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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

To investigate the effect of arthritis development and progression on the integrity and function of lubricin and the relationship of lubricin to cartilage damage in a rat antigen-induced arthritis model.

Methods

Arthritis was induced in the right knee joints, using methylated bovine serum albumin and Freund's complete adjuvant. Whole joint friction measurements were performed ex vivo with a modified Stanton pendulum configuration, and coefficients of friction (μ) were determined. Levels of messenger RNA (mRNA) for lubricin, cathepsin B, and interleukin-1β (IL-1β) in synovial tissue from control and affected joints were determined by quantitative polymerase chain reaction. Lubricin staining in cartilage was performed using a lubricin-specific monoclonal antibody.

Results

The μ values in excised right joints following arthritis induction were significantly (P < 0.001) higher than those in excised left joints. Lubricin mRNA expression levels in synovial tissue on days 4 and 7 after arthritis induction were significantly (P < 0.001) lower in the right joints compared with the left joints, whereas levels of cathepsin B and IL-1β mRNA expression on days 4, 7, and 14 were significantly (P < 0.001) higher in the right joints than the left joints. Lubricin staining was diminished in cartilage from the right joints compared with the left joints.

Conclusion

Elevated coefficients of friction in arthritic joints occur concurrently with enhanced proteolytic degradation by up-regulated cathepsin B and other proteases, as well as decreased lubricin synthesis by synovial fibroblasts and superficial zone chondrocytes. Loss of joint lubrication is an early event in inflammatory arthropathy. Restoring chondroprotection and preventing potential wear-induced cartilage degradation may require lubricin supplementation in synovial fluid.

Rheumatoid arthritis (RA) is characterized by persistent joint inflammation and synovial hyperplasia. Classic features of RA include overgrowth of synovial tissue and formation of an aggressive cell mass (pannus) (1, 2), initiated by infiltrating monocytes and macrophages (3). Destruction of articular cartilage and bone is attributed to a host of proteases, e.g., serine proteases, matrix metalloproteases, and cathepsins, secreted by the pannus tissue (4, 5). In induced arthritis in animals the pannus formation and infiltration of macrophages and granulocytes is recapitulated (6), making this a useful model for studying RA pathogenesis and screening candidate therapeutic interventions (7–9).

Lubricin, a mucinous glycoprotein secreted by synovial fibroblasts (10), is the factor responsible for lubrication of apposed and pressurized articular surfaces (11). Structurally, lubricin is composed of 12 exons. The mucin domain, encoded by exon 6, constitutes >70% of the primary amino acid composition of lubricin. This domain contains a degenerate KEPAPTT repeat, where threonine residues are likely O-linked to β(1-3)Gal-GalNAc (12). The O-linked glycosylations and the integrity of the mucin domain are associated with lubricin's boundary-lubricating properties (12).

Previously, we demonstrated that synovial fluid (SF) aspirated from the joints of patients with RA lacked chondroprotective properties conferred by lubricin (13), and inhibition of cysteine protease activity resulted in preservation of the lubricating ability of RA SF aspirates following lubricin supplementation (14). The loss of boundary-lubricating properties in RA SF was associated with increased cartilage damage as assessed by measurement of type II collagen (15). In the present study, we investigated the hypothesis that proteolytic degradation of lubricin by cysteine proteases (e.g., cathepsin B) and decreased lubricin expression can contribute to the loss of SF lubricating ability and elevation of the coefficient of friction (μ) of articular joints in RA, leading to increased risk of cartilage damage. To demonstrate that lubricin has proteolytic susceptibility, purified human lubricin was incubated with cathepsin B. The effect of cathepsin B on the integrity of lubricin was also determined by Western blot and in vitro friction assays. An antigen-induced arthritis (AIA) model was utilized to assess temporal changes in whole joint μ values following arthritis induction and their relationship to synovial expression of lubricin, markers of inflammation, and cathepsin B.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Protease digestion of human lubricin.

