IL-6, leukemia inhibitory factor (LIF), and oncostatin M (OSM) are IL-6-type cytokines that stimulate osteoclast formation and function. In the present study, the resorptive effects of these agents and their regulation of receptor activator of NF-κB ligand (RANKL), RANK, and osteoprotegerin (OPG) were studied in neonatal mouse calvaria. When tested separately, neither human (h) IL-6 nor the human soluble IL-6R (shIL-6R) stimulated bone resorption, but when hIL-6 and the shIL-6R were combined, significant stimulation of both mineral and matrix release from bone explants was noted. Semiquantitative RT-PCR showed that hIL-6 plus shIL-6R enhanced the expression of RANKL and OPG in calvarial bones, but decreased RANK expression. Human LIF, hOSM, and mouse OSM (mOSM) also stimulated 45Ca release and enhanced the mRNA expression of RANKL and OPG in mouse calvaria, but had no effect on the expression of RANK. In agreement with the RT-PCR analyses, ELISA measurements showed that both hIL-6 plus shIL-6R and mOSM increased RANKL and OPG proteins. 1,25-Dihydroxyvitamin D3 (D3) also increased the RANKL protein level, but decreased the protein level of OPG. OPG inhibited 45Ca release stimulated by RANKL, hIL-6 plus shIL-6R, hLIF, hOSM, mOSM, and D3. An Ab neutralizing mouse gp130 inhibited 45Ca release induced by hIL-6 plus shIL-6R. These experiments demonstrated stimulation of calvarial bone resorption and regulation of mRNA and protein expression of RANKL and OPG by D3 and IL-6 family cytokines as well as regulation of RANK expression in preosteoclasts/osteoclasts of mouse calvaria by D3 and hIL-6 plus shIL-6R.

Immune cells are thought to play an important role in bone loss caused by inflammation (1, 2). Disabling joint destruction in rheumatoid arthritis and the loss of teeth in periodontal disease are examples of skeletal loss that can occur with inflammation. Resorption of bone by osteoclasts represents the primary mechanism responsible for the loss of bone caused by inflammatory disease.Key factors regulating osteoclastogenesis include macrophage colony-stimulating factor (M-CSF) 2 and receptor activator of nuclear factor-B ligand (RANKL). M-CSF is a secreted product of stromal cells/ osteoblasts that enhances colony expansion of monocyte/osteoclast progenitor cells. RANKL exists both as a transmembrane protein in osteoblasts/stromal cells and as a soluble protein (3-6). RANKL directs the expanded progenitor cell population to the osteoclast lineage by activation of receptor activator of nuclear factor-B (RANK). The interaction between RANKL and RANK can be inhibited by osteoprotegerin (OPG), a decoy receptor released from stromal cells/osteoblasts.RANKL stimulation of RANK causes receptor trimerization and recruitment of tumor necrosis factor receptor-associated factors (TRAFs). TRAF1, 2, 3, and 5 bind to the carboxyl-terminal end of the RANK trimer, whereas TRAF6 binds more closely to the membrane. Downstream intracellular signaling mediated by RANK in osteoclast progenitor cells includes TRAF6-dependent activation of NF-B, mitogen-activated protein kinases (MAP kinases) and AP-1, and activation of c-Src and the phosphatidylinositol 3-kinase/Akt pathway (5-7). In addition, immunoreceptor tyrosine-based activation motif-mediated costimulatory signals have been shown to be required for expression of nuclear factor of activated T-cells 2 (NFAT2), the transcription factor believed to be crucial for osteoclast differentiation (8, 9). Several of these intracellular signaling molecules, including p50/p65, c-Fos, NFAT2, Fc receptor common ␥ subunit (FcR␥)/DNAX-activation protein 12 kD (DAP 12), and TRAF6, as well as RANK, RANKL, OPG, M-CSF, and the M-CSF receptor c-Fms, have been shown by gene deletion studies to be essential for osteoclastogenesis (2, 7, 10 -14).* This work was supported by grants from the Swedish Science Council (project 07525), the Swedish Rheumatism Association, the Royal 80 Year Fund of King Gustav V, the Swedish Dental Society, and the County Council of Västerbotten. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed. Tel.: 46-90-785-6290; Fax: 46-90-139289; E-mail: ulf.lerner@odont.umu.se.2 The abbreviations used are: M-CSF, macrophage colony-stimulating factor; ALP, alkaline phosphatase; ␣-MEM, ␣-modification of minimum essential medium; BMM, bone marrow macrophages; CT, calcitonin; CTR, calcitonin receptor; DAP12, DNAX-activating protein 12; D3, 1,25(OH) 2 -vitamin D 3 ; ERK, e...

