The impact of anterior cruciate ligament injury on lubricin metabolism and the effect of inhibiting tumor necrosis factor α on chondroprotection in an animal model

Objective

To examine the effects of anterior cruciate ligament transection (ACLT) in a rat model on lubricin metabolism and its relationship to markers of inflammation and cartilage damage, and to determine whether blocking the metabolic effects of tumor necrosis factor α (TNFα) by etanercept increases the chondroprotection provided by lubricin.

Methods

Unilateral ACLT was performed in Lewis rats. Levels of lubricin, TNFα, interleukin‐1β (IL‐1β), and sulfated glycosaminoglycans (sGAG) in synovial fluid (SF) lavage specimens and synovial tissue lubricin gene expression were evaluated at 1 week and 4 weeks following ACLT. Histologic evaluation of articular cartilage included staining with lubricin‐specific monoclonal antibody 9G3 and Safranin O. The percentage of lubricin staining on the surface of articular cartilage in weight‐bearing areas was estimated by digital imaging. Blocking of TNFα was performed using etanercept, which was administered subcutaneously at a dose of 0.5 mg/kg around the ACL‐transected joints, using different dosing strategies. The ACL‐transected and contralateral joints of these rats were harvested 4 weeks following surgery.

Results

Four weeks following ACLT, SF lubricin concentrations and the percentage of cartilage surface lubricin staining were significantly lower in the injured joints compared with the contralateral joints. A significant decrease in synovial tissue lubricin gene expression was associated with elevated TNFα and IL‐1β concentrations in SF lavage samples. With all of the etanercept treatment strategies, blocking of TNFα significantly increased the amount of lubricin bound to cartilage, coupled with a significant decrease in sGAG release. However, changes in the concentrations of lubricin in SF were variable.

Conclusion

Blocking TNFα resulted in a chondroprotective effect, exemplified by increased lubricin deposition on articular cartilage and a decrease in sGAG release from articular cartilage in an animal model of posttraumatic arthritis.

     

Prevention of cartilage degeneration in a rat model of osteoarthritis by intraarticular treatment with recombinant lubricin

Objective

Lubricin, also referred to as superficial zone protein and PRG4, is a synovial glycoprotein that supplies a friction‐resistant, antiadhesive coating to the surfaces of articular cartilage, thereby protecting against arthritis‐associated tissue wear and degradation. This study was undertaken to generate and characterize a novel recombinant lubricin protein construct, LUB:1, and to evaluate its therapeutic efficacy following intraarticular delivery in a rat model of osteoarthritis (OA).

Methods

Binding and localization of LUB:1 to cartilage surfaces was assessed by immunohistochemistry. The cartilage‐lubricating properties of LUB:1 were determined using a custom friction testing apparatus. A cell‐binding assay was performed to quantify the ability of LUB:1 to prevent cell adhesion. Efficacy studies were conducted in a rat meniscal tear model of OA. One week after the surgical induction of OA, LUB:1 or phosphate buffered saline vehicle was administered by intraarticular injection for 4 weeks, with dosing intervals of either once per week or 3 times per week. OA pathology scores were determined by histologic analysis.

Results

LUB:1 was shown to bind effectively to cartilage surfaces, and facilitated both cartilage boundary lubrication and inhibition of synovial cell adhesion. Treatment of rat knee joints with LUB:1 resulted in significant disease‐modifying, chondroprotective effects during the progression of OA, by markedly reducing cartilage degeneration and structural damage.

Conclusion

Our findings demonstrate the potential use of recombinant lubricin molecules in novel biotherapeutic approaches to the treatment of OA and associated cartilage abnormalities.

     

Association of articular cartilage degradation and loss of boundary‐lubricating ability of synovial fluid following injury and inflammatory arthritis

Objective

To study the relationship between the boundary‐lubricating ability of synovial fluid (SF) and articular cartilage damage in a rabbit knee injury model, to correlate collagen markers of such damage with SF boundary‐lubricating ability and elastase activity, and to examine the lubricating ability of SF, together with collagen markers of articular cartilage damage, under the inflammatory conditions of knee joint synovitis (KJS) and rheumatoid arthritis (RA).

