Study On The MCP-1Expression Induced By IL-1β In HPDLCs And Its Effect On The Recruitment Of Monocytes

Chemokines are bioactive peptides that regulate leukocyte activation and migration.As a potent chemokine for monocytes, MCP-1is implicated in regulating many immuneresponses and inflammation characterized by monocytic infiltrates. The present studiesshow that a large number of monocytes are recruited to the local inflammatory periodontalligament in the process of orthodontically induced inflammatory root resorption (OIIRR).But, the molecular mechanisms involved in the recruitment of monocytes have not beenelucidated in detail. The recent studies demonstrate that the expression of MCP-1inperiodontal ligament (PDL) cells is increased significantly during the process of OIIRR.For this reason, the expression of MCP-1in PDL cells is considered to have an importantrole in the recruitment of monocytes in the local inflammatory periodontal ligament ofOIIRR. Interleukin-1β (IL-1β) is an important proinflammatory cytokine. Evidence thus farsupports a role of IL-1β in molecular events that play a central role in the process of rootresorption. Studies indicate that IL-1β can favor osteoclastogenesis, and play a role incontrolling the function of odontoclasts. Additionally, IL-1β can induce the secretion ofmany cytokines in PDL cells. However, it remains unclear whether IL-1β regulates theexpression of MCP-1in hPDL cells, and contributes to the recruitment of monocytesthrough the chemotactic function of MCP-1.According to the issue of recruitment of monocytes in the periodontal tissue above,the present study was undertaken to observe the effect of inflammatory cytokine IL-1β onthe expression of MCP-1in hPDLCs, and then observe the effect of IL-1β stimulatinghPDLCs to release MCP-1in recruiting THP-1cells. The objective of this study was topreliminary analyse and discuss the molecular mechanisms involved in the recruitment ofmonocytes in periodontal tissue under the effect of inflammatory cytokine IL-1β.Methods:1. Effects of IL-1β on MCP-1expression in hPDLCshPDLCs were exposed to various concentrations of IL-1β (0,1,5,10,25ng/ml) for24h, RT-PCR, immunofluorescence staining and western blot were used to detect themRNA and protein expression of MCP-1in hPDLCs; hPDLCs were exposed to10ng/mlIL-1β at various times (12,24,48,72h), RT-PCR and western blot were used to detect themRNA and protein expression of MCP-1in hPDLCs.2. Study on IL-1β stimulating hPDLCs to release MCP-1in recruiting THP-1cellshPDLCs were exposed to various concentrations of IL-1β (0,1,5,10,25ng/ml), after24h expose, various cell culture supernatants of hPDLCs were collected, the effect ofvarious supernatants of hPDLCs on THP-1migration was assayed in Transwell chambers.hPDLCs were exposed to0or10ng/ml IL-1β. After24h expose, the cell culturesupernatants of hPDLCs were collected, and then were randomly divided intoexperimental group and control group; In the experimental group, the supernatants wereincubated with anti-MCP-1neutralizing antibodies; In the parallel control group, the supernatants were not incubated with anti-MCP-1neutralizing antibodies. After that, theeffect of supernatants of hPDLCs on THP-1migration was assayed in Transwellchambers.Results:1. Results of the effect of IL-1β on MCP-1expression in hPDLCs:By RT-PCR, the mRNA expression of MCP-1has been detected at weak level in thecontrol group; lower concentrations of IL-1β (1ng/ml and5ng/ml) had little effect onMCP-1mRNA expression in hPDLCs compared with the control group (P>0.05); Highconcentrations of IL-1β (10ng/ml and25ng/ml) increased the mRNA expression ofMCP-1in hPDLCs (P<0.05); After24h of pretreated,10ng/ml IL-1β increased the mRNAexpression of MCP-1in hPDLCs compared with the first12h (P<0.05); However, thelevels of MCP-1mRNA remained constant between48h and72h compared with24h.Immunofluorescence staining showed that, the expression of MCP-1was negative orweakly positive in control hPDLCs; After pretreated with10ng/ml IL-1β, the expressionof MCP-1was strong positive in hPDLCs, and the MCP-1-positive signals were locatedmainly in cytoplasm.By western blot, lower concentrations of IL-1β (1ng/ml and5ng/ml) had little effecton MCP-1protein expression in hPDLCs compared with the control group (P>0.05); TheMCP-1protein expression was enhanced in experimental hPDLCs pretreated with10ng/ml and25ng/ml IL-1β (P<0.05). After24h of pretreated,10ng/ml IL-1β increasedthe protein expression of MCP-1in hPDLCs compared with the first12h (P<0.05);However, the levels of MCP-1protein remained constant between48h and72h comparedwith24h.2. Results of the effect of IL-1β stimulating hPDLCs to release MCP-1in recruitingTHP-1cells:Migration assay showed that, there was no direct migration effects on THP-1cells, inwhich the cell culture medium simply contained IL-1β; The migration effects on THP-1cells were increased significantly by supernatants of various IL-1β-stimulated hPDLCs compared with the normal control group (P<0.05); However, the migration effect onTHP-1cells was inhibited by anti-MCP-1neutralizing antibodies significantly comparedwith the control group (P<0.05).Conclusions:The results of this study show that IL-1β can increase the expression of MCP-1fromhPDLCs; IL-1β has the potential to intensify the migratory response of monocytes throughstimulating hPDLCs to release MCP-1. These results imply that in the process of rootresorption, increased local inflammatory cytokine such as IL-1β, may up-regulate theexpression and secretion of MCP-1in hPDLCs, and this maybe the molecular mechanisminvolved in the recruitment of monocytes in the process of root resorption.