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, 2019, 8672604
eCollection

The Possible Role of Neutrophils in the Induction of Osteoclastogenesis

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The Possible Role of Neutrophils in the Induction of Osteoclastogenesis

Carolyn G J Moonen et al. J Immunol Res.

Abstract

The ligand of the receptor activator of NF-κB (RANKL) is a key molecule in the formation of osteoclasts, the key cells that cause the disease-associated alveolar bone resorption in periodontitis. We hypothesized that polymorphonuclear leukocytes (PMNs), found as the most prominent cells of inflamed periodontal tissues, could play an important role in providing signals to trigger osteoclastogenesis and thus activating pathological bone resorption in periodontitis. RANKL expression was investigated on circulatory PMNs (cPMNs) and oral PMNs (oPMNs) taken from both controls and periodontitis patients. On average, 2.3% and 2.4% RANKL expression was detected on the cPMNs and oPMNs from periodontitis patients, which did not differ significantly from healthy controls. Since cPMNs may acquire a more osteoclastogenesis-facilitating phenotype while migrating into the inflamed periodontium, we next investigated whether stimulated (with LPS, TNF-α, or IL-6) cPMNs have the capacity to contribute to osteoclastogenesis. Enduring surface expression of RANKL for short-lived cells as cPMNs was achieved by fixating stimulated cPMNs. RANKL expression on stimulated cPMNs, as assessed by flow cytometry and immunohistochemistry, was limited (6.48 ± 0.72%, mean expression ± SEM) after 24 and 48 hours of stimulation with LPS. Likewise, stimulation with TNF-α and IL-6 resulted in limited RANKL expression levels. These limited levels of expression did not induce osteoclastogenesis when cocultured with preosteoclasts for 10 days. We report that, under the aforementioned experimental conditions, neither cPMNs nor oPMNs directly induced osteoclastogenesis. Further elucidation of the key cellular players and immune mediators that stimulate alveolar bone resorption in periodontitis will help to unravel its pathogenesis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
RANKL expression on (unstimulated) PMNs from controls and periodontitis patients. RANKL expression was measured on circulatory (cPMNs) and oral PMNs (oPMNs) from healthy controls (white bars) and periodontitis patients (grey bars). Bars represent the percentage of RANKL expression corrected for IgG isotype control expression on CD66b-positive PMNs. Whiskers demonstrate standard errors of means. The employed gating strategy is shown in Supplementary . No significant differences were found between conditions (unpaired t-test). n = 13 controls, n = 9 periodontitis patients.
Figure 2
Figure 2
Immunohistochemical visualization of RANKL expression on LPS-stimulated cPMNs. Unstimulated (a) and LPS-stimulated (b, c) cPMNs were stained with the nuclear stain propidium iodide (PI, visualized in red) and RANKL (visualized in green). Unstimulated PMNs do not express RANKL (a). Isotype staining of stimulated cPMNs did not show any green signal (b). RANKL expression is shown here on stimulated cPMNs (indicated with arrows in (c)). Scale bars represent 37.5 μm. Here, representative micrographs of 3 independent experiments are shown. PI is visualized as red fluorescence and RANKL is visualized as green fluorescence.
Figure 3
Figure 3
RANKL expression on LPS-stimulated cPMNs. Percentages of RANKL expression by cPMNs after 24 (white bars) and 48 hours (grey bars). Different stimulatory conditions are shown on the x-axis. Percentages (mean + standard error of means, n = 3) of RANKL expression on live, CD66b-positive cells are shown on the y-axis. Overall, no significant differences (paired one-way ANOVA) were observed between unstimulated and stimulated conditions. The employed gating strategy is shown in Supplementary .
Figure 4
Figure 4
LPS-activated cPMNs do not stimulate osteoclastogenesis in cocultures with osteoclast precursors. Cells were stained for tartrate-resistant acid phosphatase (TRACP, green) and counterstained with the permeable nuclei dye 4′,6-diamidino-2-phenylindole (DAPI, blue). (a) Preosteoclasts (monocytes) were cultured for 10 days with M-CSF and RANKL. Formed osteoclasts are indicated with arrows. (b) Preosteoclasts (monocytes) were cultured for 10 days with M-CSF and unstimulated cPMNs. No osteoclasts were formed in this condition. (c) Preosteoclasts (monocytes) were cocultured with LPS-activated (100 ng/mL, 48 hours) cPMNs. In this condition, no osteoclasts were observed. Representative micrographs of three independent experiments are shown. Scale bars represent 100 μm.
Figure 5
Figure 5
RANKL expression on IL-6- or TNF-α-stimulated cPMNs. Percentages of RANKL expression by cPMNs stimulated with IL-6 (a) or TNF-α (b) after 24 (white bars) and 48 (grey bars) are shown on the y-axes. Concentrations of stimulants are shown on the x-axes. Percentages (mean + standard error of means, n = 3) of RANKL expression on live, CD66b-positive cells are shown on the y-axes. Overall, no significant differences (paired one-way ANOVA) were observed between unstimulated and IL-6-stimulated conditions. The significant difference (p < 0.05) was compared (paired one-way ANOVA) to the unstimulated condition. The employed gating strategy is shown in Supplementary .
Figure 6
Figure 6
Activated cPMNs do not stimulate osteoclastogenesis in cocultures with osteoclast precursors. Nuclei are stained with DAPI (visualized in blue), and TRACP expression is shown in green. Preosteoclasts (monocytes) were cultured for 10 days with RANKL and M-CSF. (a) TRACP-positive, multinucleated cells were formed (depicted by arrows). (b) Monocytes were cultured with unstimulated cPMNs. (c) Monocytes were cultured with stimulated cPMNs (IL-6, 100 ng/mL for 48 hours) and did not differentiate into TRACP-positive multinucleated cells. (d) Monocytes were cocultured with TNF-α-stimulated cPMNs (50 ng/mL for 24 hours) and did not differentiate into osteoclasts. (e) Monocytes were cocultured with TNF-α-stimulated cPMNs (50 ng/mL, 48 hours) and did not differentiate into osteoclasts. Representative micrographs of three independent experiments are shown. Scale bars represent 200 μm.

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