Proteoglycan Depletion Research Articles

The effects of storage temperature on the composition, metabolism and biomechanical properties of human articular cartilage

Osteochondral tibial specimens were stored for up to 84 days in DMEM +20% FCS at 4 or 37°C. Articular cartilage from specimens stored at 4°C showed significant proteoglycan depletion, with 78% loss in 84 days. Proteoglycan synthesis declined markedly during the first 3 days in storage. Mechanical analysis revealed cartilage stiffness to be reduced by 60% by the end of the storage period. By contrast, cartilage from specimens stored at 37°C showed no significant depletion of glycosaminoglycan and retained 66% of its initial mechanical stiffness. Proteoglycan synthesis was again reduced but the onset and magnitude of the decline was less than in samples stored at 4°C. Storage at 37°C promises to be useful in banking cartilage prior to clinical use.

Mechanisms of chondrocyte apoptosis

This study addresses the occurrence and significance of chondrocyte apoptosis in the pathogenesis of cartilage destruction. Chondrocyte apoptosis can be inducedin vitroby nitric oxide donors, but not by pro-inflammatory cytokines, such as IL-1 or TNF. A subset of chondrocytes, located in the superficial zone of cartilage, expresses the Fas antigen. Activation of the Fas receptor triggers apoptosis in these cells. In human and experimental osteoarthritis (OA) induced in rabbits by anterior cruciate ligament transection increased numbers of chondrocytes were undergoing apoptosis. Cartilage areas that contained apoptotic cells showed proteoglycan depletion and the number of apoptotic cells was significantly correlated with the levels of nitric oxide production and with the severity of OA. Articular cartilage is not vascularized and does not contain mononuclear phagocytes. There is, thus, no apparent mechanism for the clearance of apoptotic bodies. Chondrocyte-derived apoptotic bodies produced pyrophosphate and precipitated calcium. These results suggest that chondrocyte-derived apoptotic bodies express functional properties that may contribute to the pathologic cartilage degradation and calcification. Inhibition of chondrocyte apoptosis may be of therapeutic value after cartilage injury and in arthritis.

Animal models of arthritis in NOS2-deficient mice

ObjectiveTo study the role of nitric oxide (NO) in cartilage destruction in murine models of arthritis and osteoarthritis.MethodsJoint inflammation was induced in the knee joint by intraarticular injection of Zymosan. Osteoarthritis was induced by local injection of bacterial collagenase, causing joint instability. The effect of NO deficiency was studied by comparing the effects in normal mice and mice with genetically disrupted NOS2 (inducible NO synthase). Impact on articular cartilage was evaluated by histology and measurement of chondrocyte 35S-proteoglycan synthesis.ResultsNOS2 deficiency prevented chondrocyte proteoglycan synthesis inhibition in the arthritic cartilage and restored normal responsiveness to IGF-1. Net cartilage proteoglycan depletion was markedly reduced in the absence of NOS2, although inflammation was hardly affected. Osteoarthritic joint pathology was also significantly reduced, including diminished cartilage lesions and osteophyte formation.ConclusionNO plays a major role in cartilage damage in both arthritic and osteoarthritic conditions.

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

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. 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). 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. 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.

MRI visualization of proteoglycan depletion in articular cartilage via intravenous administration of Gd-DTPA

The effect of intravenous administration of gadolinium diethylenetriamine-pentaacetic acid (Gd-DTPA) on MR images was studied in vitro, using pathologic osteochondral specimens removed during surgery for total endoprosthesis, and in vivo, on a group of volunteers. In ex vivo specimens, lesions of different shape having lower T 1 were detected which corresponded to areas with depleted proteoglycans found histologically. In vivo experiments on young volunteers showed that the time course of cartilage enhancement was different for different anatomies. The time for maximum enhancement ranged from 45 min for the ventral femoral condyle to 270 min for patellar cartilage.

