M-CSF priming of osteoclast precursors can cause osteoclastogenesis-insensitivity, which can be prevented and overcome on bone.

Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. Such M-CSF primed cells expressed the receptor RANK, but lacked the crucial osteoclastogenic transcription factor NFATc1. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. Osteoclastogenesis-insensitive precursors grown in the absence of bone regained their osteoclastogenic potential when transferred to bone. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone.

Immune complex-induced inhibition of osteoclastogenesis is mediated via activating but not inhibitory Fcγ receptors on myeloid precursor cells.

OBJECTIVE: To investigate the role of Fcγ receptors (FcγRs) in osteoclastogenesis and osteoclast function. METHODS: Bone destruction was analysed in arthritic knee joints of several FcγR-knockout mouse strains. Unfractionated bone marrow cells were differentiated in vitro towards osteoclasts in the absence or presence of immune complexes (ICs) and stimulated thereafter for 24 h with tumour necrosis factor α (TNFα) or lipopolysaccharide (LPS). In addition, mature osteoclasts were stimulated with ICs. Experiments were analysed for osteoclast formation, bone resorption and the expression of FcγRs and osteoclast markers. RESULTS: Bone destruction was significantly increased in arthritic knee joints of FcγRIIB-deficient mice. All FcγR classes were highly expressed on osteoclast precursors. Expression of the inhibitory FcγRIIB was similar on mature osteoclasts compared to macrophages, whereas activating FcγR levels were significantly lower. IC stimulation of mature osteoclasts did not affect their number or their bone resorptive capacity. ICs significantly inhibited differentiation of unfractionated bone marrow cells towards osteoclasts, bone resorption and expression of osteoclast markers. In the presence of ICs, osteoclastogenesis of FcγRIIB(-/-) precursors and bone resorption remained inhibited. In contrast, ICs could not inhibit osteoclast formation or bone resorption of FcRγ-chain(-/-) precursors. When IC-inhibited osteoclastogenesis was followed by stimulation with TNFα or LPS, the inhibitory effects of ICs were overruled. CONCLUSION: Activating FcγRs mediate IC-induced inhibition of osteoclastogenesis, which might be overruled in the presence of proinflammatory mediators. This suggests that the balance of FcγR-mediated inflammation, through proinflammatory cytokine production, as well as the direct inhibitory effect of ICs on osteoclastogenesis determines the net effect on bone loss.

Antiinflammatory and chondroprotective effects of intraarticular injection of adipose-derived stem cells in experimental osteoarthritis.

OBJECTIVE: In experimental collagenase-induced osteoarthritis (OA) in the mouse, synovial lining macrophages are crucial in mediating joint destruction. It was recently shown that adipose-derived stem cells (ASCs) express immunosuppressive characteristics. This study was undertaken to explore the effect of intraarticular injection of ASCs on synovial lining thickness and its relation to joint pathology in experimental mouse OA. METHODS: ASCs were isolated from fat surrounding the inguinal lymph nodes and cultured for 2 weeks. Experimental OA was induced by injection of collagenase into the knee joints of C57BL/6 mice. OA phenotypes were measured within 8 weeks after induction. Histologic analysis was performed, and synovial thickening, enthesophyte formation, and cartilage destruction were measured in the knee joint. RESULTS: ASCs were injected into the knee joints of mice 7 days after the induction of collagenase-induced OA. On day 1, green fluorescent protein-labeled ASCs were attached to the lining layer in close contact with macrophages. Thickening of the synovial lining, formation of enthesophytes associated with medial collateral ligaments, and formation of enthesophytes associated with cruciate ligaments were significantly inhibited on day 42 after ASC treatment, by 31%, 89%, and 44%, respectively. Destruction of cartilage was inhibited on day 14 (65%) and day 42 (35%). In contrast to early treatment, injection of ASCs on day 14 after OA induction showed no significant effect on synovial activation or joint pathology on day 42. CONCLUSION: These findings indicate that a single injection of ASCs into the knee joints of mice with early-stage collagenase-induced OA inhibits synovial thickening, formation of enthesophytes associated with ligaments, and cartilage destruction.

Intravenously delivered glucocorticoid liposomes inhibit osteoclast activity and bone erosion in murine antigen-induced arthritis.

UNLABELLED: The objective of this study was to determine the effect of systemic delivery of prednisolone phosphate (PLP) encapsulated within long circulating 'stealth' liposomes on bone erosion and osteoclast activity during experimental antigen-induced arthritis (AIA). Liposomal PLP strongly suppressed knee joint swelling, synovial infiltrate and bone erosion in antigen-induced arthritis. The number of active osteoclasts was not only suppressed in bone lesions near inflamed synovium, but also within the trabecular bone of the tibia, suggesting a systemic suppression of osteoclast activation. Furthermore, liposomal PLP directly blocked osteoclast differentiation and bone resorption in vitro while it also suppressed expression of osteoclast differentiation factors M-CSF and RANKL in the synovium. Targeting studies showed that liposomes are most efficiently phagocytosed by macrophages and early precursors of osteoclasts in the bone marrow rather than by mature osteoclasts, indicating a possible inhibition of osteoclast differentiation from an early stage. CONCLUSION: Liposomal glucocorticoid delivery rather than free PLP offers a more efficacious way to inhibit both inflammation and bone erosion in rheumatoid arthritis.

