Rheumatoid arthritis (RA) is a chronic autoimmune disease that is considered to be one of the major public health problems worldwide. The development of therapies that target tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and co-stimulatory pathways that regulate the immune system have revolutionized the care of patients with RA. Despite these advances, many patients continue to experience symptomatic and functional impairment. To address this issue, more recent therapies that have been developed are designed to target intracellular signaling pathways involved in immunoregulation. Though this approach has been encouraging, there have been major challenges with respect to off-target organ side effects and systemic toxicities related to the widespread distribution of these signaling pathways in multiple cell types and tissues. These limitations have led to an increasing interest in the development of strategies for the macromolecularization of anti-rheumatic drugs, which could target them to the inflamed joints. This approach enhances the efficacy of the therapeutic agent with respect to synovial inflammation, while markedly reducing non-target organ adverse side effects. In this manuscript, we provide a comprehensive overview of the rational design and optimization of macromolecular prodrugs for treatment of RA. The superior and the sustained efficacy of the prodrug may be partially attributed to their Extravasation through Leaky Vasculature and subsequent Inflammatory cell-mediated Sequestration (ELVIS) in the arthritic joints. This biologic process provides a plausible mechanism, by which macromolecular prodrugs preferentially target arthritic joints and illustrates the potential benefits of applying this therapeutic strategy to the treatment of other inflammatory diseases.
Rheumatoid arthritis; Macromolecular prodrug; ELVIS; Targeting inflammation; Drug delivery
Research into the pathophysiology of osteoarthritis (OA) has focused on cartilage and peri-articular bone, but there is increasing recognition that OA affects all of the joint tissues, including the synovium (SM). Under normal physiological conditions the synovial lining consists of a thin layer of cells with phenotypic features of macrophages and fibroblasts. These cells and the underlying vascularized connective tissue stroma form a complex structure that is an important source of synovial fluid (SF) components that are essential for normal cartilage and joint function. The histological changes observed in the SM in OA generally include features indicative of an inflammatory “synovitis”; specifically they encompass a range of abnormalities, such as synovial lining hyperplasia, infiltration of macrophages and lymphocytes, neoangiogenesis and fibrosis. The pattern of synovial reaction varies with disease duration and associated metabolic and structural changes in other joint tissues. Imaging modalities including Magnetic Resonance (MRI) and ultrasound (US) have proved useful in detecting and quantifying synovial abnormalities, but individual studies have varied in their methods of evaluation. Despite these differences, most studies have concluded that the presence of synovitis in OA is associated with more severe pain and joint dysfunction. In addition, synovitis may be predictive of faster rates of cartilage loss in certain patient populations. Recent studies have provided insights into the pathogenic mechanisms underlying the development of synovitis in OA. Available evidence suggests that the inflammatory process involves engagement of Toll-like receptors and activation of the complement cascade by degradation products of extracellular matrices of cartilage and other joint tissues. The ensuing synovial reaction can lead to synthesis and release of a wide variety of cytokines and chemokines. Some of these inflammatory mediators are detected in joint tissues and SF in OA and have catabolic effects on chondrocytes. These inflammatory mediators represent potential targets for therapeutic interventions designed to reduce both symptoms and structural joint damage in OA.