Methods

SF was aspirated weekly from the affected knee joints of 10 adult rabbits following transection of the anterior and posterior cruciate ligaments. The boundary‐lubricating ability of SF was determined in vitro using a previously described friction apparatus. Lubricin concentrations and type II collagen (CII) peptides were quantified by sandwich enzyme‐linked immunosorbent assays (ELISAs). Levels of the C‐terminal neoepitope 9A4 (derived from collagenase degradation of CI, CII, and CIII) and of epitope 5‐D‐4 of keratan sulfate (a marker of proteoglycan depletion) were quantified by inhibition ELISAs. Elastase activity was measured spectrophotometrically. The sensitivity of purified human lubricin to digestion by neutrophil elastase (NE) was examined by Western blotting.

Results

The lubricating ability of SF from injured rabbit knees was significantly decreased at weeks 2 and 3 compared with week 1 after injury. Lubricin concentrations were significantly higher at week 1 than at weeks 2 and 3. CII peptide concentrations increased significantly at weeks 2 and 3 compared with week 1, while 9A4 neoepitope concentrations increased significantly at week 3 compared with weeks 1 and 2. There were no significant differences in epitope 5‐D‐4 concentrations among the 3 weeks. Elastase activity in SF increased significantly at weeks 2 and 3 compared with week 1. Elastase activity correlated significantly with diminishing lubrication at weeks 1, 2, and 3. SF from patients with KJS or RA exhibited deficient lubrication and elevated levels of CII peptides compared with SF from normal controls. NE was shown to completely degrade purified human lubricin in vitro.

Conclusion

Loss of boundary‐lubricating ability of SF after injury is associated with damage to the articular cartilage matrix. This can be attributed to inflammatory processes resulting from the injury, particularly in the early phases. This association also exists in patients with acute knee injuries or progressive chronic inflammatory arthritis.

     

Prevention of cartilage degeneration and restoration of chondroprotection by lubricin tribosupplementation in the rat following anterior cruciate ligament transection

Objective

To investigate whether cartilage degeneration is prevented or minimized following intraarticular injections of lubricin derived from human synoviocytes in culture, recombinant human PRG4 (rhPRG4), or human synovial fluid (SF) in a rat model of anterior cruciate ligament (ACL) injury.

Methods

Unilateral ACL transection (ACLT) was performed in Lewis rats (n = 45). Nine animals were left untreated. The remaining rats were given intraarticular injections (50 μl/injection) of either phosphate buffered saline (PBS) (n = 9), human synoviocyte lubricin (200 μg/ml; n = 9), rhPRG4 (200 μg/ml; n = 9), or human SF lubricin (200 μg/ml; n = 9) twice weekly beginning on day 7 after injury. Joints were harvested on day 32 after injury. Histologic analysis was performed using Safranin O–fast green staining, and articular cartilage degeneration was graded using the Osteoarthritis Research Society International (OARSI)–modified Mankin criteria. Histologic specimens were immunoprobed for lubricin and sulfated glycosaminoglycans. A 24‐hour urine collection was performed on days 17 and 29 postinjury, and urinary C‐terminal telopeptide of type II collagen (CTX‐II) levels were measured.

Results

Treatment with human synoviocyte lubricin resulted in significantly lower OARSI scores for cartilage degeneration compared with no treatment or PBS treatment (P < 0.05). Increased immunostaining for lubricin in the superficial zone chondrocytes and on the surface of cartilage was observed in lubricin‐treated, but not untreated or PBS‐treated, joints. On day 17, urinary CTX‐II levels in human synoviocyte lubricin– and human SF lubricin–treated animals were significantly lower than those in untreated animals (P = 0.005 and P = 0.002, respectively) and in PBS‐treated animals (P = 0.002 and P < 0.001, respectively).

Conclusion

After treatment with any of the 3 types of lubricin evaluated in this study, a reduction in cartilage damage following ACLT was evident, combined with a reduction in type II collagen degradation. Our findings indicate that intraarticular lubricin injection following an ACL injury may be beneficial in retarding the degeneration of cartilage and the development of posttraumatic OA.

     

Inhibition of toll‐like receptor 4 breaks the inflammatory loop in autoimmune destructive arthritis

Objective

Degeneration of extracellular matrix of cartilage leads to the production of molecules capable of activating the immune system via Toll‐like receptor 4 (TLR‐4). The objective of this study was to investigate the involvement of TLR‐4 activation in the development and progression of autoimmune destructive arthritis.

