Effects of histamine on human periodontal ligament fibroblasts under simulated orthodontic pressure

Abstract

As type-I-allergies show an increasing prevalence in the general populace, orthodontic patients may also be affected by histamine release during treatment. Human periodontal ligament fibroblasts (PDLF) are regulators of orthodontic tooth movement. However, the impact of histamine on PDLF in this regard is unknown. Therefore PDLF were incubated without or with an orthodontic compressive force of 2g/cm2 with and without additional histamine. To assess the role of histamine-1-receptor (H1R) H1R-antagonist cetirizine was used. Expression of histamine receptors and important mediators of orthodontic tooth movement were investigated. PDLF expressed histamine receptors H1R, H2R and H4R, but not H3R. Histamine increased the expression of H1R, H2R and H4R as well as of interleukin-6, cyclooxygenase-2, and prostaglandin-E2 secretion even without pressure application and induced receptor activator of NF-kB ligand (RANKL) protein expression with unchanged osteoprotegerin secretion. These effects were not observed in presence of H1R antagonist cetirizine. By expressing histamine receptors, PDLF seem to be able to respond to fluctuating histamine levels in the periodontal tissue. Increased histamine concentration was associated with enhanced expression of proinflammatory mediators and RANKL, suggesting an inductive effect of histamine on PDLF-mediated osteoclastogenesis and orthodontic tooth movement. Since cetirizine inhibited these effects, they seem to be mainly mediated via histamine receptor H1R.

Fig 1. In vitro simulation of compressive force application to periodontal ligament fibroblasts (PDLF) occurring during orthodontic tooth movement.

After a preincubation time of 24 h with or without histamine (100 μM, 50/100/200 μM in receptor expression experiments) and an H1R/H2R/H4R antagonist (100 μM), a pressure of 2g/cm² was applied to PDLF by means of a sterile glass disc (ø33cm, 17.1g) for 48 h according to an established and published in vitro model.

Fig 2. Histamine-dependent expression of histamine receptors and COX-2 by periodontal ligament fibroblasts.

(a) Representative pictures of gene expression of histamine receptors in PDLF. Expression of histamine-3-receptor (H3R) could not be determined. Densitometric analysis of semiquantitative PCR for histamine-1-receptor (H1R, b), histamine-2-receptor (H2R, c) or histamine-4-receptor (H4R, d) after treatment with different histamine concentrations. (e) COX-2 gene expression after stimulation with histamine and inhibition of histamine receptor interaction using different inhibitors. AU: arbitrary units; *p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Statistics: Welch-corrected ANOVA with Games-Howell posthoc tests. Each symbol in figures represents a data point. Horizontal lines represent the mean ± standard error of mean.

Fig 3

Assessment of cell number (a), cytotoxicity (b) and viability (c) after compression with or without histamine or inhibition with cetirizine. AU: arbitrary units; *p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Statistics: Welch-corrected ANOVA with Games-Howell posthoc tests. Each symbol in figures represents a data point. Horizontal lines represent the mean ± standard error of mean.

Fig 4

COX-2 gene expression (a), PG-E2 secretion (b), IL-6 gene expression (c) and IL-6 secretion (d) after compression with or without histamine or inhibition with cetirizine. AU: arbitrary units; *p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Statistics: Welch-corrected ANOVA with Games-Howell posthoc tests. Each symbol in figures represents a data point. Horizontal lines represent the mean ± standard error of mean.

Fig 5

Effects of histamine and H1R antagonist cetirizine on OPG (a,b) and RANKL (c-e) gene and protein expression as well as on RANKL/OPG mRNA ratio (f). AU: arbitrary units; *p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Statistics: Welch-corrected ANOVA with Games-Howell posthoc tests. Each symbol in figures represents a data point. Horizontal lines represent the mean ± standard error of mean.