Giant cell tumor of bone (GCTB) is a benign, locally destructive neoplasm, with tumors comprised of mesenchymal fibroblast-like stromal cells; monocytic, mononuclear cells of myeloid lineage; and the characteristic osteoclast-like, multinucleated giant cells. Hampering the study of the complex interaction of its constituent cell types is the propensity of longstanding, repeatedly passaged cell cultures to undergo phenotypic alteration and loss of osteoclast-inducing capacities. In this study, we employed a novel, single-step technique to purify freshly harvested stromal cells using a CD14-negative selection column. Using 9 freshly harvested GCTB specimens and the purified stromal cell component, we performed analyses for markers of osteoblast lineage and analyzed the capacity of the stromal cells to undergo osteoblastic differentiation and induce osteoclastogenesis in co-cultures with monocytic cells. Successful purification of the CD14-negative stromal cells was confirmed via flow cytometric analysis and immunocytochemistry. Osteogenic media upregulated the expression of osteocalcin, suggesting an osteoblastic lineage of the GCTB stromal cells. The effects of the Wnt pathway agonist, SB415286, and recombinant human bone morphogenetic protein (BMP)-2 on osteoblastogenesis varied among samples. Notably, osteogenic media and SB415286 reversed the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) expression ratio resulting in diminished osteoclastogenic capacity. Recombinant human BMP2 had the opposite effect, resulting in enhanced and sustained support of osteoclastogenesis. Targeting the giant cell tumor stromal cell may be an effective adjunct to existing anti-resorptive strategies.
The seronegative spondyloarthopathies (SpA) share certain common articular and peri-articular features that differ from rheumatoid arthritis (RA) and other forms of inflammatory arthritis. These include the tendency of the SpAs to involve the axial skeleton in addition to the diarthrodial joints, and the prominent involvement of the extra-articular entheses (sites of ligamentous and tendon insertion), which are not common sites of primary pathology in RA and other inflammatory arthropathies. The differential anatomic sites of bone pathology in the SpAs in comparison to the other forms of arthritis suggest that the underlying pathogenic processes and cellular and molecular mechanisms that account for the peri-articular bone pathology involve different underlying disease mechanisms. This review will highlight the molecular and cellular processes that are involved in the pathogenesis of the skeletal pathology in the SpAs, and provide evidence that many of the factors involved in regulation of bone cell function exhibit potent immune-regulatory activity, providing support for the general concept of osteoimmunology.
Osteoimmunology; Bone homeostasis; Spondyloarthritis; Ankylosing spondylitis; Psoriatic arthritis; Osteoclast; Osteoblast; Osteocyte; Bone remodeling; Inflammation; Cytokine; Bone morphogenic proteins; Wnt/βcatenin; Sclerostin
This study examined whether patients with rheumatoid arthritis (RA) demonstrate different patterns of prosthetic wear or cellular responses to implant wear debris compared to patients without inflammatory joint disease.
Thirty-eight patients who had a primary revision of a total elbow arthroplasty (TEA) for aseptic loosening between 1996 and 2008 were identified. Twenty-five had RA and 13 had no inflammatory arthritis. Clinical data, gross wear patterns of the removed prostheses, and histopathological analyses of peri-implant tissue were compared between RA and non-RA patients.
Evaluation of the retrieved prostheses showed that conformational change of the humeral polyethylene bushing was associated with the generation of polyethylene and metal particles. The amount and type of wear debris in peri-prosthetic tissues was similar in RA and non-RA patients. RA patients not on anti-tumor necrosis factor (TNF) therapy exhibited a histologic pattern of interstitial and sheet-like lymphocytic infiltrates associated with a high plasma cell composition, which was different from the predominantly perivascular infiltrates with few plasma cells seen in non-RA patients (p-value = 0.04). RA patients on anti-TNF therapy fell in between these two groups.
RA patients exhibit a distinct cellular response to implant wear debris compared with non-RA patients. This reaction was unrelated to differences in the type or amount of wear debris and was mitigated by anti-TNF therapy. These results suggest an intrinsic alteration in immunoregulation in RA and have implications for potential immunologic treatment of osteolysis in these patients.
In their recent study, Sohn and colleagues identify multiple plasma proteins in the synovial fluid of patients with osteoarthritis (OA) and demonstrate the capacity of several of the proteins to activate macrophages via the innate immune receptor Toll-like receptor-4 (TLR-4). The authors speculate that the plasma proteins transit into the synovial compartment at sites of tissue damage where the endothelial barrier may be compromised, thus bringing them into contact with the articular surface and cells within the synovium. These results demonstrate a novel mechanism by which synovial inflammation can be initiated in patients with OA and how this process may contribute to the pathogenesis of OA joint pathology.