Esculetin suppresses proteoglycan metabolism by inhibiting the production of matrix metalloproteinases in rabbit chondrocytes

The possible mechanism of the chondroprotective effect of 6,7-dihydroxycoumarin (esculetin) was investigated using primary cultures of rabbit articular chondrocytes. Esculetin (EST) significantly suppressed the proteoglycan depletion and the release of pulse-labeled [ 35 S] proteoglycan from the matrix layer of rabbit chondrocytes treated with recombinant human interleukin-1α. The matrix metalloproteinase inhibitor, 1,10-phenanthroline, also blocked the proteoglycan depletion and [ 35 S] proteoglycan release. From these results, it is likely that recombinant human interleukin-1α-induced proteoglycan depletion is mediated by matrix metalloproteinases. Although esculetin did not directly inhibit collagenolytic activity in the culture media, it significantly suppressed the production of pro-matrix metalloproteinase-1/interstitial procollagenase and pro-matrix metalloproteinase-3/prostromelysin 1, accompanied by a decrease in the steady-state levels of their mRNAs. These results suggest that esculetin is a therapeutically effective candidate for inhibition of cartilage destruction in osteoarthritis and rheumatoid arthritis.

Collagen type I antisense and collagen type IIA messenger RNA is expressed in adult murine articular cartilage

Objective: Articular cartilage has only limited capacities for repair and it is not known what is the exact mechanism of matrix restoration. It was investigated whether the reparative process in murine articular cartilage after moderate proteoglycan depletion is accompanied by a change in the chondrocyte phenotype either to hypertrophy or to a less differentiated phenotype as assayed by the expression of specific collagen subtypes.Design: Moderate proteoglycan depletion was induced by injection of papain whereafter the expression of collagen type I mRNA, collagen IIA and IIB mRNA and type X collagen mRNA in patellar cartilage, as markers for chondrocyte phenotype, was investigated by RT-PCR during normal cartilage physiology and matrix restoration. In addition,in-situexpression of collagen subtypes was assayed by immunolocalisation.Results: In normal articular cartilage collagen I, collagen IIB and collagen type X transcripts were easily detected. Surprisingly, collagen type I sense as well as antisense mRNA was detected and in addition to IIB transcripts collagen IIA transcripts were detected in a number of samples. During cartilage matrix restoration no change in the expression of collagen I, collagen IIA or IIB or collagen type X mRNA transcripts could be detected. Immunolocalization demonstrated the presence of type I (pericellular) and type II collagen in the extracellular matrix. The pericellular matrix of hypertrophic chondrocytes showed collagen type X staining in the calcified cartilage in normal and papain-injected knee joints. Increased staining for collagen type X was found in the upper cartilage layer in the interterritorial matrix from day 7 after papain injection.Conclusion: The absence of changes in collagen mRNA expression indicates that alteration of chondrocyte phenotype does not occur during the successful repair process after moderate proteoglycan depletion. Collagen type X appears to be deposited in the upper cartilage layer during this process.

Linkage of chondrocyte apoptosis and cartilage degradation in human osteoarthritis.

To examine the occurrence of apoptosis in human osteoarthritis (OA) cartilage, and to determine its relationship to cartilage degradation. Knee cartilage was obtained from subjects at autopsy, from a tissue bank, and from OA patients undergoing total joint replacement surgery. Chondrocytes were isolated and the number of apoptotic cells was analyzed by flow cytometry. Apoptotic cells in cartilage sections were identified by the detection of DNA strand breaks. Electron microscopy was applied to demonstrate morphologic changes, and Safranin O staining was performed to analyze the relationship between apoptosis and proteoglycan depletion. Flow cytometry on cell suspensions prepared from collagenase digests of cartilage showed that approximately 22.3% of OA chondrocytes and 4.8% of normal chondrocytes were undergoing apoptosis. Staining of cartilage sections demonstrated the presence of apoptotic cells in the superficial and middle zones. Cartilage areas that contained apoptotic cells showed proteoglycan depletion, and the number of apoptotic cells was significantly correlated with the OA grade. These observations demonstrate increased chondrocyte apoptosis in OA cartilage. Chondrocyte apoptosis and proteoglycan depletion are anatomically linked and may be mechanistically related.

