The pro-apoptotic and pro-inflammatory effects of calprotectin on human periodontal ligament cells

Abstract

Calprotectin, a heterodimer of S100A8 and S100A9 subunits, is associated with inflammatory disorders such as rheumatoid arthritis and cystic fibrosis. Although calprotectin levels are increased significantly in the gingival crevicular fluid (GCF) of periodontitis patients, its effects on periodontal ligament cells (PDLCs) remain largely unknown. The aim of this study was to evaluate calprotectin levels in the GCF of generalized aggressive periodontitis (AgP) patients and to investigate the effects of recombinant human calprotectin (rhS100A8/A9) and its subunits (rhS100A8 and rhS100A9) in PDLCs. Both the concentration and amount of crevicular calprotectin were significantly higher in the AgP group compared with healthy controls. In addition, the GCF calprotectin levels were correlated positively with clinical periodontal parameters including bleeding index, probing depth, and clinical attachment loss. rhS100A8/A9 promoted cell apoptosis, whereas rhS100A8 and rhS100A9 individually exerted little effect on apoptosis in PDLCs. rhS100A9 and rhS100A8/A9 increased the activation of nuclear factor-κB (NF-κB) by promoting the nuclear translocation of p65 in PDLCs, subsequently inducing expression of the pro-inflammatory cytokines IL-6, IL-8, TNFα, and COX2. Treatment with an NF-κB inhibitor partially reversed the rhS100A9- and rhS100A8/A9-induced upregulation of the pro-inflammatory cytokines. rhS100A9, and not rhS100A8, was mainly responsible for the pro-inflammatory role of calprotectin. Collectively, our results suggest that calprotectin promotes apoptosis and the inflammatory response in PDLCs via rhS100A9. These findings might help identify novel treatments for periodontitis.

Figure 1. Analysis of PDLC apoptosis using annexin V/PI staining.

PDLCs were treated with 10⁻⁶ M rhS100A8, rhS100A9, rhS100A8/A9 or heat-inactivated rhS100A8, rhS100A9, rhS100A8/A9 for 48 h. Apoptosis was then assessed using flow cytometry after annexin V/PI staining. The fluorescence intensity of 10,000 cells was measured and the number of annexin-V-FITC-positive cells was calculated. Statistical analysis revealed that there were significantly more apoptotic cells in the rhS100A8/A9-treated group (10.2475%±2.019%, p<0.05) compared with the control group (5.653%±2.914%). There were no differences between the rhS100A8- and rhS100A9-treated groups. * p<0.05 vs. the control group.

Figure 2. Morphology of PDLCs after DAPI staining.

The morphological changes of PDLCs treated with rhS100A8, rhS100A9, rhS100A8/A9, or heat-inactivated rhS100A8, rhS100A9, or rhS100A8/A9 were evaluated by fluorescence microscopy after DAPI staining. Viable cells displayed a normal-sized nucleus and blue fluorescence, whereas apoptotic cells exhibited condensed chromatin and fragmented nuclei. Treatment with rhS100A8/A9 increased the percentage of apoptotic cells significantly compared with the control (24.5%±5.1% vs. 6.7%±3.1%, p<0.05) and heat-inactivated rhS100A8/A9 (24.5%±5.1% vs. 5.273%±1.14%, p<0.05). Fewer apoptotic cells were observed in rhS100A8-treated cultures whereas more apoptotic cells were present in rhS100A9-treated cells. * p<0.05 vs. the control or heat-inactivated groups.

Figure 3. rhS100A8, rhS100A9 and rhS100A8/9 upregulate pro-inflammatory cytokines in PDLCs.

(A). PDLCs were treated with rhS100A8, rhS100A9 or rhS100A8/A9 for 12 h, and the mRNA levels of cytokines were analyzed. rhS100A9 and rhS100A8/A9 upregulated the expression of IL-6, IL-8, TNFα, and COX2 significantly compared with rhS100A8 and the control group (p<0.05). (B). PDLCs were treated with rhS100A8, rhS100A9 or rhS100A8/A9 for 12 h, and the protein levels of IL-6 and IL-8 in PDLCs culture supernatants were detected using ELISA. rhS100A9 promoted the protein expression of IL-6 and IL-8 significantly (p<0.05). * p<0.05 vs. the control or heat-inactivated groups.

Figure 4. rhS100A8, rhS100A9 and rhS100A8/9 increase NF-κB signaling in PDLCs.

(A). PDLCs were treated with rhS100A8, rhS100A9 or rhS100A8/A9 for 6 h, fixed, and then incubated with NF-κB p65 antibodies. The translocation of p65 was observed in cells treated with rhS100A9 and rhS100A8/A9, whereas p65 remained mainly in the cytoplasm of cells treated with rhS100A8. (B). NF-κB activation was assessed using EMSA 12 h after treatment with rhS100A8, rhS100A9, or rhS100A8/A9. Compared with lane F, which contains oligonucleotides without nuclear extracts, a band representing binding was visible after the addition of PDLCs nuclear extracts from cells treated with rhS100A8 (lanes 2), rhS100A9 (lane 3), or rhS100A8/A9 (lane 4). A competition assay was then used to confirm the binding specificity (lane C). The addition of rhS100A8 attenuated the rhS100A9-induced DNA-binding activity of NF-κB. (C). PDTC reversed rhS100A9- and rhS100A8/A9-induced cytokines upregulation. * p<0.05 vs. the control or heat-inactivated groups.