Wear particle-induced inflammation is considered to be the major cause of aseptic implant loosening and clinical failure after total joint replacement. Due to the frequent absence of symptoms, early detection and intervention prior to implant failure presents a significant challenge. To address this issue, a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based optical imaging contrast agent (P-IRDye) was developed and used for the detection of wear particle-induced inflammation employing a murine calvaria osteolysis model. The particle-induced osteolysis of calvaria was evaluated by H&E, tartrate-resistant acid phosphatase (TRAP) staining and μ-CT after necropsy. One-day post particles implantation, P-IRDye was administrated to the mice via tail vein injection. Live imaging of the animals 6 days after implantation revealed the preferential distribution and sustained retention of the macromolecular contrast agent at the site of particle implantation. Immunohistochemical staining and FACS analyses of the calvaria-associated soft tissue revealed extensive uptake of the HPMA copolymer by F4/80, Ly-6G (Gr1) and CD11c positive cells, which accounts for the retention of the macromolecular probes at the inflammatory sites. To test the potential of the system for therapeutic intervention, an acid-labile HPMA copolymer-dexamethasone conjugate (P-Dex) was prepared and shown to prevent the particle-induced inflammation and bone damage in the calvaria osteolysis model.
HPMA copolymer; aseptic orthopedic implant loosening; theranostics; orthopedic wear particle; inflammation targeting
The articular cartilage and the subchondral bone form a biocomposite that is uniquely adapted to the transfer of loads across the diarthrodial joint. During the evolution of the osteoarthritic process biomechanical and biological processes result in alterations in the composition, structure and functional properties of these tissues. Given the intimate contact between the cartilage and bone, alterations of either tissue will modulate the properties and function of the other joint component. The changes in periarticular bone tend to occur very early in the development of OA. Although chondrocytes also have the capacity to modulate their functional state in response to loading, the capacity of these cells to repair and modify their surrounding extracellular matrix is relatively limited in comparison to the adjacent subchondral bone. This differential adaptive capacity likely underlies the more rapid appearance of detectable skeletal changes in OA in comparison to the articular cartilage. The OA changes in periarticular bone include increases in subchondral cortical bone thickness, gradual decreases in subchondral trabeular bone mass, formation of marginal joint osteophytes, development of bone cysts and advancement of the zone of calcified cartilage between the articular cartilage and subchondral bone. The expansion of the zone of calcified cartilage contributes to overall thinning of the articular cartilage. The mechanisms involved in this process include the release of soluble mediators from chondrocytes in the deep zones of the articular cartilage and/or the influences of microcracks that have initiated focal remodeling in the calcified cartilage and subchondral bone in an attempt to repair the microdamage. There is the need for further studies to define the pathophysiological mechanisms involved in the interaction between subchondral bone and articular cartilage and for applying this information to the development of therapeutic interventions to improve the outcomes in patients with OA.
osteoarthritis; bone remodeling; articular cartilage; biomechanics
Traumatic and degenerative meniscal tears have different anatomic features and different proposed etiologies, yet both are associated with development or progression of osteoarthritis (OA). In established OA, synovitis is associated with pain and progression, but a relationship between synovitis and symptoms in isolated meniscal disease has not been reported. Accordingly, we sought to characterize synovial pathology in patients with traumatic meniscal injuries and determine the relationships between inflammation, meniscal and cartilage pathology, and symptoms.
Thirty-three patients without evidence of OA undergoing arthroscopic meniscectomy for meniscal injuries were recruited. Pain and function were assessed preoperatively; meniscal and cartilage abnormalities were documented at the time of surgery. Inflammation in synovial biopsies was scored and associations between inflammation and clinical outcomes determined. Microarray analysis of synovial tissue was performed and gene expression patterns in patients with or without inflammation compared.
Synovial inflammation was present in 43% of patients and was associated with worse pre-operative pain and function scores, independent of age, gender, or cartilage pathology. Microarray analysis and real-time PCR revealed a chemokine signature in synovial biopsies with increased inflammation scores.