DIFFERENT ROLES OF TUMOUR NECROSIS FACTOR α AND INTERLEUKIN 1 IN MURINE STREPTOCOCCAL CELL WALL ARTHRITIS

In this study two different aspects of tumour necrosis factor α (TNF-α) and interleukin 1 (IL-1) in locally induced murine streptococcal cell wall arthritis (SCW) were investigated. First, the kinetics and interdependence of TNF-α and IL-1 release; and second; their involvement in inflammation and cartilage destruction. Kinetic studies showed that the TNF-α peak level preceded the IL-1 peak level. However, in vivo neutralization of TNF-α did not result in decreased IL-1 bioactivity or immunoreactivity, suggesting that there is no dominant TNF-α-dependent IL-1 release in this model. Inflammation was studied by measuring knee joint swelling and inflammatory cell influx. Impact on cartilage was studied by measuring chondrocyte proteoglycan synthesis and cartilage proteoglycan depletion. The role of TNF-α in these phenomena was investigated using anti-TNF-α antibodies and tumour necrosis factor binding protein (TNFbp). Similarly, the role of IL-1 was studied using anti-IL-1 antibodies or IL-1 receptor antagonist (IL-1Ra). Anti-TNF-α treatment significantly reduced joint swelling, whereas this effect was not found by using anti-IL-1 or IL-1Ra. In contrast, neutralization of IL-1, but not TNF-α, resulted in a significant decrease of chondrocyte proteoglycan synthesis inhibition. Moreover, histology revealed that anti-IL-1 treatment reduced cartilage proteoglycan depletion and inflammatory cell influx. Combined anti-TNF-α/anti-IL-1 treatment significantly suppressed both inflammation and cartilage damage. However, the impact on these separate parameters did not exceed the effects of either anti-TNF-α or anti-TNF-1. It can be concluded that both TNF-α and IL-1 exert specific activities in SCW arthritis. The involvement of TNF-α in this model is limited to joint swelling, whereas IL-1 plays a dominant role in cartilage destruction and inflammatory cell influx.

Sodium MRI of human articular cartilage in vivo.

Preliminary results from in vivo sodium MRI of human patellar articular cartilage are presented. Sodium images generated of an in vitro bovine patella clearly distinguish the region of proteoglycan depletion from the region of healthy cartilage. This provides the first evidence that sodium imaging may be used to detect changes due to osteoarthritis in vivo. The process of optimizing imaging time and signal-to-noise ratio, as well as potential implications in the detection of osteoarthritic change, are discussed.

Interleukin‐1 receptor antagonist prevents expression of the metalloproteinase‐generated neoepitope VDIPEN in antigen‐induced arthritis

Objective To investigate the relationship between occurrence of the matrix metalloproteinase-generated neoepitope VDIPEN and proteoglycan (PG) loss in arthritis, and to examine the role of interleukin-1 (IL-1) in VDIPEN expression. Methods VDIPEN expression was investigated in murine antigen-induced arthritis by immunolocalization studies on joint sections. The involvement of IL-1 in VDIPEN expression was studied by blocking of IL-1 using IL-1 receptor antagonist (IL-1Ra). Results Profound PG loss was evident early in arthritis, without significant VDIPEN expression. Full expression of the neoepitope appeared after a few days, when PG depletion was severe, and disappeared at late stages when cartilage showed recovery from PG depletion. At sites where chondrocyte death occurred and cartilage did not recover from the initial cartilage depletion, VDIPEN expression remained present. Prophylactic IL-1Ra treatment of arthritic mice resulted in almost complete prevention of VDIPEN expression. However, IL-1Ra had only a minor effect on PG depletion, emphasizing that there is no correlation between VDIPEN and early PG depletion. Conclusion This study indicates that IL-1 is involved in VDIPEN expression. Although VDIPEN-inducing metalloproteinases do not seem to be involved in early PG depletion during antigen-induced arthritis, metalloproteinase neoepitopes are present when PG depletion is severe.

