Peter L. E. M. van Lent

Synovial macrophage depletion with clodronate-containing liposomes in rheumatoid arthritis

OBJECTIVE To assess whether intraarticular (IA) administration of clodronate liposomes results in local macrophage depletion in patients with rheumatoid arthritis (RA). Primary goals were to address both the immunohistologic and potential toxic effects of this approach. Moreover, the correlation between immunohistologic findings and clinical assessments of disease activity and cartilage damage were assessed. METHODS An open study was conducted in consecutive RA patients who were scheduled for knee joint replacement in our department. Synovial biopsy tissue was obtained from the knee joint at 2 weeks before and at the time of surgery. This protocol was controlled for safety and immunohistologic concordance in 6 patients. One week before surgery, 10 patients received a single IA dose of clodronate liposomes. Staining of synovial tissue for cell markers (CD68, CD14, CD3, CD38) and adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1], intercellular adhesion molecule 1 [ICAM-1]) was assessed by 2 blinded observers. Local and systemic parameters of disease activity were measured before each intervention. Cartilage damage was scored using standard radiologic techniques at baseline and during surgery. RESULTS A single IA dose of clodronate liposomes significantly reduced the number of CD68-positive cells (P = 0.005) and the expression of ICAM-1 and VCAM-1 in the synovial lining (P = 0.013 and P = 0.039, respectively). The intervention did not affect fibroblast-like synoviocytes, T cells, or plasma cells. No immunohistologic changes were observed in the control group. The procedure was well tolerated. The levels of ICAM-1 and VCAM-1 in the sublining layers correlated with the extent of macroscopic synovitis (P < 0.0005 and P < 0.005, respectively). The expression of ICAM-1 and CD14 in the sublining correlated with the levels of C-reactive protein (P < 0.0005 and P < 0.01, respectively). Cartilage destruction was correlated only with the expression of CD68 in the sublining (P = 0.02). CONCLUSION A single IA administration of clodronate liposomes leads to macrophage depletion and decreased expression of adhesion molecules in the synovial lining in patients with longstanding RA. The procedure is well tolerated, and its therapeutic potential is currently under investigation. The expression of adhesion molecules in the sublining layers reflects ongoing inflammation.

Involvement of the Wnt signaling pathway in experimental and human osteoarthritis: Prominent role of Wnt‐induced signaling protein 1

OBJECTIVE Wnt signaling pathway proteins are involved in embryonic development of cartilage and bone, and, interestingly, developmental processes appear to be recapitulated in osteoarthritic (OA) cartilage. The present study was undertaken to characterize the expression pattern of Wnt and Fz genes during experimental OA and to determine the function of selected genes in experimental and human OA. METHODS Longitudinal expression analysis was performed in 2 models of OA. Levels of messenger RNA for genes from the Wnt/beta-catenin pathway were determined in synovium and cartilage, and the results were validated using immunohistochemistry. Effects of selected genes were assessed in vitro using recombinant protein, and in vivo by adenoviral overexpression. RESULTS Wnt-induced signaling protein 1 (WISP-1) expression was strongly increased in the synovium and cartilage of mice with experimental OA. Wnt-16 and Wnt-2B were also markedly up-regulated during the course of disease. Interestingly, increased WISP-1 expression was also found in human OA cartilage and synovium. Stimulation of macrophages and chondrocytes with recombinant WISP-1 resulted in interleukin-1-independent induction of several matrix metalloproteinases (MMPs) and aggrecanase. Adenoviral overexpression of WISP-1 in murine knee joints induced MMP and aggrecanase expression and resulted in cartilage damage. CONCLUSION This study included a comprehensive characterization of Wnt and Frizzled gene expression in experimental and human OA articular joint tissue. The data demonstrate, for the first time, that WISP-1 expression is a feature of experimental and human OA and that WISP-1 regulates chondrocyte and macrophage MMP and aggrecanase expression and is capable of inducing articular cartilage damage in models of OA.

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.

