Lilyanne C. Grevers

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.

Myeloid-related proteins S100A8/S100A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis

Objective: To study the active involvement of Myeloid-related proteins S100A8 and S100A9 in joint inflammation and cartilage destruction during antigen-induced arthritis (AIA). Methods: Joint inflammation and cartilage destruction was measured with 99mTc uptake and histology. The role of S100A8/A9 was investigated by inducing AIA in S100A9–/– mice that also lack S100A8 at protein level, or after intra-articular injection of rS100A8 in mouse knee joints. Cartilage destruction was measured using immunolocalisation of the neoepitope VDIPEN or NITEGE. mRNA levels of matrix metalloproteinases (MMPs) and cytokines were measured using reverse transcriptase (RT)-PCR. Results: Immunisation of S100A9–/– mice with the antigen mBSA induced normal cellular and humoral responses, not different from wild type (WT) controls. However, joint swelling measured at day 3 and 7 after AIA induction was significantly lower (36 and 70%, respectively). Histologically, at day 7 AIA, cellular mass was much lower (63–80%) and proteoglycan depletion from cartilage layers was significantly reduced (between 50–95%). Cartilage destruction mediated by MMPs was absent in S100A9–/– mice but clearly present in controls. MMP3, 9 and 13 mRNA levels were significantly lowered in arthritic synovia of S100A9–/–. In vitro stimulation of macrophages by the heterodimer S100A8/A9 or S100A8 elevated mRNA levels of MMP3, 9 and in particular MMP13. Intra-articular injection of S100A8 caused prominent joint inflammation and depletion of proteoglycans at day 1. Significant upregulation of mRNA levels of S100A8/A9, cytokines (interleukin 1 (IL1)), MMPs (MMP3, MMP13 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4) was found in the synovium and correlated with strong upregulation of NITEGE neoepitopes within the cartilage layers. Conclusions: S100A8/A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis.

Stimulation of chondrocyte‐mediated cartilage destruction by S100A8 in experimental murine arthritis

OBJECTIVE To investigate whether S100A8 is actively involved in matrix metalloproteinase (MMP)-mediated chondrocyte activation. METHODS S100A8 and S100A9 proteins were detected in inflamed knee joints from mice with various forms of murine arthritis, using immunolocalization. Murine chondrocyte cell line H4 was stimulated with proinflammatory cytokines or recombinant S100A8. Messenger RNA (mRNA) and protein levels were measured using reverse transcriptase-polymerase chain reaction and intracellular fluorescence-activated cell sorting (FACS). Breakdown of aggrecan on the pericellular surface of the chondrocytes was measured using VDIPEN and NITEGE antibodies and FACS, and breakdown in patellar cartilage was measured by immunolocalization. RESULTS S100A8 and S100A9 proteins were abundantly expressed in and around chondrocytes in inflamed knee joints after induction of antigen-induced arthritis or onset of spontaneous arthritis in interleukin-1 (IL-1) receptor antagonist-knockout mice. Stimulation of chondrocytes by the proinflammatory cytokines tumor necrosis factor alpha, IL-1beta, IL-17, and interferon-gamma caused strong up-regulation of S100A8 mRNA and protein levels and up-regulation to a lesser extent of S100A9 levels. Stimulation of chondrocytes with S100A8 induced significant up-regulation of MMP-2, MMP-3, MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5 mRNA levels (up-regulated 4, 4, 3, 16, 8, and 4 times, respectively). VDIPEN and NITEGE neoepitopes were significantly elevated in a concentration-dependent manner in chondrocytes treated with 0.2, 1, or 5 microg/ml of S100A8. (VDIPEN levels were elevated 17%, 67%, and 108%, respectively, and NITEGE levels were elevated 8%, 33%, and 67%, respectively.) S100A8 significantly increased the effect of IL-1beta on MMP-3, MMP-13, and ADAMTS-5. Mouse patellae incubated with both IL-1beta and S100A8 had elevated levels of NITEGE within the cartilage matrix when compared with patellae incubated with IL-1beta or S100A8 alone. CONCLUSION These findings indicate that S100A8 and S100A9 are found in and around chondrocytes in experimental arthritis. S100A8 up-regulates and activates MMPs and aggrecanase-mediated pericellular matrix degradation.

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.

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 effects of both, shared mouse bone marrow precursor myeloid blast was precultured 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. J. Cell. Physiol. 230: 210–225, 2015.

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.

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.