S100A8 enhances osteoclastic bone resorption in vitro through activation of Toll-like receptor 4: implications for bone destruction in murine antigen-induced arthritis.

OBJECTIVE: Rheumatoid arthritis, which is associated with elevated levels of S100A8 and S100A9, is characterized by severe bone erosions caused by enhanced osteoclast formation and activity. The aim of the present study was to investigate the role of S100A8 and S100A9 in osteoclastic bone destruction in murine antigen-induced arthritis (AIA). METHODS: Bone destruction was analyzed in the arthritic knee joints of S100A9-deficient mice in which S100A8 protein expression was also lacking, and in wild-type (WT) controls. Osteoclast precursors from S100A9-deficient and WT mice were differentiated into osteoclasts in vitro. Additionally, precursors were stimulated with S100A8, S100A9, or S100A8/A9 during osteoclastogenesis. Receptor involvement was investigated using an anti-receptor for advanced glycation end products (anti-RAGE)-blocking antibody, soluble RAGE, or Toll-like receptor 4 (TLR-4)-deficient osteoclast precursors. The formation of osteoclasts and actin rings, the regulation of osteoclast markers, and bone resorption were analyzed. RESULTS: Bone erosions and cathepsin K staining were significantly suppressed in S100A9-deficient mice after AIA induction. However, osteoclast precursors from S100A9-deficient mice developed normally into functional osteoclasts, which excludes a role for intrinsic S100A8/A9. In contrast to the results observed with S100A9 and S100A8/A9, the addition of S100A8 during osteoclastogenesis resulted in stimulation of osteoclast formation in conjunction with enhanced actin ring formation and increased bone resorption. Analysis of the putative receptor for S100A8 in osteoclastogenesis revealed that osteoclast differentiation and function could not be inhibited by blocking RAGE, whereas the increase in osteoclast numbers and enhanced bone resorption were completely abrogated using TLR-4-deficient osteoclast precursors. CONCLUSION: These results demonstrate that S100A8 stimulated osteoclast formation and activity and suggest that both S100A8 and TLR-4 are important factors in mediating osteoclastic bone destruction in experimental arthritis.

S100A8 causes a shift toward expression of activatory Fcγ receptors on macrophages via toll-like receptor 4 and regulates Fcγ receptor expression in synovium during chronic experimental arthritis.

OBJECTIVE: The levels of both Fcγ receptor (FcγR) and the alarmins S100A8 and S100A9 are correlated with the development and progression of cartilage destruction during antigen-induced arthritis (AIA). This study was undertaken to study the active involvement of S100A8, S100A9, and S100A8/S100A9 in FcγR regulation in murine macrophages and synovium during AIA. METHODS: Recombinant murine S100A8 (rS100A8) was injected into normal mouse knee joints, and the synovium was isolated for analysis of FcγR messenger RNA (mRNA) expression by reverse transcription-polymerase chain reaction (RT-PCR). Macrophages, including bone marrow macrophages derived from Toll-like receptor 4-deficient (TLR-4(-/-)) mice, and polymorphonuclear cells (PMNs) were stimulated with S100 proteins, and levels of FcγR mRNA and protein were measured using RT-PCR and fluorescence-activated cell sorting analyses. AIA was induced in the knee joints of S100A9-deficient (S100A9(-/-)) mice, compared with wild-type (WT) controls, and the extent of cartilage destruction was determined using immunohistochemical analysis. RESULTS: Intraarticular injection of rS100A8 into the knee joints of normal mice caused a strong up-regulation of mRNA levels of activating FcγRI (64-fold increase) and FcγRIV (256-fold increase) in the synovium. Stimulation of macrophages with rS100A8 led to significant up-regulation of mRNA and protein levels of FcγRI and FcγRIV, but not FcγRIII, while the effects of S100A9 or S100A8/S100A9 complexes were less potent. Stimulation of PMNs (32Dcl3 cell line) with S100 proteins had no effect on FcγR expression. Up-regulation of FcγRI and FcγRIV was abrogated in rS100A8-stimulated macrophages from TLR-4(-/-) mice, indicating that the induction of FcγR expression by S100A8 is mediated by TLR-4. FcγR expression in the inflamed synovium of S100A9(-/-) mice was significantly lower on day 14 after arthritis induction when compared with WT controls, and these findings correlated with reduced severity of matrix metalloproteinase-mediated cartilage destruction. CONCLUSION: S100A8 is a strong promoter of activating FcγRI and FcγRIV in macrophages through the activation of TLR-4, and acts as a regulator of FcγR expression in inflamed synovium in chronic experimental arthritis.