Methods

A naturally occurring TLR‐4 antagonist, highly purified lipopolysaccharide (LPS) from Bartonella quintana, was first characterized using mouse macrophages and human dendritic cells (DCs). Mice with collagen‐induced arthritis (CIA) and mice with spontaneous arthritis caused by interleukin‐1 receptor antagonist (IL‐1Ra) gene deficiency were treated with TLR‐4 antagonist. The clinical score for joint inflammation, histologic characteristics of arthritis, and local expression of IL‐1 in joints were evaluated after treatment.

Results

The TLR‐4 antagonist inhibited DC maturation induced by Escherichia coli LPS and cytokine production induced by both exogenous and endogenous TLR‐4 ligands, while having no effect on these parameters by itself. Treatment of CIA using TLR‐4 antagonist substantially suppressed both clinical and histologic characteristics of arthritis without influencing the adaptive anti–type II collagen immunity crucial for this model. Treatment with TLR‐4 antagonist strongly reduced IL‐1β expression in articular chondrocytes and synovial tissue. Furthermore, such treatment inhibited IL‐1–mediated autoimmune arthritis in IL‐1Ra^−/− mice and protected the mice against cartilage and bone pathology.

Conclusion

In the present study, we demonstrate for the first time that inhibition of TLR‐4 suppresses the severity of experimental arthritis and results in lower IL‐1 expression in arthritic joints. Our data suggest that TLR‐4 might be a novel target in the treatment of rheumatoid arthritis.

     

Synovial fibroblasts self‐direct multicellular lining architecture and synthetic function in three‐dimensional organ culture

Objective

To define the intrinsic capacity of fibroblast‐like synoviocytes (FLS) to establish a 3‐dimensional (3‐D) complex synovial lining architecture characterized by the multicellular organization of the compacted synovial lining and the elaboration of synovial fluid constituents.

Methods

FLS were cultured in spherical extracellular matrix (ECM) micromasses for 3 weeks. The FLS micromass architecture was assessed histologically and compared with that of dermal fibroblast controls. Lubricin synthesis was measured via immunodetection. Basement membrane matrix and reticular fiber stains were performed to examine ECM organization. Primary human and mouse monocytes were prepared and cocultured with FLS in micromass to investigate cocompaction in the lining architecture. Cytokine stimuli were applied to determine the capacity for inflammatory architecture rearrangement.

Results

FLS, but not dermal fibroblasts, spontaneously formed a compacted lining architecture over 3 weeks in the 3‐D ECM micromass organ cultures. These lining cells produced lubricin. FLS rearranged their surrounding ECM into a complex architecture resembling the synovial lining and supported the survival and cocompaction of monocyte/macrophages in the neo–lining structure. Furthermore, when stimulated by cytokines, FLS lining structures displayed features of the hyperplastic rheumatoid arthritis synovial lining.

Conclusion

This 3‐D micromass organ culture method demonstrates that many of the phenotypic characteristics of the normal and the hyperplastic synovial lining in vivo are intrinsic functions of FLS. Moreover, FLS promote survival and cocompaction of primary monocytes in a manner remarkably similar to that of synovial lining macrophages. These findings provide new insight into inherent functions of the FLS lineage and establish a powerful in vitro method for further investigation of this lineage.

     

Early elevation in circulating levels of C‐telopeptides of type II collagen predicts structural damage in articular cartilage in the rodent model of collagen‐induced arthritis

Objective

To investigate changes in the circulating levels of the C‐telopeptide of type II collagen (CTX‐II) with relation to disease onset and structural damage of cartilage in a rodent model of collagen‐induced arthritis (CIA), and to investigate immunolocalization of the CTX‐II epitope in the articular cartilage of affected joints.

Methods

Seven‐week‐old female Lewis rats were immunized with type II collagen and monitored using blood sampling at weekly intervals. At study termination (day 23), the animals were killed, synovial fluid was collected, and the affected joints were scored macroscopically for disease severity and underwent immunohistochemical evaluation.

Results

At the time of disease onset (day 15), which was characterized by redness and swelling of the affected joints (mean ± SD macroscopic severity score 9.1 ± 1.6), there was a 355% increase in serum CTX‐II levels. The early change in serum CTX‐II from day 0 to day 15 showed a significant association with the severity of cartilage damage (r = 0.61, P < 0.01). Immunostaining revealed extensive presence of the CTX‐II epitope in the damaged, uncalcified cartilage tissue.