Interleukin-1 receptor antagonist prevents expression of the metalloproteinase-generated neoepitope VDIPEN in antigen-induced arthritis.

To investigate the relationship between occurrence of the matrix metalloproteinase-generated neoepitope VDIPEN and proteoglycan (PG) loss in arthritis, and to examine the role of interleukin-1 (IL-1) in VDIPEN expression. VDIPEN expression was investigated in murine antigen-induced arthritis by immunolocalization studies on joint sections. The involvement of IL-1 in VDIPEN expression was studied by blocking of IL-1 using IL-1 receptor antagonist (IL-1Ra). Profound PG loss was evident early in arthritis, without significant VDIPEN expression. Full expression of the neoepitope appeared after a few days, when PG depletion was severe, and disappeared at late stages when cartilage showed recovery from PG depletion. At sites where chondrocyte death occurred and cartilage did not recover from the initial cartilage depletion, VDIPEN expression remained present. Prophylactic IL-1Ra treatment of arthritic mice resulted in almost complete prevention of VDIPEN expression. However, IL-1Ra had only a minor effect on PG depletion, emphasizing that there is no correlation between VDIPEN and early PG depletion. This study indicates that IL-1 is involved in VDIPEN expression. Although VDIPEN-inducing metalloproteinases do not seem to be involved in early PG depletion during antigen-induced arthritis, metalloproteinase neoepitopes are present when PG depletion is severe.

Exposure of cartilage to a fibronectin fragment amplifies catabolic processes while also enhancing anabolic processes to limit damage.

The addition of fibronectin fragments to cultured cartilage causes an initial suppression of proteoglycan synthesis, induction of matrix metalloproteinases, and resultant decrease in proteoglycan content by about 50% during the first few days in culture. Because the proteoglycan loss appears to be limited, we investigated whether the fibronectin fragments induce anabolic responses that might counter the damage. The effects of various lengths of exposure of cultured cartilage to the fibronectin fragment on proteoglycan content, proteoglycan synthesis rates, stromelysin-1 release, and tumor necrosis factor-alpha, interleukin-1alpha, and interleukin-6 release were investigated. The results showed that about 7 days of exposure of cultured cartilage to the fibronectin fragment was required for maximal cytokine release, proteoglycan depletion, and stromelysin-1 release. However, nearly maximal suppression of proteoglycan synthesis occurred within 1 day of the addition of the fibronectin fragment and, after its removal, the rates increased to supernormal levels. Decreasing exposure to 3 days caused only a small decrease in cartilage proteoglycan content, although stromelysin-1 release still occurred. Decreasing exposure to 1 day caused an immediate increase in proteoglycan synthesis and an increase to supernormal proteoglycan contents. The effect of first treating cartilage with the fibronectin fragment for various periods and then allowing a recovery was to make the cartilage more resistant to secondary exposures. This study shows that cartilage damage can be caused by short exposures to the fibronectin fragment and that exposures either optimal or suboptimal for damage additionally amplify anabolic processes to make the cartilage resistant to further damage and, thus, condition it against pending amplification of damage.