Human lubricin was purified from pooled SF aspirates from patients undergoing total knee replacement, as described previously (12). Aliquots of purified human lubricin (1 ml; 250 μg/ml) were subjected to protease digestion using purified cathepsin B (Sigma-Aldrich, St. Louis, MO) at a final concentration of 0.5 units/ml (1 unit liberates 1 nmole of 7-amino-4-methylcoumarin from Z-Arg-Arg 7-amido-4-methylcoumarin per minute at pH 6.0 at 40°C), reconstituted in 0.25M sodium acetate buffer (pH 5.5). Aliquots of digested human lubricin (200 μl) were removed after 2, 4, 6, 12, and 24 hours, and the reaction was stopped by adding E-64 (Sigma-Aldrich), to a final concentration of 100 μM.

Western blot analysis of protease digestion of human lubricin.

Electrophoresis was performed on precast 4–15% sodium dodecyl sulfate polyacrylamide gels (Bio-Rad, Hercules, CA), under reducing conditions. High molecular weight standards (BRL, Gaithersburg, MD) were electrophoresed simultaneously with digested human lubricin and control human lubricin. Electrophoresis was performed at 150V for 90 minutes, until the wave front exited from the bottom of the gel. Western blot transfer to nitrocellulose was carried out under semidry conditions at 20V for 40 minutes. The blot was blocked overnight at 4°C with 2% (weight/volume) bovine serum albumin (BSA) in phosphate buffered saline (PBS).

For probing of human lubricin, peanut agglutinin (PNA) from Arachis hypogaea was conjugated to peroxidase (Sigma-Aldrich) at a concentration of 0.5 mg/ml in PBS–2% Tween 20, and incubated for 60 minutes at room temperature. Following extensive washing with PBS and PBS–2% Tween 20, chemiluminescence substrate (Pierce, Rockford, IL) was added. Immunopositive bands on BioMax film (Eastman Kodak, Rochester, NY) were detected in a darkroom.

In vitro friction assay of digested human lubricin.

The boundary-lubricating ability of protease-treated human lubricin was measured using a friction apparatus on which latex was oscillated against polished glass, as reported by Davis et al (16) and as previously described (13). Lubrication was manifested as a reduction in μ with the lubricant relative to that of normal saline. Negative Δμ values (−Δμ) indicate lubrication, whereas positive Δμ values indicate friction.

Animals.

Male Lewis rats ranging in age from 7 to 8 weeks and weighing from 180 to 200 gm were obtained from Harlan (Indianapolis, IA). The animals were housed 2 per cage under conditions of 12 hours light/12 hours darkness, were allowed food and water ad libitum, and were kept for 3 days before immunization. Approval from the Institutional Animal Care and Use Committee (IACUC) at the University of Rhode Island was obtained prior to initiation of the study.

Arthritis induction.

Arthritis was induced in the right femorotibial joint of each rat as previously described (17), with modifications. Briefly, on 2 occasions 2 weeks apart, the joint was injected subcutaneously with an emulsion (1.0 cc) of an equal mixture of methylated BSA (mBSA) (0.5 mg; Sigma-Aldrich) and Freund's complete adjuvant (0.25 mg Mycobacterium tuberculosis; Sigma-Aldrich). Six days following the second injection, 50 μg of mBSA was injected into the right joint. Drinking water was supplemented with acetaminophen at a dose of 3 gm/liter as recommended by the IACUC; acetaminophen supplementation may have exerted some weak antiinflammatory effect, thereby decreasing the extent of inflammation in this model. The left knee joints of the rats received a sham injection of saline and were used as controls.

Assessment of inflammation.

The progression of inflammation was monitored 7, 14, 21, 24, and 28 days following induction of arthritis, in 6 rats at each time point, by measuring the diameters of the right and left knee joints using a high-precision micrometer. The diameter measurement was consistently performed on the medial to lateral aspect of the joint. The mean diameter in millimeters was calculated as the average of 2 consecutive measurements. The mean diameters of the right and left knee joints of 6 untreated control rats were also recorded.

Determination of ex vivo μ values in the excised knee joints of control and arthritic rats.

