Boundary lubrication by synovial fluid (SF) refers to the ability of SF to reduce friction between apposed and pressurized cartilaginous surfaces independently of viscosity. Lubricin (∼227.5 kd), encoded by the gene PRG4, is a heavily glycosylated (50% weight/weight) mucinous glycoprotein secreted by synovial fibroblasts (1). It is considered the factor responsible for boundary lubrication of diarthrodial joints (2–4). Superficial-zone protein (SZP; ∼345 kd), another PRG4 gene product (1, 5), is expressed by chondrocytes located in the superficial zone of articular cartilage (6, 7). SZP is a component of the lamina splendens. The carbohydrate moieties of lubricin, and by extrapolation those of SZP, are considered to play an important role in lubrication, since glycosidase digestion of lubricin causes loss of boundary lubrication (3, 8).
Individuals with a history of knee joint injury are more likely to develop osteoarthritis (OA) than are those without a history of knee injury (9, 10). We recently reported that early signs of articular cartilage degradation, as indicated by type II collagen (CII) peptide release, appear in the SF from patients following knee injury (11). Presumably, the extent of articular damage depends, among other factors, on the friction and wear between the cartilaginous surfaces in the joint. Compromising boundary lubrication will cause increased friction, load amplification, and ultimately, greater cartilage damage.
We used the rabbit knee injury model, as described by Hulth et al (12), to determine the relationship between the boundary-lubricating ability of SF and articular cartilage damage. In vitro boundary-lubricating ability of SF, lubricin concentration, markers of cartilage degradation, and elastase activity in SF were measured, and correlations among these factors were determined. In particular, the in vitro lubricating ability was compared with the lubricin concentration as determined by a sandwich enzyme-linked immunosorbent assay (ELISA). The chosen epitopes reside in the functional elements of a boundary lubricant. The boundary-lubricating ability of SF was further examined in 2 populations of patients with inflammatory knee joint conditions: acute knee joint synovitis (KJS) and chronic inflammatory rheumatoid arthritis (RA). The boundary lubrication provided by SF under these 2 conditions was compared, as were collagen markers of articular cartilage degradation. The sensitivity of lubricin to neutrophil elastase (NE) was determined using Western blotting to analyze the time-dependent digestion of lubricin.
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- MATERIALS AND METHODS
In this study, we examined the temporal pattern of changes in boundary-lubricating ability of SF and in markers of cartilage degradation in rabbit knees following a surgically induced injury. The early biochemical changes that may precipitate OA secondary to acute knee injury are not entirely understood. We hypothesized that subsequent to injury, damage to the articular cartilage and loss of boundary-lubricating ability are early interrelated events. The loss of boundary lubrication by SF following injury would contribute to premature wear as a result of increased friction. Previous studies of either in vitro or in vivo cartilage injury models have established cartilage damage to be a consequence of excessive loading (20–24). Investigators of these models of injury did not examine changes to the boundary-lubricating ability of SF or its role in mitigating cartilage damage. Given the important chondroprotective properties that SF confers on articular cartilage, it is essential to understand the effects of acute injury on boundary lubrication.
The boundary-lubricating ability of SF was determined in an artificial bearing system under load, which isolated boundary lubricant conditions. This system has the advantage of reproducibility compared with cartilage-upon-cartilage bearing, and it mimics the results of this bearing, although μ values were higher overall (14). The lubricating ability of rabbit SF diminished after injury, as shown by the elevation in μ values at weeks 2 and 3 after injury. When measured by ELISA, lubricin concentrations in SF at weeks 2 and 3 after injury decreased compared with those at week 1 and compared with those in normal rabbit SF. Furthermore, μ values in SF correlated significantly with the lubricin concentration determined by sandwich ELISA.
Deterioration of the boundary-lubricating ability of SF could result from decreased lubricin synthesis or increased lubricin degradation. Support for the hypothesis of increased degradation of lubricin in the injured joint derives from measures of elastase and matrix metalloproteinase (MMP) activity in the SF. NE, the levels of which are elevated in inflammation, can degrade purified human lubricin, as demonstrated by the diminishing immunopositive bands of human lubricin following incubation with NE. This observation is supported by previous studies that established the sensitivity of SZP to degradation by NE (25).
Digestion of the N-terminal domain prior to the mucin-like domain, as evidenced in Figure 6, supports the utility of a sandwich ELISA. The assay of either epitope alone would be insensitive or could falsely show the complete digestion of lubricin. This may have occurred in our Western blot assays of SF aspirated from rabbits 2, 4, 8, 9, and 10, none of which showed reactivity, but all of which continued to lubricate at week 2. Similarly, SF from rabbit 1 continued to lubricate at week 2, but lubricin was undetectable by Western blot analysis using PNA–peroxidase (Figure 2D).