In patients with traumatic meniscal injury undergoing arthroscopic meniscectomy without clinical or radiographic evidence of OA, synovial inflammation occurs frequently and is associated with increased pain and dysfunction. Synovia with increased inflammation scores exhibit a unique chemokine signature. Chemokines may contribute to the development of synovial inflammation in patients with meniscal pathology; they also represent potential therapeutic targets for reducing inflammatory symptoms.
Meniscectomy; meniscal injury; inflammation; synovium; synovitis
During granulomatous inflammatory reactions, myeloid cells can differentiate into activated phagocytic macrophages, wound-healing macrophages, foreign body giant cells, and bone-resorbing osteoclasts. Although it is appreciated that a variety of stimuli, including cytokines, cell–matrix interactions, and challenge with foreign materials can influence myeloid cell fate, little is known of how these signals integrate during this process. In this study, we have investigated the cross talk between receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and particle phagocytosis-induced activation of human monocytes. Understanding interconnected signals is of particular importance to disorders, such as periprosthetic osteolysis, in which granulomatous inflammation is initiated by particle phagocytosis in proximity to bone and leads to inflammatory bone loss. Using cell-based osteoclastogenesis and phagocytosis assays together with expression analysis of key regulators of osteoclastogenesis, we show in this study that phagocytosis of disease-relevant particles inhibits RANKL-mediated osteoclastogenesis of human monocytes. Mechanistically, phagocytosis mediates this effect by downregulation of RANK and c-Fms, the receptors for the essential osteoclastogenic cytokines RANKL and M-CSF. RANKL pretreatment of monocytes generates preosteoclasts that are resistant to RANK downregulation and committed to osteoclast formation, even though they retain phagocytic activity. Thus, the relative timing of exposure to phagocytosable particulates and to osteoclastogenic cytokines is critically important in the determination of myeloid cell fate.
The interest in the relationship between articular cartilage and the structural and functional properties of peri-articular bone relates to the intimate contact that exists between these tissues in joints that are susceptible to the development of osteoarthritis (OA). The demonstration in several animal models that osteoporosis and decreased bone tissue modulus leads to an increased propensity for the development of post-traumatic OA is paradoxical in light of the extensive epidemiological literature indicating that individuals with high systemic bone mass, assessed by bone mineral density, are at increased risk for OA. These observations underscore the need for further studies to define the pathophysiological mechanisms involved in the interaction between subchondral bone and articular cartilage and for applying this information to the development of therapeutic interventions to improve the outcomes in patients with OA.
TLRs have been implicated in promoting osteoclast-mediated bone resorption associated with inflammatory conditions. TLRs also activate homeostatic mechanisms that suppress osteoclastogenesis and can limit the extent of pathologic bone erosion associated with infection and inflammation. We investigated mechanisms by which TLRs suppress osteoclastogenesis. In human cell culture models, TLR ligands suppressed osteoclastogenesis by inhibiting expression of receptor activator of NF-κB (RANK), thereby making precursor cells refractory to the effects of RANKL. Similar but less robust inhibition of RANK expression was observed in murine cells. LPS suppressed generation of osteoclast precursors in mice in vivo, and adsorption of LPS onto bone surfaces resulted in diminished bone resorption. Mechanisms that inhibited RANK expression were down-regulation of RANK transcription, and inhibition of M-CSF signaling that is required for RANK expression. TLRs inhibited M-CSF signaling by rapidly down-regulating cell surface expression of the M-CSF receptor c-Fms by a matrix metalloprotease- and MAPK-dependent mechanism. Additionally, TLRs cooperated with IFN-γ to inhibit expression of RANK and of the CSF1R gene that encodes c-Fms, and to synergistically inhibit osteoclastogenesis. Our findings identify a new mechanism of homeostatic regulation of osteoclastogenesis that targets RANK expression and limits bone resorption during infection and inflammation.