Conclusion

The elevation in serum CTX‐II concomitant with the onset of disease and proportional to cartilage damage demonstrates that CTX‐II is a sensitive diagnostic tool for monitoring joint disease in the rodent model of CIA. Furthermore, the immunohistochemical findings are consistent with the concept that the major source of serum CTX‐II is the damaged articular cartilage.

     

Cyclic loading increases friction and changes cartilage surface integrity in lubricin‐mutant mouse knees

Objective

To investigate the effects of lubricin gene dosage and cyclic loading on whole joint coefficient of friction and articular cartilage surface integrity in mouse knee joints.

Methods

Joints from mice with 2 (Prg4^+/+), 1 (Prg4^+/−), or no (Prg4^−/−) functioning lubricin alleles were subjected to 26 hours of cyclic loading using a custom‐built pendulum. Coefficient of friction values were measured at multiple time points. Contralateral control joints were left unloaded. Following testing, joints were examined for histologic evidence of damage and cell viability.

Results

At baseline, the coefficient of friction values in Prg4^−/− mice were significantly higher than those in Prg4^+/+ and Prg4^+/− mice (P < 0.001). Cyclic loading continuously increased the coefficient of friction in Prg4^−/− mouse joints. In contrast, Prg4^+/− and Prg4^+/+ mouse joints had no coefficient of friction increases during the first 4 hours of loading. After 26 hours of loading, joints from all genotypes had increased coefficient of friction values compared to baseline and unloaded controls. Significantly greater increases occurred in Prg4^−/− and Prg4^+/− mouse joints compared to Prg4^+/+ mouse joints. The coefficient of friction values were not significantly associated with histologic evidence of damage or loss of cell viability.

Conclusion

Our findings indicate that mice lacking lubricin have increased baseline coefficient of friction values and are not protected against further increases caused by loading. Prg4^+/− mice are indistinguishable from Prg4^+/+ mice at baseline, but have significantly greater coefficient of friction values following 26 hours of loading. Lubricin dosage affects joint properties during loading, and may have clinical implications in patients for whom injury or illness alters lubricin abundance.

     

Lectin‐like oxidized low‐density lipoprotein receptor 1 mediates leukocyte infiltration and articular cartilage destruction in rat zymosan‐induced arthritis

Objective

The relationship between rheumatoid arthritis and atherosclerosis has been recognized for >20 years. This study aimed to elucidate the roles of oxidized low‐density lipoprotein (ox‐LDL; one of the main pathogenic factors of atherosclerosis) and its endothelial receptor, lectin‐like ox‐LDL receptor 1 (LOX‐1), in arthritic joints using a rat zymosan‐induced arthritis (ZIA) model.

Methods

LOX‐1 expression and ox‐LDL accumulation in arthritic joints were detected by immunohistochemistry using specific mouse anti–LOX‐1 and anti–ox‐LDL monoclonal antibodies, respectively. To elucidate the effects of the expressed LOX‐1 on arthritis, ZIA rats were treated with anti–LOX‐1 antibody or normal mouse IgG. The severity of arthritis was analyzed by joint swelling. Cell infiltration, synovial hyperplasia, and proteoglycan losses were also determined by histologic scoring. Proinflammatory cytokine and nitrite levels in serum and joint fluid were also measured.

Results

Immunohistochemical study of ZIA demonstrated LOX‐1 expression on synovial endothelium and postcapillary venules at 6 hours after the induction of inflammation, with maximum expression detected at 24 hours. LOX‐1 was also expressed weakly on both joint cartilage and synovium. Ox‐LDL, a ligand of LOX‐1, was also detected in articular chondrocytes. Administration of anti–LOX‐1 antibody, which blocks LOX‐1 activity, suppressed joint swelling (by 33.5%), leukocyte infiltration, and joint nitrite accumulation at 24 hours, as well as cartilage destruction at 7 days, compared with control rats.

Conclusion

LOX‐1 induction in arthritic joints might play a role in promoting joint inflammation and cartilage destruction by mediating leukocyte infiltration into the arthritic joints of ZIA rats.

     

Host metalloproteinases in Lyme arthritis

Objective

: To assess the role of matrix metalloproteinases (MMPs) in cartilage and bone erosions in Lyme arthritis

Methods

We examined synovial fluid from 10 patients with Lyme arthritis for the presence of MMP‐2, MMP‐3, MMP‐9, and “aggrecanase” activity using gelatinolytic zymography and immunoblot analysis. We developed an in vitro model of Lyme arthritis using cartilage explants and observed changes in cartilage degradation in the presence of Borrelia burgdorferi and/or various protease inhibitors.