In addition to changes in boundary lubrication, we also found evidence of damage to the articular cartilage matrix following injury, as assessed by the release of CII peptides and the creation of neoepitope 9A4. The release of CII peptides in the SF indicates that CII degradation is an early event in damage of the matrix. Elevated 9A4 epitope levels point to active metalloproteinase activity following injury (26). Epitope 9A4 is produced when CI, CII, and CIII are degraded by the collagenase subfamily of metalloproteinases (MMPs) (26). Increased expression of collagenases has been reported following injury (27) and in traumatized synovial membranes (28). The SF from injured rabbit knees contained elevated levels of epitope 5-D-4 of keratan sulfate compared with lavage fluid from the sham-operated knees, providing corroborating evidence of the damage to articular cartilage, which may also be indicative of severity. Released CII peptides first increased and then decreased over weeks 1–3, while levels of proteoglycans (as detected via epitope 5-D-4 of keratan sulfate) did not change significantly, suggesting that the damage to articular cartilage in this model is perhaps superficial, but could evolve over time into arthritic lesions clinically consistent with OA of traumatic origin (10).
Elastase activity due to NE was compared with elastase activity due to metal-dependent metalloproteinases to further understand the biochemical factors that lead to deterioration of SF boundary-lubricating ability and matrix damage in the injury model. The significant and early contribution of metal-dependent elastase activity to the total elastase activity, represented by significant lowering of elastase activity with EDTA pretreatment (19), indicates a contribution by MMPs to the damage of the collagen matrix. This role was supported by the finding of elevated 9A4 epitope levels in the SF following injury. Purified NE has been shown to damage cartilage explants in vitro (29). NE, and not MMPs, can destroy the superficial layer of cartilage, which leads to MMPs having better access to CII molecules in less superficial layers of cartilage (30). We speculate that the convergent effects of NE and MMPs lead to the damage of chondroprotective mechanisms provided by lubricin, which then leads to damage of the superficial zone of cartilage and release of CII peptides into the SF following injury.
The significant correlation between elastase activity and diminishing boundary lubrication (increasing μ values) supports the notion of a causal relationship between inflammation and proteolytic degradation of lubricin, leading to damage to articular cartilage. In the later stages of injury, other factors, such as joint utilization and loss of interarticular surface congruence, contribute to the extent of damage, which may in turn complicate our ability to understand these cascading catabolic events.
The KJS patient population represents a clinical extension of the rabbit injury model. These patients have experienced a knee insult as a result of a blunt injury, a sprain, or a strain, or they may have had subclinical trauma. These patients had an elevated SF nucleated cell count indicative of active inflammation, which correlated with SF boundary-lubricating ability and was also associated with CII peptide release (15). At the opposite end of this spectrum is the population of patients with RA (representing progressive chronic inflammatory arthritis), in whom SF boundary-lubricating ability was completely lost and articular cartilage damage was evident. These findings indicate that the deterioration of SF boundary-lubricating ability (and its possible association with matrix damage) is not a unique feature of early stages of a knee injury, but is instead a common feature in inflammatory conditions, both acute and chronic. Furthermore, wear-induced damage, which adheres to a classic tribologic model, may also occur, but it probably does not adequately explain CII peptide release.
The results of this study delineate early changes to the boundary-lubricating ability of SF and to articular cartilage integrity following injury. The loss of boundary-lubricating ability of SF and the release of CII peptides into SF are both early and distinct events following injury. NE released from infiltrating nucleated cells and MMPs released from the articular matrix appear to be associated with these early changes in boundary lubrication by SF and release of CII peptides. In this model, we could not isolate the wear effects and pattern of cartilage degradation created by the loss of boundary lubrication. Injury models in general display contemporaneous inflammation, which can affect many extracellular matrix proteins digested by a myriad of enzymes. However, our efforts in this study were restricted to NE and MMPs.
A sandwich ELISA proved to be a sensitive and specific method for determining lubricin concentration. Rational epitope selection was directed toward a non–alternatively spliced N-terminal region and O-linked glycosylations. These moieties play roles in molecular adhesion to a surface and in repulsion force generation, respectively. Sandwich ELISA of these posited functional motifs in lubricin correlated highly with results of mechanical assays across SF samples with varying lubricin concentrations.
Another limitation of this study was the lack of normal rabbit SF from the contralateral control joints to establish normal lubricating ability. Scant SF aspirates from control joints also necessitated lavaging of the joint cavity with the resulting dilution of its components of interest, which could have rendered them undetectable. However, detection limits for the assays were established by internal controls, by our previous assays of normal human SF, and by retesting on normal rabbit SF from excised limbs. Given the high lubricin concentration by sandwich ELISA, normal rabbit SF should exhibit efficient lubricating ability, better than the SF from the injured rabbit knees.
We undertook the present study to clarify and develop our recent findings concerning traumatic synovial effusions observed in patients in an emergency department (15). The clinical population of patients in emergency departments has not previously been a focus of rheumatologic and orthopedic investigation, and these patients frequently do not receive subspecialty followup. Further study is needed to determine the long-term consequences of trauma resulting in monarticular knee joint effusions judged to be clinically significant and requiring arthrocentesis in the absence of radiographic evidence of fracture. Support for a classic lubrication and wear (tribologic) explanation of OA will necessarily await studies using animal models in which endogenous lubricin levels can be manipulated and/or in which purified lubricin can be therapeutically administered.