Hyaluronic acid suppresses fibronectin fragment mediated cartilage chondrolysis: I. In vitro

A commercial preparation of 800-kDa hyaluronic acid (HA), (ARTZ from Seikagaku, Inc.), has been used as a therapeutic intervention in the treatment of osteoarthritis (OA). We tested the effect of this HA form, HA/800, in an in vitro cartilage chondrolytic system in which a specific amino-terminal 29-kDa fragment of fibronectin (Fn-f) penetrates cartilage tissue to activate chondrocytes to amplify two major chondrolytic activities: suppression of proteoglycan (PG) synthesis and induction of matrix metalloproteinases. We report that HA/800 did not block damage by Fn-f in serum free cartilage cultures. However, HA/800 was effective in blocking the ability of 100 nM Fn-f to cause the degradation and release of half of the total cartilage PG from cartilage in 10% serum/DMEM cultures. While the Fn-f caused a half-time for PG release of 3 days, continuous exposure to 0.1 or 1 mg/ml HA/800 slowed the half-time to 12 days. Further, a single 1 day pre-incubation with 0.1 or 1 mg/ml HA/800 was sufficient to decrease the half-time of 100 nM Fn-f mediated PG depletion to 7 and 12 days, respectively. HA/800 completely blocked the effect of 10 nM Fn-f. Blocking of Fn-f-mediated cartilage PG depletion was associated with a decreased concentration of Fn-f on the superficial cartilage surface and decreased penetration into the cultured cartilage tissue. Further, the two major chondrolytic activities of the Fn-f, suppression of synthesis of PG and enhanced release of stromelysin-1, were suppressed by HA/800. HA/800 also partially restored PG in cartilage first damaged with the Fn-F. We conclude that HA/800 slows Fn-f-mediated cartilage chondrolysis in vitro and has some reparative potential. The damage blocking activity appears to be associated with the ability of HA/800 to block penetration of the Fn-f, rather than with direct effects on cartilage tissue.

Effect of proteoglycan removal on solute mobility in articular cartilage

Transport of nutrients, cytokines, pharmacologic agents, and matrix components through articular cartilage is critical for the viability and structural integrity of the tissue. To understand the role of the extracellular matrix in regulating this process, we measured the diffusivity of three uncharged solutes of different molecular size (glucose, MW 180; inulin, MW 5000; dextran, MW 70,000) into intact cartilage and cartilage that had its proteoglycan (PG) component removed. Solute diffusivity was measured by performing transient (nonsteady state) one-dimensional diffusion tests using radiolabelled solutes. Compared to intact cartilage, the diffusivity of glucose was unchanged after PG removal, inulin was unchanged but dextran inereased by 1.7 times after 71% PG removal, and both inulin and dextran increased by 1.6 and 2.0 times, respectively, after 93% PG removal. The diffusivities of inulin and dextran were inversely proportional to the PG content. While no change was found in the tissue's bulk fluid content. PG depletion resulted in an increase in fluid content in the upper regions of the tissue and a decrease in the lower regions. These results indicate that in intact tissue small uncharged solutes have free mobility through the inter-molecular and intra-molecular PG volumes, larger molecules have limited intra-molecular mobility, and very large molecules are excluded from the intra-molecular space.

Lipoprotein Lipase Reduces Secretion of Apolipoprotein E from Macrophages

Macrophages are a significant source of lipoprotein lipase (LPL) and apolipoprotein E (apo E) in the developing arterial wall lesion, and each of these proteins can importantly modulate lipid and lipoprotein metabolism by arterial wall cells. LPL and apo E share a number of cell surface binding sites, including proteoglycans, and we have previously shown that proteoglycans are important for modulating net secretion of apoprotein E from macrophages. We therefore evaluated a potential role for LPL in modulating net secretion of macrophage-derived apo E. In pulse-chase experiments, addition of LPL during the chase period produced a decrease in secretion of apoprotein E from human monocyte-derived macrophages, from the human monocytic THP1 cell line, and from J774 cells transfected to constitutively express a human apo E cDNA. LPL similarly reduced apo E secretion when it was prebound to the macrophage cell surface at 4 degrees C. A native LPL particle was required to modulate apo E secretion; addition of monomers and aggregates did not produce the same effect. Depletion of cell surface proteoglycans by a 72-h incubation in 4-methylumbelliferyl-beta-D-xyloside did not attenuate the ability of LPL to reduce apo E secretion. However, addition of receptor-associated protein attenuated the effect of LPL on apo E secretion. Although LPL could mediate removal of exogenously added apo E from the culture medium, detailed pulse-chase analysis suggested that it primarily prevented release of newly synthesized apo E from the cell layer. Cholesterol loading of cells or antibodies to the low density lipoprotein receptor attenuated LPL effects on apo E secretion. We postulate that LPL sequesters endogenously synthesized apo E at the cell surface by a low density lipoprotein receptor-dependent mechanism. Such post-translational regulation of macrophage apo E secretion by LPL could significantly influence apo E accumulation in arterial vessel wall lesions.

