Effect Of IL-6 And Soluble IL-6 Receptor On Osteoclast Differentiation And Activity Of Mice Osteoclast Precursors Induced By Varying Concentrations Of RANKL

Introduction:Periodontitis is one of the most common diseases in dental clinic and the major cause of age-related tooth loss.Dental plaque has been well proved to be the primary pathogenic factor for periodontal inflammation.Multiple pathogenic bacteria and their exotoxin aggregate in local periodontal tissue and trigger immunoresponse and infiltration of inflammation cells,resulting in destruction of periodontal supportive tissue(including parodontium,cementum and alveolar bone)featured by deep periodontal pocket,severe alveolar bone resorption and even tooth loosening or loss.During the process of periodontitis,persistent and significant inflammation is critical for alveolar bone destruction.A large number of mononuclear macrophages and lymphocytes recruited by bacterial stimulation produce inflammatory factors including IL-6,TNF-?,IL-1 etc.which potentiate the commitment of osteoclast precusors towards mature osteoclasts,leading to pathological alveolar bone resorption.IL-6 is a lymphokine secretes by activated T lymphocyte and fibroblast which has strong pro-resorption activity.Clinical studies showed that the level of IL-6 in periodontal tissue and gingival crevicular fluid from people with periodontitis was much higher than that of healthy control and this increase in IL-6 level was reversed after periodontal sequential treatment,which indicated that IL-6 level in local periodontal tissue is positively correlated with inflammatory response and alveolar bone resorption.Scholars previously hold the opinion that the stimulatory effect of IL-6 on osteoclastogenesis is indirectly achieved by acting on osteoblast/stromal cells to promote RANKL production,the most critical stimulator for osteoclast differentiation.However,recent studies revealed that IL-6 can also mediate osteoclastic differentiation through binding with IL-6R expressed on osteoclast precusors and this effect is inhibitory rather than stimulatory.This controversy is obvious and can not be explained yet.sIL-6R is the soluable form of IL-6R.Like membrane-bound IL-6R,sIL-6R can activate IL-6 signal transduction by inducing dimerization of gp-130 through binding with IL-6.Thus,sIL-6R plays a complementary role to classic IL-6-IL-6R system.It has been reported that only in the presence of sIL-6R can IL-6 fully function.For instance,in the absence of RANKL,the combined use of IL-6 and sIL-6R slightly induced osteoclastic differentiation of osteoclast precursor,but IL-6 alone did not.Therefore,we postulated that sIL-6R is required for the full activation of IL-6 transduction and it must be included in the researches in related to IL-6 and osteoclastogenesis.In conclusion,to investigate the exact mechanism underling the regulation of RANKL-induced osteoclastogenensis by IL-6,we established in vitro osteoclast differerntiation model using two lineages of osteoclast precusors:mouse bone marrow monocytes and RAW264.7 cells.Full dose of IL-6/sIL-6R was used to interfere series concentrations of RANKL-induced osteoclast differentiation.TRAP staining and Pit formation assays were performed to evaluate the effect of IL-6/sIL-6R on formation and resorption ablility of induced mature osteoclast.At the meantime,we also analyzed the changes of RANKL-activated intracellular signaling cascades in response to IL-6/sIL-6R treatment to uncover the potential mechanism by which IL-6 participates in regulation of osteoclast differentiantion.Materials and methods:1.The establishment of in vitro model of osteoclastic induction.Primary bone marrow macrophages were isolated from the whole bone marrow of four to six-week-old C57BL/6 mice.Murine RAW264.7 monocytic cells were purchased form the Shanghai Cell Center(Shanghai,China).The cells were seeded in 6-well plate(5X105 cells/well)or 24-well plates(3×104 cells/well)and cultured for 6 days in ?