The knee joint friction measurements were performed ex vivo with a modified Stanton pendulum configuration as previously described (18). Rats were killed on days 0, 7, 21, 24, and 28 following arthritis induction, and supporting connective tissue and musculature was stripped from excised joints, while the synovium was left intact. The femur and tibia were severed midlength and covered with connecting Plexiglas tubing. The center of the joint was used as the axis of rotation of a 1-Hz pendulum, which weighed 170 gm. The pendulum was set in motion at a 30° angle off the perpendicular axis and allowed to oscillate. The pendulum motion was videotaped, and post hoc analysis was performed to establish the baseline μ value. The deceleration of the pendulum, a = dv/dt, was used to calculate μ (a = acceleration; t = time). Velocity (v) was calculated from the equation v = (2gh)½, where h is the height at which the pendulum reached apogee to the point of maximum velocity at 0° off the perpendicular axis. The μ of rat joints was calculated to equal a/g. Presently this calculation does not account for aerodynamic drag, and g (the earth's acceleration constant) is assumed to equal 9.81 m/s2.

Measurement of sulfated glycosaminoglycan (GAG) content in patellar cartilage.

Patellae from the knee joints of 6 rats were carefully isolated on days 0, 7, 21, 24, and 28 following arthritis induction and placed in 5% formic acid overnight for decalcification. The cartilage was subsequently stripped from the underlying bone, and sulfated GAG content was determined by 1,9-dimethylmethylene blue assay using a procedure similar to one previously described (19). The proteoglycan content was expressed as micrograms per patella.

Isolation of total RNA from control and arthritic knee joint synovial tissue and conversion of messenger RNA (mRNA) to complementary DNA (cDNA).

Synovial tissue was removed from the right and left knee joints of 6 rats 4, 7, 14, and 28 days following arthritis induction and of 6 control rats, and was quickly snap-frozen in liquid nitrogen and stored at −80°C. Total RNA was isolated from synovial tissue, using a Ribopure kit (Ambion, Austin, TX). Contaminating genomic DNA was removed by DNase I treatment of isolated RNA. Messenger RNA was converted to cDNA using a Cells-to-cDNA II kit (Ambion).

Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) primer design and testing.

The mRNA sequences of genes of interest were obtained from the National Center for Biotechnology Information (Bethesda, MD) using accession numbers NW_047397, NW_047487, and NW_047658, corresponding to lubricin, cathepsin B, and interleukin-1 β (IL-1β), respectively. Primer 3 (Whitehead Institute, Cambridge, MA) was used to develop cDNA primers for quantitation of levels of mRNA for the above-mentioned proteins. The primer pairs used were as follows: lubricin 5′-GGAACCGATCTCTTGGTTGA-3′ (forward), 3′-ATCCACTGGCTTACCATTGC-5′ (reverse); cathepsin B 5′-ACCCTACTGGCTGGTAGCAA-3′ (forward), 3′-TCCAGCCACGATTTCTGATT-5′ (reverse); and IL-1β 5′-AGCAACGACAAAATCCCTGT-3′ (forward), 3′-GAAGACAAACCGCTTTTCCA-5′ (reverse). These resulted in 96-bp, 112-bp, and 150-bp products, respectively. The cDNA primers for lubricin were designed to specifically amplify a sequence that spans exons 6 and 7, the cDNA primers for cathepsin B were designed to specifically amplify a sequence that spans exons 8 and 9, and the cDNA primers for IL-1β were designed to specifically amplify a sequence that spans exons 6 and 7. The oligonucleotide primer sequences were manufactured by Bioserve Biotechnologies (Laurel, MD). A BLAST search was conducted to determine the likelihood that mRNA sequences other than those of interest would be amplified using our designed primers. Lubricin, cathepsin B, and IL-1β primers were designed in such a way as to prevent interference of genomic DNA in the quantitation of these genes' expression in the synovial tissue from rats with AIA and control rats. The PCR product sizes obtained using these primers were identical to the expected PCR product sizes predicted with Primer 3 software. The PCR product identities of genes of interest were further confirmed by PCR product sequencing.