Different amplifying mechanisms of interleukin-17 and interferon-gamma in Fcgamma receptor-mediated cartilage destruction in murine immune complex-mediated arthritis.

OBJECTIVE: Previously, we reported that interferon-gamma (IFNgamma) aggravates cartilage destruction in immune complex (IC)-mediated arthritis via up-regulation of activating Fcgamma receptors (FcgammaR). Recently, we found that interleukin-17 (IL-17) also aggravates cartilage destruction in arthritis models in which ICs are involved, but the underlying mechanism remains unknown. This study was undertaken to determine the role of IL-17 in FcgammaR-mediated cartilage destruction in IC-mediated arthritis and to compare its effect with that of IFNgamma. METHODS: IC-mediated arthritis was passively induced in gamma-chain(-/-) mice, which lack functional activating FcgammaR, and in wild-type controls. AdIL-17 or a control vector was injected into the knee joints 1 day prior to induction of IC-mediated arthritis. Knee joints were isolated for histologic analysis, and synovium samples were obtained for reverse transcriptase-polymerase chain reaction (RT-PCR). Macrophage (RAW 264.7) cell lines and polymorphonuclear cell (PMN; 32Dcl3) lines were stimulated with IFNgamma or IL-17 for analysis of FcgammaR expression using RT-PCR and fluorescence-activated cell sorting. RESULTS: IL-17 overexpression prior to induction of IC-mediated arthritis significantly aggravated cartilage destruction and inflammation, characterized by a massive influx of PMNs, which adhered to the cartilage surface. Although IL-17 overexpression increased FcgammaR messenger RNA levels in the synovium, in vitro stimulation of macrophages and PMNs revealed that, in contrast to IFNgamma, IL-17 did not directly regulate FcgammaR expression. Despite similar inflammation in AdIL-17-enhanced IC-mediated arthritis in gamma-chain(-/-) mice and wild-type controls, severe cartilage destruction and PMN adherence were completely absent in gamma-chain(-/-) mice. CONCLUSION: Our findings indicate that IL-17-mediated aggravation of cartilage destruction in IC-mediated arthritis is FcgammaR dependent. However, in contrast to IFNgamma, which directly up-regulates FcgammaR expression on macrophages and PMNs, IL-17 enhances cartilage destruction by increasing the local amount of FcgammaR-bearing neutrophils.

Fcgamma receptors directly mediate cartilage, but not bone, destruction in murine antigen-induced arthritis: uncoupling of cartilage damage from bone erosion and joint inflammation.

OBJECTIVE: To determine the relationship between synovial inflammation and the concomitant occurrence of cartilage and bone erosion during conditions of variable inflammation using various Fcgamma receptor knockout (FcgammaR(-/-)) mice. METHODS: Antigen-induced arthritis (AIA) was introduced in the knee joints of various FcgammaR(-/-) mice and wild-type controls. Joint inflammation and cartilage and bone destruction levels were determined by histologic analysis. Cathepsin K, RANKL, and osteoprotegerin (OPG) levels were detected by immunolocalization. RESULTS: In FcgammaRIIb(-/-) mice, which lack the inhibiting Fcgamma receptor IIb, levels of joint inflammation and cartilage and bone destruction were significantly higher (infiltrate 93%, exudate 200%, cartilage 100%, bone 156%). AIA in mice lacking activating FcgammaR types I, III, and IV, but not FcgammaRIIb (FcR gamma-chain(-/-) mice), prevented cartilage destruction completely. In contrast, levels of bone erosion and joint inflammation were comparable with their wild-type controls. Of great interest, in arthritic mice lacking activating FcgammaR types I, II, and III, but not IV (FcgammaRI/II/III(-/-) mice), levels of joint inflammation were highly elevated (infiltrate and exudate, 100% and 188%, respectively). Cartilage destruction levels were decreased by 92%, whereas bone erosion was increased by 200%. Cathepsin K, a crucial mediator of osteoclasts, showed a strong correlation with the amount of inflammation but not with the amount of activating FcgammaR, which was low in osteoclasts. RANKL, but not OPG, levels were higher in the inflammatory cells of arthritic knee joints of FcgammaRI/II/III(-/-) mice versus wild-type mice. CONCLUSION: Activating FcgammaR are crucial in mediating cartilage destruction independently of joint inflammation. In contrast, FcgammaR are not directly involved in bone erosion. Indirectly, FcgammaR drive bone destruction by regulating joint inflammation.