-MEM supplemented with 10%FBS,30 ng/ml M-CSF and series concentrations of RANKL.The culture medium was changed to fresh medium every other day.After 4 days of induced culture,TRAP staining was used to evaluate osteoclast differentiation and multinucleated TRAP-positive cells with at least 3 nuclei were scored as osteoclasts.Pit formation assay was performed to determine osteoclastic activity of induced osteoclast following 7 days of induction.The percentage of the resorbed areas and the number of resorption pits in three random resorption sites were measured under microscopic examination using Image-Pro Plus 6.2 software.2.The effect of IL-6/sIL-6R on series concentrations of RANKL-induced osteoclast differentiation and resorptive activity.BMMs and RAW264.7 ells were cultured with series levels of IL-6,sIL-6R or IL-6/sIL-6R in the absence or presence of RANKL and subjected to TRAP staining after 4 days.Multinucleated TRAP-positive cells with more than 3 nuclei were identified as osteoclast and scored.Pit formation assay was conducted to determine osteoclastic activity of osteoclast.Quantitative real time PCR was used to analyze mRNA expression levels of TRAP,CK,CTR and MMP-9.Protein expression levels of two critical transcriptional factors highly associated with osteoclastic differentiation including NFATc1 and c-fos were determined by Western blot.3.The potential mechanisms by which IL-6/sIL-6R differentially regulate varying concentrations of RANKL-induced osteoclast differentiation.BMMs were pretreated with 100 ng/ml IL-6/sIL-6R prior to osteoclastic induction with 10 ng/ml or 50 ng/ml RANKL.Cell lysates were collected at day 2 and 4 and subjected to western blot using anti-TRAF6 antibody.Co-immunoprecipitation assay was used to evaluate the effect of 100 ng/ml IL-6/sIL-6R pretreatment on RANK-TRAF6 interaction after 0,5,15 and 30 mins of RANKL incubation.After 0,15,30 and 60 mins of culture as above mentioned,cell lysates were obtained and subjected to western blot analysis using anti-p-38,anti-p-p38;anti-p-ERK,anti-ERK;anti-p-JNK,anti-JNK;anti-p-Akt,anti-Akt;anti-p-NF-?B,anti-NF-?B antibodies.Results:1.The establishment of in vitro model of osteoclastic induction.Both BMMs and RAW264.7 cells can differentiate into TRAP-positive multinucleated cells with the ability of bone resorption.There were many differences between osteoclasts derived from BMMs and RAW264.7 cells in cell number;size,morphology as well as resorption ability.Both the number,size and resorptive ability of the osteoclast-like cells increased with increased concentration of RANKL.50 ng/ml was considered as the saturation concentration of RANKL in osteoclastic induction.2.The effect of IL-6/sIL-6R on series concentrations of RANKL-induced osteoclast differentiation and resorptive activity.Neither IL-6 nor sIL-6R alone stimulated TRAP-positive multinucleated osteoclasts.However,when IL-6 and sIL-6R were simultaneously applied to the BMM cultures,a small but significant increase in osteoclast formation was observed at concentrations of 100 ng/ml.Exposure to sIL-6R did not affect the number of osteoclast-like cells induced by different concentrations of RANKL.High dose of IL-6(>10 ng/ml)suppressed 50 ng/ml RANKL-induced osteoclastogenesis but had little effect on lower concentrations of RANKL-induced osteoclast formation(?20 ng/ml).When IL-6 and sIL-6R were used in combination,10 ng/ml RANKL-induced osteoclast formation was strongly favored while that induced by 50 ng/ml RANKL was significantly inhibited.Bone resorption pit formation assay,expression of osteoclastic marker genes(CTR,CK,TRAP and MMP-9)and transcription factors(NFATc1 and c-fos)confirmed differential regulation of RANKL-induced osteoclastogenesis by IL-6/sIL-6R.3.The potential mechanisms by which IL-6/sIL-6R differentially regulate varying concentrations of RANKL-induced osteoclast differentiation.IL-6/sIL-6R neither changed expression profile of TRAF6 nor affected RANK-TRAF6 interaction induced by RANKL stimulation.Intracellular signaling transduction analysis revealed IL-6/sIL-6R specifically upregulated and downregulated the phosphorylation of NF-?B,ERK and JNK induced by low-and high level of RANKL,respectively.Conclusions:1.Mouse BMMs and RAW264.7 cells are appropriate osteoclast precursors for establishment of in vitro model of osteoclastic differentiation.2.IL-6/sIL-6R significantly promote and suppress osteoclast differentiation induced by low-(10 ng/ml)and high-level(50 ng/ml)of RANKL,respectively.3.IL-6/sIL-6R differentially regulate varing concertrations of RANKL-induced osteoclast differentiation via specifically upregulating and downregulating the phosphorylation of NF-?B,ERK and JNK induced by low-and high level of RANKL,respectively.