IL-1alpha (interleukin-1alpha); IL-1beta (interleukin-1beta); IL-6 (interleukin-6); IL-11 (interleukin-11); LIF (leukemia inhibitory factor); OSM (oncostatin M); alpha(1)-coll. I (alpha(1)-collagen I); ALP (alkaline phosphatase); BMP-2 (bone morphogenetic protein-2); OC (osteocalcin); BSP (bone sialoprotein); TNFR I (tumor necrosis factor receptor I); TNFR II (tumor necrosis factor receptor II); IL-1R1 (interleukin-1 receptor 1); GAPDH (glyceraldehyde-3-phosphate dehydrogenase); RPL13A (ribosomal protein L13A); mRNA (messenger ribonucleic acid); cDNA (complementary deoxyribonucleic acid); PCR (polymerase chain-reaction); BCA (bicinchoninic acid); ELISA (enzyme-linked immunosorbent assay); alpha-MEM (alpha modification of Minimum Essential Medium); and FCS (fetal calf serum).

Periodontitis is a chronic inflammatory disease of tooth supporting tissues resulting in periodontal tissue destruction, which may ultimately lead to tooth loss. The disease is characterized by continuous leukocyte infiltration, likely mediated by local chemokine production but the pathogenic mechanisms are not fully elucidated. There are no reliable serologic biomarkers for the diagnosis of periodontitis, which is today based solely on the degree of local tissue destruction, and there is no available biological treatment tool. Prompted by the increasing interest in periodontitis and systemic inflammatory mediators we mapped serum cytokine and chemokine levels from periodontitis subjects and healthy controls. We used multivariate partial least squares (PLS) modeling and identified monocyte chemoattractant protein-1 (MCP-1) and eotaxin as clearly associated with periodontitis along with C-reactive protein (CRP), years of smoking and age, whereas the number of remaining teeth was associated with being healthy. Moreover, body mass index correlated significantly with serum MCP-1 and CRP, but not with eotaxin. We detected higher MCP-1 protein levels in inflamed gingival connective tissue compared to healthy but the eotaxin levels were undetectable. Primary human gingival fibroblasts displayed strongly increased expression of MCP-1 and eotaxin mRNA and protein when challenged with tumor necrosis factor-α (TNF-α and interleukin-1β (IL-1β), key mediators of periodontal inflammation. We also demonstrated that the upregulated chemokine expression was dependent on the NF-κΒ pathway. In summary, we identify higher levels of CRP, eotaxin and MCP-1 in serum of periodontitis patients. This, together with our finding that both CRP and MCP-1 correlates with BMI points towards an increased systemic inflammatory load in patients with periodontitis and high BMI. Targeting eotaxin and MCP-1 in periodontitis may result in reduced leukocyte infiltration and inflammation in periodontitis and maybe prevent tooth loss.

The osteotropic interleukin-6 (IL-6) types of cytokines IL-6, IL-11, and leukemia inhibitory factor (LIF), but not oncostatin M, are expressed by human gingival fibroblasts, and their expressions are regulated by IL-1 and tumor necrosis factor-alpha (TNF-alpha). In the present study, we investigated whether signaling through Toll-like receptor 2 (TLR2) can affect the expression of these cytokines in human gingival fibroblasts. Lipopolysac-charide (LPS) from P. gingivalis was found to stimulate IL-6 and LIF mRNA and protein, but not IL-11 or OSM mRNA. Using two synthetic ligands acting specifically at TLR2 and siRNA knockdown of TLR2, we demonstrated the important role of TLR2 in the stimulation of IL-6 and LIF in gingival fibroblasts. Analysis of these data suggests that signaling through the innate immune system controls the expression of osteotropic cytokines in human gingival fibroblasts.

Our discovery of the presence of ILCs both in gingivitis and periodontitis and concomitant expression of RANKL on a fraction of the ILC1 population suggest that these cells may be of importance in periodontal disease. In addition, our findings provide a new insight into the field of oral immunology.

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Marginal jawbone loss preceded the clinical onset of RA symptoms, but this was observed only in nonsmokers. Moreover, marginal jawbone loss was significantly greater in RANKL-positive presymptomatic subjects compared with RANKL-negative presymptomatic subjects and was highest in presymptomatic subjects positive for both ACPA and RANKL.