Results

Synovial fluid from patients with Lyme arthritis was found to contain at least 3 MMPs: gelatinase A (MMP‐2), stromelysin (MMP‐3), and gelatinase B (MMP‐9). In addition, there was evidence in 2 patients of “aggrecanase” activity not accounted for by the above enzymes. Infection of cartilage explants with B burgdorferi resulted in induction of MMP‐3, MMP‐9, and “aggrecanase” activity. Increased induction of these enzymes by B burgdorferi alone was not sufficient to cause cartilage destruction in the explants as measured by glycosaminoglycan (GAG) and hydroxyproline release. However, addition of plasminogen, which can act as an MMP activator, to cultures resulted in significant GAG and hydroxyproline release in the presence of B burgdorferi. The MMP inhibitor batimastat significantly reduced the GAG release and completely inhibited the collagen degradation.

Conclusion

MMPs are found in synovial fluids from patients with Lyme arthritis and are induced from cartilage tissue by the presence of B burgdorferi. Inhibition of MMP activity prevents B burgdorferi–induced cartilage degradation in vitro.

     

Mammalian target of rapamycin signaling is crucial for joint destruction in experimental arthritis and is activated in osteoclasts from patients with rheumatoid arthritis

Objective

Activation of the mammalian target of rapamycin (mTOR) pathway is important for immune cell activation and bone metabolism. To date, the contribution of mTOR signaling to joint inflammation and structural bone and cartilage damage is unknown. The aim of this study was to investigate the potential of inhibiting mTOR as a treatment of inflammatory arthritis.

Methods

Human tumor necrosis factor–transgenic mice in which inflammatory arthritis was developing were treated with 2 different mTOR inhibitors, sirolimus or everolimus. The effects of treatment on clinical disease activity, inflammation, and localized joint and cartilage destruction were studied. In addition, the effects of mTOR inhibition on osteoclast survival and expression of key molecules of osteoclast function were analyzed in vitro. Moreover, synovial tissue from patients with rheumatoid arthritis (RA) was assessed for activation of the mTOR pathway.

Results

Inhibition of mTOR by sirolimus or everolimus reduced synovial osteoclast formation and protected against local bone erosions and cartilage loss. Clinical signs of arthritis improved after mTOR inhibition, and histologic evaluation showed a decrease in synovitis. In vitro, mTOR inhibition down‐regulated the expression of digestive enzymes and led to osteoclast apoptosis. Moreover, mTOR signaling was shown to be active in the synovial membrane of patients with RA, particularly in synovial osteoclasts.

Conclusion

Signaling through mTOR is an important link between synovitis and structural damage in inflammatory arthritis. Current pharmacologic inhibitors of mTOR could be effective in protecting joints against structural damage.

     

Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritis

Objective

A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage, and this process requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen‐degrading matrix metalloproteinases (MMPs), membrane type 1 MMP (MT1‐MMP), in synovial pannus invasiveness.

Methods

The expression and localization of MT1‐MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemical analysis of RA joint specimens. The functional role of MT1‐MMP was analyzed by 3‐dimensional (3‐D) collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP‐1), TIMP‐2, or GM6001. The effect of adenoviral expression of a dominant‐negative MT1‐MMP construct lacking a catalytic domain was also examined.

Results

MT1‐MMP was highly expressed at the pannus–cartilage junction in RA joints. Freshly isolated rheumatoid synovial tissue and isolated RA synovial fibroblasts invaded into a 3‐D collagen matrix in an MT1‐MMP–dependent manner. Invasion was blocked by TIMP‐2 and GM6001 but not by TIMP‐1. Invasion was also inhibited by the overexpression of a dominant‐negative MT1‐MMP, which inhibits collagenolytic activity and proMMP‐2 activation by MT1‐MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1‐MMP–dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix.

Conclusion

MT1‐MMP serves as an essential collagen‐degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1‐MMP–dependent invasion may represent a novel therapeutic strategy for RA.