The transcription factor NFATc1 plays an essential role in transducing signals from RANKL in osteoclast differentiation. To date, however, the specific transcriptional targets of NFATc1 are unknown. Expression of the β3 integrin is required for normal osteoclast function. We therefore examined the role of NFATc1 in human β3 integrin expression in osteoclast differentiation. Analysis of the mouse and human β3 gene promoters revealed considerable sequence homology across a 1.3 kb region upstream of the transcription start site (TSS), with conserved NFAT binding elements present. The region −1242 to +29 (relative to the TSS) was cloned as a luciferase reporter construct (pB3-1.3) and a deletion construct removing to −997 (pB3-1) made. The deletion of 245 bp 5′ removed three conserved NFAT sites including a consensus NFAT:AP-1 site. The pB3-1.3 reporter construct was induced by treatment with RANKL in the range 2.5–40 ng/ml and dose-dependently induced by co-transfection with human NFATc1 in RAW264.7 cells. The pB3-1 deletion construct was minimally induced with RANKL treatment and unresponsive to co-transfected NFATc1. Direct NFAT binding to two of the consensus NFAT sites within this 245 bp 5′ region was demonstrated by EMSA and supershift with anti-NFAT antibodies. Mutation of two of the conserved NFAT sites in the −1242 to −997 fragment was required to prevent binding. The double NFAT mutant, in the context of the full-length promoter was unresponsive to RANKL treatment or co-transfected NFATc1. We generated cell-permeable TAT-dominant-negative (dn)NFATc1 fusion proteins to assess the effect of blockade of NFAT signaling. Transduction with dnNFAT inhibited RANKL induction of the human β3 integrin promoter. Involvement of the NFATc1-calcineurin pathway in regulating the human β3 integrin promoter was further confirmed using the calcineurin pathway inhibitory peptide 11R-VIVIT. Together these results establish the β3 gene as a direct target of NFATc1 in RANKL-dependent osteoclast formation.
Transcriptional regulation; Beta 3; Bone; RANKL; BLAST, basic local alignment search tool; mBMM, mouse bone marrow macrophage; bp, base pairs; CTR, calcitonin receptor; cath K, cathepsin K; dn, dominant negative; TBE, Tris Buffered EDTA; EMSAs, electrophoretic mobility shift assays; HA, hemagglutinin; IPTG, isopropyl-β-d-thiogalactopyranoside; luc, luciferase; NFAT, nuclear factor of activated T cells; OSCAR, osteoclast associated receptor; PBS, phosphate buffered saline; PMA, phorbol 12-myristate 13-acetate; RANKL, receptor activator NFκB ligand; S.D., standard deviation; TSS, transcription start site; WT, wild type
Rheumatoid arthritis (RA) is a chronic autoimmune disease of unknown etiology. Effective treatment of this disorder has been hampered by the lack of availability of agents that selectively target affected joint tissue. We developed a novel pH-sensitive drug delivery system of dexamethasone (Dex) based on an N-(2-hydroxypropyl)methacrylamide copolymer (P-Dex) and have shown that the delivery system specifically accumulates in inflamed joints in an animal model of arthritis. We hypothesize that the arthrotropism of the delivery system and the local acidosis-mediated drug release provide superior therapeutic efficacy and potentially reduced side effects in RA treatment. The initial in vitro drug-release study confirmed that the Dex release is indeed dependent upon the environmental pH. At pH 5, 37°C, the conjugate shows the highest level of drug release. When administered systemically in an adjuvant-induced arthritis rat model, P-Dex offers superior and longer-lasting anti-inflammatory effects compared with systemically administered free Dex. In addition, greater bone and cartilage preservation was observed with the P-Dex treatment compared with free Dex treatment. Our data indicate that the differential effect of the conjugate is related to its selective accumulation, potential macrophage-mediated retention, and pH-sensitive drug release (extracellular and intracellular) in arthritic joints. This newly developed drug delivery system provides a unique method for selective targeting of glucocorticoids to inflamed joints which may potentially reduce systemic side effects.
