrophil elastase (vii and viii). (b) Human inflamed gingiva stained with anti-S100A8. Shown are negative (omission of primary antibody and replacement with nonimmune rabbit immunoglobulin G) (i) and positive (ii) controls. Field widths: for i and ii of panel a, 479 μm; for iii to viii of panel a, 241 μm; for i and ii of panel b, 241 μm.

FIG. 6.

Gene expression analysis of S100 family members, cytokines, collagen-1α, and osteopontin in monocytes (Mo), macrophages (Mφ), peripheral blood derived lymphocytes (PBL), neutrophils (N), and E. coli LPS-stimulated neutrophils (N+). Cycle number at which PCR samples were analyzed is shown in parenthesis. A representative image of duplicate analyses is shown.