     

Microbubble‐enhanced ultrasound exposure promotes uptake of methotrexate into synovial cells and enhanced antiinflammatory effects in the knees of rabbits with antigen‐induced arthritis

Objective

To evaluate whether microbubble‐enhanced ultrasound (US) treatment promotes the delivery of methotrexate (MTX) into synovial cells and the enhanced antiinflammatory effects of intraarticular MTX therapy in a rabbit arthritis model.

Methods

Arthritis was induced in both knees of 53 rabbits by immunization with ovalbumin. MTX including a microbubble agent was then injected into the left and right knee joints, and the right knees were exposed to US (MTX+/US+ group), while the left knees were not (MTX+/US− group). The knee joints were evaluated histologically in 7 rabbits at 5 time points up to day 56. Quantitative gene expression of interleukin‐1β (IL‐1β) in synovial tissue was measured on days 7 and 28. Eight rabbits were used for the measurement of MTX concentration in synovial tissue 12 hours after treatment. To evaluate the effect of microbubble‐enhanced US treatment in the absence of MTX, only the microbubble agent was injected into the left and right knee joints of 10 rabbits with or without US exposure, and these animals were evaluated histologically on days 7 and 28.

Results

The MTX concentration in synovial tissue was significantly higher in the MTX+/US+ group than in the MTX+/US− group. Synovial inflammation was less prominent in the MTX+/US+ group compared with the MTX+/US− group, judging from the results of the histologic evaluation and the gene expression levels of IL‐1β in synovial tissue. It also appeared that microbubble‐enhanced US exposure itself did not affect inflammation.

Conclusion

Microbubble‐enhanced US exposure promoted the uptake of MTX into synovial cells, which resulted in enhancement of the antiinflammatory effects of the intraarticular MTX injection. These results suggest that application of this technique may have clinical benefit.

     

Crucial role of macrophages in matrix metalloproteinase–mediated cartilage destruction during experimental osteoarthritis : Involvement of matrix metalloproteinase 3

Objective

To explore the involvement of synovial macrophages in early cartilage damage in osteoarthritis (OA), and to identify the role of matrix metalloproteinase 3 (MMP‐3) in the pathology of early and late OA.

Methods

The role of synovial macrophages in MMP‐mediated damage in OA was studied by depleting synovial macrophages prior to elicitation of a collagenase‐induced instability model of OA. The expression of MMP in synovium and cartilage was monitored using TaqMan analysis. In spontaneous and induced OA, cartilage pathology was scored in MMP‐3–knockout mice and control mice, by histologic assessment and VDIPEN staining.

Results

On day 14 following induction of OA, MMP‐mediated neoepitopes were detected in cartilage from mice with mild experimental OA (mean ± SD positively stained surface area 20 ± 3.2%). Remarkably, by depleting synovial macrophages prior to induction of OA, the generation of MMP‐induced neoepitopes was largely prevented (mean ± SD positively stained surface area 5 ± 1%; P< 0.001), indicating an important role for synovial macrophages in the occurrence of MMP‐mediated cartilage damage. We observed a strong decrease in MMP‐3 and MMP‐9 expression in synovial but not cartilage tissue in macrophage‐depleted joints. Among 2‐year‐old mice, spontaneous OA–like changes in the lining layer were significantly decreased in MMP‐3–knockout mice compared with control mice. Even more striking was the 67% reduction in the occurrence of severe cartilage damage in MMP‐3–knockout mice. In addition, MMP‐mediated VDIPEN expression was significantly decreased, indicating reduced MMP‐mediated cartilage breakdown.

Conclusion

The results of this study prove that MMP‐3 is involved in the generation of severe cartilage damage in murine OA. Synovial macrophages are crucial in early MMP activity and appear to mediate MMP production in synovium rather than cartilage.

     

Induction of cartilage damage by overexpression of T cell interleukin‐17A in experimental arthritis in mice deficient in interleukin‐1

Objective

To examine the capacity of T cell interleukin‐17A (IL‐17A; referred to hereinafter as IL‐17) to induce cartilage damage during experimental arthritis in the absence of IL‐1.