Prosthetic wear debris-induced peri-implant osteolysis is a major cause of aseptic loosening after total joint replacement. In this condition, wear particles released from the implant components induce a granulomatous inflammatory reaction at the interface between implant and adjacent bone, leading to progressive bone resorption and loss of fixation. The present study was undertaken to characterize definitively the phenotype of osteoclast-like cells associated with regions of peri-implant focal bone resorption and to compare the phenotypic features of these cells with those of mononucleated and multinucleated cells associated with polyethylene wear particles. Peri-implant tissues were obtained from patients undergoing hip revision surgery for aseptic loosening after total joint replacement. Cells were examined for the expression of several markers associated with the osteoclast phenotype using immunohistochemistry, histochemistry, and/or in situ hybridization. CD68 protein, a marker expressed by multiple macrophage lineage cell types, was detected in mononucleated and multinucleated cells associated with polyethylene particles and the bone surface. Cathepsin K and tartrate-resistant acid phosphatase were expressed highly in both mononucleated and multinucleated cells associated with the bone surface. Levels of expression were much lower in cells associated with polyethylene particles. High levels of β3 integrin protein were detected in cells in contact with bone. Multinucleated cells associated with polyethylene particles exhibited faint positive staining. Calcitonin receptor mRNA expression was detected solely in multinucleated cells present in resorption lacunae on the bone surface and was absent in cells associated with polyethylene particles. Our findings provide further evidence that cells expressing the full repertoire of osteoclast phenotypic markers are involved in the pathogenesis of peri-implant osteolysis after total joint replacement. They also demonstrate that foreign body giant cells, although believed to be phenotypically and functionally distinct from osteoclasts, express many osteoclast-associated genes and gene products. However, the levels and patterns of expression of these genes in the two cell types differ. We speculate that, in addition to the role of cytokines and growth factors, the substrate with which these cells interact plays a critical role in their differential phenotypic and functional properties.
Amylin is a member of the calcitonin family of hormones cosecreted with insulin by pancreatic β cells. Cell culture assays suggest that amylin could affect bone formation and bone resorption, this latter function after its binding to the calcitonin receptor (CALCR). Here we show that Amylin inactivation leads to a low bone mass due to an increase in bone resorption, whereas bone formation is unaffected. In vitro, amylin inhibits fusion of mononucleated osteoclast precursors into multinucleated osteoclasts in an ERK1/2-dependent manner. Although Amylin +/− mice like Amylin-deficient mice display a low bone mass phenotype and increased bone resorption, Calcr +/− mice display a high bone mass due to an increase in bone formation. Moreover, compound heterozygote mice for Calcr and Amylin inactivation displayed bone abnormalities observed in both Calcr +/− and Amylin +/− mice, thereby ruling out that amylin uses CALCR to inhibit osteoclastogenesis in vivo. Thus, amylin is a physiological regulator of bone resorption that acts through an unidentified receptor.
osteoclast; islet amyloid polypeptide; CTR; CALCR; mouse models
The contribution of osteoclasts to the process of bone loss in inflammatory arthritis has recently been demonstrated. Studies in osteoclast biology have led to the identification of factors responsible for the differentiation and activation of osteoclasts, the most important of which is the receptor activator of NF-κB ligand/osteoclast differentiation factor (RANKL/ODF), a tumor necrosis factor (TNF)-like protein. The RANKL/ODF receptor, receptor activator of NF-κB (RANK), is a TNF-receptor family member present on both osteoclast precursors and mature osteoclasts. Like other TNF-family receptors and the IL-1 receptor, RANK mediates its signal transduction via TNF receptor-associated factor (TRAF) proteins, suggesting that the signaling pathways activated by RANK and other inflammatory cytokines involved in osteoclast differentiation and activation are interconnected.