Activating FCγ RECEPTORS mediate immune complex-induced inhibition of osteoclastogenesis

Background Rheumatoid arthritis is characterised by osteoclast-mediated bone loss. Co-stimulatory signalling via ITAM- and ITIM-coupled receptors is essential for osteoclast formation and function. The ITAM- and ITIM-coupled Fcγ receptors (FcγR) play a crucial role in mediating inflammation and cartilage destruction in experimental arthritis, but their role in osteoclast-mediated bone loss is unknown. Objectives To investigate the role of FcγRs in osteoclastogenesis and osteoclast function. Materials and methods Bone destruction was analysed in arthritic knee joints of FcγRIIB-deficient, FcRγ-chain−/− (lacking expression of activating FcγRs), and wild type mice. Bone marrow-derived osteoclast precursors were differentiated in vitro towards osteoclasts in the absence or presence of immune complexes (ICs) and stimulated thereafter for 24 h with or without TNFα or LPS. Additionally, mature osteoclasts were stimulated with ICs. Experiments were analysed for osteoclast formation, bone resorption, and the expression of FcγRs and osteoclast markers. Results Bone erosions and cathepsin K-positive osteoclast numbers were significantly increased during antigen-induced arthritis in the knee joints of FcγRIIB-deficient mice. All FcγR classes were highly expressed on bone marrow-derived osteoclast progenitors. On mature osteoclasts, as compared to macrophages, expression of the inhibitory FcγRIIB was similar, whereas expression of activating FcγRs was significantly lower. IC stimulation of mature osteoclasts neither affected their number nor their bone resorptive capacity. Differentiation of bone marrow-derived precursors in the presence of ICs significantly inhibited osteoclast formation, bone resorption, and expression of the osteoclast markers cathepsin K, CTR, DC-STAMP and NFATc1. 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. Conclusions Activating FcγRs, but not the inhibitory FcγRIIB, mediate IC-induced inhibition of osteoclastogenesis. In the presence of pro-inflammatory mediators like TNFα and LPS the inhibitory effect might be overruled. This suggests that the balance of FcγR-mediated induction of inflammation, through pro-inflammatory cytokine production, as well as the direct inhibitory effect of ICs on osteoclastogenesis determines the net effect on bone loss.

S100A8 enhances osteoclast-mediated bone resorption in experimental antigen-induced arthritis through activation of toll-like receptor 4

Objective Rheumatoid arthritis (RA) is characterised by severe bone erosions caused by enhanced osteoclast formation and activity. Significantly elevated S100A8 and S100A9 levels in serum and synovial fluid of RA patients are strongly correlated to joint destruction. The aim of the present study was to investigate the role of S100A8 on osteoclastic bone resorption in murine antigen-induced arthritis (AIA). Methods Bone destruction was analysed 7 and 21 days after AIA induction in knee joints of S100A9−/− mice, also lacking S100A8 protein expression, and wild type controls. Bone marrow precursors from S100A9−/− and wild type mice were differentiated into osteoclasts in vitro. Additionally, precursors were stimulated with recombinant S100A8 during osteoclastogenesis. Receptor involvement was investigated using an anti-receptor for advanced end products (anti-RAGE) blocking antibody or toll-like receptor 4 negative (TLR4−/−) osteoclast precursors. Experiments were analysed for the formation of tartrate-resistant acid phosphatase-positive multinucleated cells (TRACP+ MNCs), actin rings, mRNA expression levels of osteoclast markers and resorption pit formation on bone. Results Bone erosions and cathepsin K staining were significantly suppressed in S100A9−/− mice after AIA induction. In vitro however, bone marrow-derived precursors from S100A9−/− mice developed normally into functional osteoclasts, excluding a role for intrinsic S100A8/A9. Addition of S100A8 during osteoclastogenesis resulted in increased osteoclast formation The mRNA expression levels of osteoclast markers were not affected by S100A8 stimulation, but the formation of actin rings, essential for the bone resorptive capacity of osteoclasts, was significantly enhanced in conjunction increased bone resorption levels. The stimulatory effects of S100A8 on osteoclast maturation and function could not be inhibited by RAGE blockade, whereas the increased osteoclast numbers, actin ring formation and resorption pit formation were completely abrogated using TLR4−/− osteoclasts. Conclusion This study demonstrates that S100A8 stimulates osteoclast formation and activity and suggests that both S100A8 and TLR4 are important factors in mediating osteoclastic bone destruction in experimental arthritis. Topic Myeloid cells and innate immune receptors.

Alarmin S100A8 causes severe cartilage damage in antigen-induced arthritis through TLR4 activation and upregulation of FcεRI and FcεRIV

The ‘alarmins’ S100 A8 and A9 have been described not only as markers for inflammation but may also be involved in induction of erosive cartilage destruction.1 S100A8 represents the active form whereas S100A9 binds to S100A8, protecting the protein from degradation. The aim of the study is to identify the mechanisms of S100 involvement in severe cartilage destruction during antigen-induced arthritis. Antigen-induced arthritis (AIA) was induced in knee joints of S100A8/A9-/- mice (myeloid cells also …