Chemonucleolysis with human stromelysin-1.

Immunohistologic analysis was performed on surgically removed samples of herniated nucleus pulposus to examine the expression of stromelysin-1. We performed in vitro and in vivo experiments to determine whether recombinant human (rh) stromelysin-1 is capable of degrading nucleus pulposus. To analyze the production of stromelysin-1 in various types of herniated nucleus pulposus, and to examine the effects of this recombinant protein on nucleus pulposus tissues. The authors previously demonstrated a progressive decrease in herniated nucleus pulposus size in some of the transligamentous and sequestration types of herniated nucleus pulposus using magnetic resonance imaging. An increased production of stromelysin-1, a cartilage proteoglycan degrading enzyme, in herniated nucleus pulposus was reported recently. The authors speculated that if stromelysin-1 is involved in the degradation of herniated nucleus pulposus, stromelysin-1 itself may be used as a chemonucleolytic agent. Immunohistologic analysis using streptoavidin-biotin method was performed on 20 herniated nucleus pulposus samples to investigate the expression of stromelysin-1. Five herniated nucleus pulposus samples were incubated in a tissue culture medium in the presence or absence of rh stromelysin-1. After 24 hours of incubation, their weight changes were measured, and the loss of proteoglycan was assessed by Safranin O staining. Rat nucleus pulposus tissues were obtained from coccygeal intervertebral discs, and autologous subcutaneous transplantation was performed. Rh stromelysin-1 was injected into the grafted materials, and the reduction in size was followed by two-dimensional measurements from the skin surface, using engineer's calipers. Immunohistologic analysis demonstrated the production of stromelysin-1 in the granulation tissues of herniated nucleus pulposus. When stromelysin-1 was injected into the murine nucleus pulposus tissues, they reduced in size more rapidly than the control group. In addition, human herniated nucleus pulposus materials obtained at surgery showed significant weight loss when treated with stromelysin-1 in an organ culture system. Safranin O staining revealed extensive depletion of proteoglycan in these herniated nucleus pulposus samples. Stromelysin-1 is a possible key enzyme in herniated nucleus pulposus resorption, and stromelysin-1 may be a good candidate for use in chemonucleolysis. Administration of human stromelysin-1 may physiologically facilitate the resorption process of herniated nucleus pulposus, increase the healing rate and decrease complications after chemonucleolysis.

Can magnetization transfer magnetic resonance imaging follow proteoglycan depletion in articular cartilage?

In this study we determined the efficiency of magnetization transfer magnetic resonance imaging (MT-MRI) to differentiate native and enzymatically degraded cartilage, using bovine sesamoid bones from the metacarpophalangeal joint as a model system. Gradual proteoglycan (PG) depletion was achieved by increasing incubation periods with testicular hyaluronidase. For native cartilage a Ms/Mo ratio of 0.303 +/- 0.09 (mean +/- SEM) was measured. Biochemically determined PG diminution up to 50% correlated strongly (r = 0.953) with changes in the Ms/Mo ratio. Further PG loss is not reflected in an equally drastic Ms/Mo increase, whereas subsequent treatment of PG-depleted cartilage samples with collagenase led to an additional rise in the Ms/Mo ratio. Proteoglycan depletion and the beginning destruction of the collagen structure were also assessed histochemically. Our study confirms that collagen contributes to the baseline MT effect observed in articular cartilage. However, the changes in the MT ratio in gradually PG-depleted cartilage with a largely intact collagen network indicate that PG contributes to the MT effect as well. Therefore MT-MRI might become a sensitive technique for the monitoring of subtle degradational changes in articular cartilage, the still inaccessible process in osteoarthritis.