Methods

Local IL‐17 gene transfer was performed in the knee joint of IL‐1–deficient mice and wild‐type controls during streptococcal cell wall (SCW)–induced arthritis. Knee joints were isolated at various time points for histologic analysis of cartilage proteoglycan (PG) depletion. Expression of messenger RNA for inducible nitric oxide synthase, matrix metalloproteinases (MMPs) 3, 9, and 13, and ADAMTS‐4 was determined by quantitative polymerase chain reaction analysis. VDIPEN staining was analyzed to study MMP‐mediated cartilage damage. In addition, systemic anti–IL‐1α/β antibody treatment was performed in mice immunized with type II collagen and injected locally with an adenoviral vector expressing IL‐17 or with control adenovirus. Knee joints were isolated and analyzed for cartilage PG depletion, chondrocyte death, and cartilage surface erosion.

Results

During SCW‐induced arthritis, local T cell IL‐17 gene transfer turned this acute, macrophage‐driven joint inflammation into a severe, chronic arthritis accompanied by aggravated cartilage damage. Of high interest, the IL‐1 dependency of cartilage PG depletion was fully abrogated when IL‐17 was locally overexpressed in the joint. Moreover, local IL‐17 gene transfer increased MMP expression without the need for IL‐1, although IL‐1 remained essential for part of the cartilage VDIPEN expression. Furthermore, when IL‐17 was overexpressed in the knee joints of mice with collagen‐induced arthritis, anti–IL‐1 treatment did not reduce the degree of chondrocyte death or cartilage surface erosion.

Conclusion

These data show the capacity of IL‐17 to replace the catabolic function of IL‐1 in cartilage damage during experimental arthritis.

     

Expression of hypoxia‐inducible factor 1α by macrophages in the rheumatoid synovium: Implications for targeting of therapeutic genes to the inflamed joint

Objective

To determine if the rheumatoid synovium is a suitable target for hypoxia‐regulated gene therapy.

Methods

Sequential sections of wax‐embedded synovial membrane samples were obtained from 10 patients with rheumatoid arthritis (RA), 10 with primary osteoarthritis (OA), and from 6 healthy controls. Membrane sections from each patient were immunostained for hypoxia‐inducible factor 1α (HIF‐1α) and CD68 (a pan–macrophage marker).

Results

HIF‐1α was expressed abundantly by macrophages in most rheumatoid synovia, predominantly close to the intimal layer but also in the subintimal zone. There was markedly lower expression of HIF‐1α in OA synovia, and it was absent from all of the healthy synovia.

Conclusion

These observations indicate that macrophages transduced with a therapeutic gene under the control of a hypoxia‐inducible promoter could be administered to RA patients systemically. Migration of these cells to synovial tissue would result in the transgene being switched on in diseased joints but not in healthy tissues.

     

Suppression of T cell responses by chondromodulin I,a cartilage‐derived angiogenesis inhibitory factor: Therapeutic potential in rheumatoid arthritis

Objective

Chondromodulin I (ChM‐I), a cartilage matrix protein, promotes the growth and proteoglycan synthesis of chondrocytes. However, it also inhibits angiogenesis. Since ChM‐I is expressed not only in cartilage, but also in the thymus, we investigated the modulation of T cell function by ChM‐I to assess its therapeutic potential in rheumatoid arthritis (RA).

Methods

The localization of ChM‐I expression in mouse thymus tissue was examined by in situ hybridization. The proliferative response of peripheral blood T cells and synovial cells obtained from patients with RA was evaluated by ³H‐thymidine incorporation assay. The effects of ChM‐I were examined using recombinant human ChM‐I (rHuChM‐I). Modulation of the antigen‐specific immune response was evaluated by the recall response of splenic T cells and the delayed‐type hypersensitivity response induced in the ear of mice primed with ovalbumin (OVA). Antigen‐induced arthritis (AIA) was induced in mice by injecting methylated bovine serum albumin into the ankle joints 2 weeks after the priming.

Results

ChM‐I was expressed in the cortex of the thymus. Recombinant human ChM‐I suppressed the proliferative response of mouse splenic T cells and human peripheral blood T cells stimulated with anti‐CD3/CD28 antibodies, in a dose‐dependent manner. Production of interleukin‐2 was decreased in rHuChM‐I–treated mouse CD4 T cells. Ten micrograms of rHuChM‐I injected intraperitoneally into OVA‐primed mice suppressed the induction of the antigen‐specific immune response. Finally, rHuChM‐I suppressed the development of AIA, and also suppressed the proliferation of synovial cells prepared from the joints of patients with RA.

Conclusion

These results suggest that ChM‐I suppresses T cell responses and synovial cell proliferation, implying that this cartilage matrix protein has a therapeutic potential in RA.