Reduced cartilage proteoglycan loss during zymosan‐induced gonarthritis in NOS2‐deficient mice and in anti‐interleukin‐1‐treated wild‐type mice with unabated joint inflammation

OBJECTIVE To investigate the role of nitric oxide (NO) and interleukin-1 in (IL-1) joint inflammation and cartilage destruction during zymosan-induced gonarthritis (ZIA). METHODS Monarticular arthritis was elicited by intraarticular injection of zymosan. The effect of NO deficiency on arthritis was studied in mice with genetically disrupted NOS2. The role of IL-1 was examined by treating wild-type mice with neutralizing anti-murine IL-1(alpha+beta) antibodies. Joint swelling was measured externally by the increased uptake of circulating 99mtechnetium pertechnetate. Proteoglycan (PG) synthesis was assessed using 35S-sulfate incorporation into patellae ex vivo. Histology evaluated exudation and infiltration of leukocytes and the extent of cartilage destruction. RESULTS The proinflammatory mediators NO, IL-1, and IL-6 were released by the articular tissues during the first hours of inflammation. Interestingly, anti-IL-1 treatment moderately reduced, and NOS2 deficiency moderately enhanced, joint swelling. However, the influx of neutrophils into the joint occurred independently of IL-1 and NOS2 activities. In the first week of inflammation, chondrocyte PG synthesis was significantly suppressed and chondrocytes became unresponsive to their essential anabolic factor, insulin-like growth factor 1 (IGF-1). Anti-IL-1 treatment or NOS2 deficiency prevented the inhibition of PG synthesis, and the chondrocytes remained IGF-1 responsive. Intraarticular injections of IL-1alpha into NOS2-deficient mice did not affect PG synthesis, thus proving that NO mediated this IL-1 effect in vivo. Furthermore, histology showed that cartilage PG loss was markedly ameliorated in NOS2-deficient and anti-IL-1-treated mice. Intermediate cartilage pathology was found in mice that were heterozygous for disrupted NOS2. CONCLUSION IL-1 and NO play a minor role in edema and neutrophil influx, but a major role in cartilage destruction of ZIA. In this model of murine arthritis, cartilage destruction was, for the most part, caused by pronounced suppression of PG synthesis and IGF-1 unresponsiveness of the chondrocytes, which were induced by de novo-synthesized IL-1 and were mediated by NOS2 activation.

Kinetics of aggrecanase- and metalloproteinase-induced neoepitopes in various stages of cartilage destruction in murine arthritis.

OBJECTIVE Two major cleavage sites, one mediated by metalloproteinases (MMPs) and the other by an as-yet unidentified enzyme termed aggrecanase, have been observed in aggrecan. To learn more about the relative contribution of these enzymes during cartilage degradation, this study assessed the occurrence of both specific neoepitopes in cartilage during murine arthritis and examined the correlation between neoepitope formation and different aspects of cartilage damage. METHODS Reversible cartilage damage was induced in mice in the zymosan-induced arthritis (ZIA) model, partly irreversible cartilage damage in the antigen-induced arthritis (AIA) model, and irreversible, destructive cartilage damage in the collagen-induced arthritis (CIA) model. Immunolocalization techniques were used to detect the specific C-terminal neoepitopes VDIPEN (MMPS) and NITEGE (aggrecanase). RESULTS In normal cartilage from young adult mice, no VDIPEN epitopes were detected, but a limited amount of NITEGE epitopes were already present. During the early phase of proteoglycan (PG) depletion, NITEGE expression was raised substantially in all arthritis models. VDIPEN epitopes were not detected in this early phase of cartilage destruction. When PG depletion progressed toward advanced cartilage damage, VDIPEN epitopes were induced. During ZIA, minimal induction of VDIPEN was observed, whereas in AIA, strong, but partly reversible, VDIPEN staining was evident, and in CIA, an extensive presence and persistence of the MMP-induced neoepitope was seen. When VDIPEN epitopes were intensely present, NITEGE epitopes were greatly reduced at that site in the cartilage. CONCLUSION Presence of VDIPEN epitopes in cartilage correlated with severe cartilage damage, but these epitopes were not detected during early PG degradation. This suggests a limited role for VDIPEN-inducing MMPs in early PG degradation during murine arthritis. In contrast, aggrecanase epitopes were induced before the appearance of VDIPEN epitopes, but they disappeared with progression of cartilage damage.

Alarmins S100A8 and S100A9 elicit a catabolic effect in human osteoarthritic chondrocytes that is dependent on Toll-like receptor 4.