osteoclasts; RANK; RANKL; TNF-α; TRAF
Rheumatoid arthritis represents an excellent model in which to gain insights into the local and systemic effects of joint inflammation on skeletal tissues. Three forms of bone disease have been described in rheumatoid arthritis. These include: focal bone loss affecting the immediate subchondral bone and bone at the joint margins; periarticular osteopenia adjacent to inflamed joints; and generalized osteoporosis involving the axial and appendicular skeleton. Although these three forms of bone loss have several features in common, careful histomorphometric and histopathological analysis of bone tissues from different skeletal sites, as well as the use of urinary and serum biochemical markers of bone remodeling, provide compelling evidence that different mechanisms are involved in their pathogenesis. An understanding of these distinct pathological forms of bone loss has relevance not only with respect to gaining insights into the different pathological mechanisms, but also for developing specific and effective strategies for preventing the different forms of bone loss in rheumatoid arthritis.
bone loss; cytokines; osteoclast; osteoporosis; rheumatoid arthritis
Osteoarthritis, characterized by the breakdown of articular cartilage in synovial joints, has long been viewed as the result of “wear and tear”1. Although low-grade inflammation is detected in osteoarthritis, its role is unclear2–4. Here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis. Through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis, we find that expression and activation of complement is abnormally high in human osteoarthritic joints. Using mice genetically deficient in C5, C6, or CD59a, we show that complement, and specifically the membrane attack complex (MAC)-mediated arm of complement, is critical to the development of arthritis in three different mouse models of osteoarthritis. Pharmacological modulation of complement in wild-type mice confirmed the results obtained with genetically deficient mice. Expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints of C5-deficient mice than C5-sufficient mice, and MAC induced production of these molecules in cultured chondrocytes. Furthermore, MAC co-localized with matrix metalloprotease (MMP)-13 and with activated extracellular signal-regulated kinase (ERK) around chondrocytes in human osteoarthritic cartilage. Our findings indicate that dysregulation of complement in synovial joints plays a critical role in the pathogenesis of osteoarthritis.
The purpose of the present manuscript is to test the hypothesis that arthrotropic localization and synovial cell internalization account for the unique capacity of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-dexamethasone conjugate (P-Dex, a macromolecular prodrug of dexamethasone) to induce sustained amelioration of joint inflammation and inhibition of tissue damage in an animal model of inflammatory arthritis.
Rats with adjuvant-induced arthritis (AA) were treated with P-Dex, free dexamethasone, saline or HPMA homopolymer. To define the biodistribution of P-Dex, conjugates with different imaging labels were given to AA rats and analyzed. Isolated joint tissues were evaluated by fluorescence-activated cell sorting (FACS) and immunohistochemical staining. Cellular uptake of P-Dex and its effects on apoptosis and production of proinflammatory cytokines were examined using human monocyte-macrophages and fibroblasts.
A single systemic administration of P-Dex completely suppressed AA for >20 days. Magnetic resonance imaging demonstrated higher HPMA copolymer influx into the inflamed joints than the normal joints. Immunohistochemistry and FACS analyses of arthritic joints revealed extensive uptake of the polymer conjugate by synovial fibroblasts and myeloid lineage cells. The capacity of P-Dex to suppress inflammation was confirmed in monocyte-macrophage cultures in which P-Dex treatment resulted in suppression of lipopolysaccharide-induced IL-6 and TNFα release. Similarly, TNFα-induced expression of matrix metalloproteinases (MMP1 and MMP3) in synovial fibroblasts from a rheumatoid arthritis patient was suppressed by P-Dex. P-Dex showed no detectable effect on monocyte apoptosis.
P-Dex provides superior and sustained amelioration of AA compared with an equivalent dose of free dexamethasone. The arthrotropism and local retention of P-Dex is attributed to the enhanced vascular permeability in arthritic joints and the internalization of P-Dex by synovial cells. The uptake and processing of P-Dex by macrophages and fibroblasts, and downregulation of proinflammatory mediators, provides an explanation for the sustained anti-inflammatory efficacy of P-Dex in this model of inflammatory arthritis.