Monocytes/macrophages rather than PMN are involved in early cartilage degradation in cationic immune complex arthritis in mice.

We investigated the involvement of polymorphonuclear granulocytes (PMN) and monocytes in cartilage degradation in immune complex-mediated arthritis (ICA). ICA induced with lysozyme-antilysozyme in the murine knee joint is characterized by a major influx of PMNs followed by monocytes and marked cartilage proteoglycan (PG) depletion develops within 2 days. Around 60% of 35S-prelabeled PG is lost at day 2. Influx of cells was manipulated using interleukin-1 (IL-1) receptor antagonist (IL-1ra) or antibodies to adhesion molecules. Cellular infiltrate was analyzed on hematoxylin-stained joint sections. Early systemic treatment with IL-1ra highly reduced PMN influx, whereas monocyte influx was hardly diminished. PG loss was not significantly reduced, declining from 62% in controls to 47% in IL-1ra-treated mice. Total blockade of cell influx was found after intravenous treatment with monoclonal antibodies 5C6 (anti-CD11b/CD18:anti-CR3) or NIMP.R14 (25-30 kDa protein mainly present on PMN) and PG loss was reduced to 5-10%. A similar reduction was observed after prior depletion of circulating PMNs with total body irradiation. Because amounts of IL-1 produced in leukopenic and control arthritic joints are comparable, this suggests that IL-1 is only marginally involved in PG loss in the first phase of ICA. This study indicates that monocytes rather than PMN might be involved in PG loss in this form of arthritis, either directly or by local activation of synovial layer cells of the joint.

Fibronectin-fragment-induced cartilage chondrolysis is associated with release of catabolic cytokines.

Fibronectin fragments have both catabolic and anabolic activities toward articular cartilage explants in vitro. Whereas a 1 nM concentration of an N-terminal 29 kDa fibronectin fragment (Fn-f) increases the proteoglycan (PG) content of cartilage without induction of matrix metalloproteinases (MMPs), 0.1-1 microM Fn-f temporarily suppresses PG synthesis and enhances MMP release. The higher concentrations cause an initially rapid PG depletion during the first week of culture, followed by much slower PG loss and gradually increasing rates of PG synthesis. To test for the involvement of mediators, human articular cartilage was cultured with Fn-f, and conditioned media were assayed for selected cytokines and factors. With 1 nM Fn-f, the release of the anabolic factors, insulin growth factor-I and transforming growth factor beta1, from cultured cartilage was enhanced by 50-100% during the entire 28-day culture period and this was associated with both supernormal rates of PG synthesis and PG content. However, the higher concentrations of Fn-f additionally enhanced release, by at least 10-fold, of the cytokines, tumour necrosis factor alpha, interleukin-1alpha, interleukin-1beta and interleukin-6 while causing depletion of cartilage PG. Release of tumour necrosis factor alpha, interleukin 1beta and interleukin 1alpha peaked at days 2, 3 and 9 during or slightly after the period of maximal PG depletion and decreased to control levels by days 7, 7 and 21 respectively, whereas release of interleukin 6 was enhanced throughout the culture period. Neutralizing antibodies to the catabolic cytokines reduced Fn-f-mediated MMP-3 release and suppression of PG synthesis. The temporal aspects of this interplay between catabolic and anabolic factors are consistent with the kinetics of Fn-f-mediated cartilage damage and attempted repair and may be relevant to cartilage damage and repair in vivo.