OBJECTIVE S100A8 and S100A9 are two Ca(2+) binding proteins classified as damage-associated molecular patterns or alarmins that are found in high amounts in the synovial fluid of osteoarthritis (OA) patients. The purpose of this study was to investigate whether S100A8 and/or S100A9 can interact with chondrocytes from OA patients to increase catabolic mediators. METHODS Using immunohistochemistry, we stained for S100A8 and S100A9 protein, matrix metalloproteinases (MMPs), and a cartilage-breakdown epitope specific for MMPs (VDIPEN) in cartilage from OA donors. Isolated chondrocytes or explants from OA and non-OA donors were stimulated with S100A8 and/or S100A9. Messenger RNA and protein levels of MMPs, cytokines, and cartilage matrix molecules were determined with quantitative reverse transcription-polymerase chain reaction and Luminex techniques, respectively. For receptor blocking studies, specific inhibitors for Toll-like receptor 4 (TLR-4), receptor for advanced glycation end products (RAGE), and carboxylated glycans were used. RESULTS In cartilage from OA patients, the expression of S100A8 and S100A9 protein close to chondrocytes was associated with proteoglycan depletion and expression of MMP-1, MMP-3, and VDIPEN. Stimulation of chondrocytes with S100A8 and S100A9 caused a strong up-regulation of catabolic markers (MMPs 1, 3, 9, and 13, interleukin-6 [IL-6], IL-8, and monocyte chemotactic protein 1) and down-regulation of anabolic markers (aggrecan and type II collagen), thereby favoring cartilage breakdown. Blocking TLR-4, but not carboxylated glycans or RAGE, inhibited the S100 effect. The catabolic S100 effect was significantly more pronounced in chondrocytes from OA patients as compared to those from non-OA patients, possibly due to higher TLR-4 expression. CONCLUSION S100A8 and S100A9 have a catabolic effect on human chondrocytes that is TLR-4 dependent. OA chondrocytes are more sensitive than normal chondrocytes to S100 stimulation.

Cleavage of aggrecan at the Asn341–Phe342 site coincides with the initiation of collagen damage in murine antigen‐induced arthritis: A pivotal role for stromelysin 1 in matrix metalloproteinase activity

OBJECTIVE The destruction of articular cartilage during arthritis is due to proteolytic cleavage of the extracellular matrix components. This study investigates the kinetic involvement of metalloproteinases (MMPs) in the degradation of the 2 major cartilage components, aggrecan and type II collagen, during murine antigen-induced arthritis (AIA). In addition, the role of stromelysin 1 (SLN-1) induction of MMP-induced neoepitopes was studied. METHODS VDIPEN neoepitopes in aggrecan and collagenase-induced COL2-3/4C neoepitopes in type II collagen were identified by immunolocalization. Stromelysin 1-deficient knockout (SLN1-KO) mice were used to study SLN-1 involvement. RESULTS In AIA, the VDIPEN epitopes in aggrecan appeared after initial proteoglycan (PG) depletion. The collagenase-induced type II collagen neoepitopes colocalized with VDIPEN epitopes. Remarkably, cartilage from arthritic SLN1-KO mice showed neither the induction of VDIPEN nor collagen cleavage-site neoepitopes during AIA, suggesting that stromelysin is a pivotal mediator in this process. PG depletion, as measured by the loss of Safranin O staining, was similar in SLN1-KO mice and wild-type strains. Furthermore, in vitro induction of VDIPEN epitopes in aggrecan and COL2-3/4C epitopes in type II collagen, on exposure of cartilage to interleukin-1, could not be accomplished in SLN1-KO mice, whereas intense staining was achieved for both epitopes in cartilage of wild-type strains. CONCLUSION This study emphasizes that SLN-1 is essential in the induction of MMP-specific aggrecan and collagen cleavage sites during AIA. It suggests that SLN-1 is not a dominant enzyme in PG breakdown, but that it activates procollagenases and is crucial in the initiation of collagen damage.

Active involvement of alarmins S100A8 and S100A9 in the regulation of synovial activation and joint destruction during mouse and human osteoarthritis

OBJECTIVE To investigate whether alarmins S100A8 and S100A9 are involved in mediating cartilage destruction during murine and human osteoarthritis (OA). METHODS Two different murine models of OA that differed in terms of synovial activation were compared. Cartilage destruction was measured histologically. Synovial biopsy and serum samples from OA patients were derived from the Cohort Hip and Cohort Knee (CHECK) patients with symptomatic early OA. Expression of mediators in the synovium was measured by reverse transcription-polymerase chain reaction analysis and immunolocalization. RESULTS In collagenase-induced OA, which showed marked synovial activation, interleukin-1β was expressed at significant levels only during the early stages of disease, whereas S100A8 and S100A9 expression remained high for a prolonged period of time (up to day 21 after induction). In S100A9-knockout mice, we found a major impact of S100A8 and S100A9 on synovial activation (62% inhibition) and OA cartilage destruction (45-73% inhibition) as compared to wild-type controls. In contrast, in the surgically induced destabilized medial meniscus model, in which synovial involvement is scant, we found no role of S100A8 and S100A9 in the focal OA cartilage destruction. Examination of arthroscopic synovial biopsy samples from patients in the early symptomatic OA CHECK cohort revealed substantial levels of S100A8 and S100A9 messenger RNA and protein, which correlated significantly with synovial lining thickness, cellularity in the subintima, and joint destruction. Levels of S100A8/A9 serum protein were significantly enhanced (19%) at baseline in patients who had pronounced progression of joint destruction after 2 years. CONCLUSION Our data suggest that the S100A8 and S100A9 proteins are crucially involved in synovial activation and cartilage destruction during OA and that high levels may predict joint destruction in humans with OA.

Tumor necrosis factor–interleukin-17 interplay induces S100A8, interleukin-1β, and matrix metalloproteinases, and drives irreversible cartilage destruction in murine arthritis: Rationale for combination treatment during arthritis

OBJECTIVE To examine whether synovial interleukin-17 (IL-17) expression promotes tumor necrosis factor (TNF)-induced joint pathologic processes in vivo, and to analyze the surplus ameliorative value of neutralizing IL-17 in addition to TNF during collagen-induced arthritis (CIA). METHODS Adenoviral vectors were used to induce overexpression of IL-17 and/or TNF in murine knee joints. In addition, mice with CIA were treated, at different stages of arthritis, with soluble IL-17 receptor (sIL-17R), TNF binding protein (TNFBP), or the combination. RESULTS Overexpression of IL-17 and TNF resulted in joint inflammation and bone erosion in murine knees. Interestingly, IL-17 strikingly enhanced both the joint-inflammatory and joint-destructive capacity of TNF. Further analysis revealed a strongly enhanced up-regulation of S100A8, IL-1β, and matrix metalloproteinase (MMP) messenger RNA, only when both TNF and IL-17 were present. Moreover, the increase in irreversible cartilage destruction was not merely the result of enhanced inflammation, but also was associated with a direct synergistic effect of these cytokines in the joint. S100A9 deficiency in mice protected against IL-17/TNF-induced expression of cartilage NITEGE neoepitopes. During established arthritis, the combination of sIL-17R and TNFBP was more effective than the anticytokine treatments alone, and significantly inhibited further joint inflammation and cartilage destruction. CONCLUSION Local synovial IL-17 expression enhances the role of TNF in joint destruction. Synergy between TNF and IL-17 in vivo results in striking exaggeration of cartilage erosion, in parallel with a synergistic up-regulation of S100A8, IL-1β, and erosive MMPs. Moreover, neutralizing IL-17 in addition to TNF further improves protection against joint damage and is still effective during late-stage CIA. Therefore, compared with anti-TNF alone, combination blocking of TNF and IL-17 may have additional therapeutic value for the treatment of destructive arthritis.

Deficiency of NADPH Oxidase Components p47phox and gp91phox Caused Granulomatous Synovitis and Increased Connective Tissue Destruction in Experimental Arthritis Models

Recent studies indicated that the nicotinamide dinucleotide phosphate oxidase (NADPH) oxidase-derived oxygen radicals plays a deleterious role in arthritis. To study this in more detail, gonarthritis was induced in NADPH oxidase-deficient mice. Mice received an intraarticular injection of either zymosan, to elicit an irritant-induced inflammation, or poly-L-lysine coupled lysozyme, to evoke an immune-complex mediated inflammation in passively immunized mice. In contrast to wild-type mice, arthritis elicited in both p47phox(-/-) and gp91(-/-) mice showed more severe joint inflammation, which developed into a granulomatous synovitis. Treatment with either Zileuton or cobra venom factor showed that the chemokines LTB4 and complement C3 were not the driving force behind the aggravated inflammation in these mice. Arthritic NADPH oxidase-deficient mice showed irreversible cartilage damage as judged by the enhanced aggrecan VDIPEN expression, and chondrocyte death. Furthermore, only in the absence of NADPH oxidase-derived oxygen radicals, the arthritic joints showed osteoclast-like cells, tartrate-resistant acid phosphatase (TRAP)-positive/multinucleated cells, extensive bone erosion, and osteolysis. The enhanced synovial gene expression of tumor necrosis factor-alpha, interleukin-1alpha, matrix metalloproteinase (MMP)-3, MMP-9 and receptor activator of NF-kappaB ligand (RANKL) might contribute to the aggravated arthritis in the NADPH oxidase-deficient mice. This showed that the involvement of NADPH oxidase in arthritis is probably far more complex and that oxygen radicals might also be important in controlling disease severity, and reducing joint inflammation and connective tissue damage.