The cDNA from control and arthritic synovial tissue (3.0 μl; 0.02 μg/μl) was mixed with 10 μl of 5× RT-PCR buffer, 4 μl of dNTP mix (10 mM), 3.5 μl of MgCl2 (25 mM), 1.0 μl of 20 μM primer solution, 0.4 μl of Taq polymerase at a concentration of 50 units/μl, and autoclaved water, to a final volume of 60 μl. All reagents were obtained from PerkinElmer Applied Biosystems (Foster City, CA). Thermal cycling and PCR product amplification were initiated by denaturation of the RNA–cDNA complex at 95°C for 15 minutes. Thermal cycling was performed for 40 cycles of 1 minute at 94°C for denaturation, followed by 1 minute at 62°C and 1.5 minutes at 72°C. At the end of 40 cycles, a 7-minute final extension at 72°C was performed. RT-PCR products were analyzed by agarose gel electrophoresis with 2% NuSieve GTG (FMC Bioproducts, Rockland, ME) and ethidium bromide staining. Bands corresponding to expected RT-PCR product sizes were excised and extracted from agarose. Amplicon sequencing was performed at the Keck Foundation Biotechnology Laboratory, Yale University School of Medicine (New Haven, CT), using an Applied Biosystems 377 DNA Sequencer.

Real-time quantitative PCR assays.

Real-time quantitative PCR assays of levels of lubricin, cathepsin B, and IL-1β mRNA in synovial tissue were performed using a SYBR Green fluorometric-based detection system. In 96-well polypropylene microplates (MJ Research, Waltham, MA), 9.5 μl of autoclaved water was mixed with 1 μl cDNA template at a concentration of 0.02 μg/μl and with 1 μl of forward and reverse primers for each gene of interest at a concentration of 20 μM. A 12.5-μl concentration of 2× DyNAmo master mix reagent (MJ Research) was added subsequently, to constitute a 25-μl reaction volume. Thermal cycling and PCR product amplification were performed with a DNA Engine Opticon 2 (MJ Research). PCR product amplification was initiated by denaturation of the RNA–cDNA complex at 95°C for 10 minutes. Thermal cycling was performed for 40 cycles of 10 seconds at 94°C for denaturation, followed by 20 seconds at 55°C and 30 seconds at 72°C. At the end of 40 cycles, a 5-minute final extension at 72°C was performed.

PCR product quantitation was based on the comparative threshold cycle (Ct) method (20). The Ct is the fractional PCR cycle at which the quantity of the amplified product reaches a predetermined threshold. The Ct equation was used to calculate the expression level of the target gene in synovial tissue from the right joints relative to the expression level of the same gene in synovial tissue from the left joints as 2−▵▵Ct, where ▵▵Ct equals the ▵Ct of target gene in right joint synovial tissue minus the ▵Ct of target gene in left joint synovial tissue. The ▵Ct is the difference between the Ct of target gene and that of 18S ribosomal RNA in the same sample. The melting curves generated after each assay were examined to ensure the presence of a single PCR species and the lack of primer-dimer formation in each well.

Histologic examination of articular cartilage specimens using monoclonal antibody (mAb) S6.89 and hematoxylin and eosin staining.

The femorotibial joints of 3 rats that were killed 1 week following induction of arthritis were examined histologically. The joints were dissected and stored in buffered neutral formalin solution (100 ml 37–40% formalin, 900 ml distilled water, 4 gm sodium phosphate monobasic, monohydrate, and 6.5 gm sodium phosphate dibasic, anhydrous [pH 7.4]). Decalcification was performed for 8 hours at room temperature, using a decalcifying solution (100 ml concentrated formic acid, 80 ml concentrated HCl, 50 gm 1,3,5-trihydroxybenzene, and 800 ml distilled water). The specimens were embedded in paraffin, and 6-μm sections were generated and stained with a 1:1,000 dilution of S6.89 (an mAb specific for lubricin/superficial zone protein (SZP) [21]) followed by peroxidase-linked goat anti-mouse IgG. In an additional 3 rats that were killed 3 weeks after arthritis induction, histologic sections were processed in a manner similar to the one described above and stained with hematoxylin and eosin.

Statistical analysis.

The change in μ values with purified human lubricin compared with normal saline was calculated as the mean ± SD, and joint diameters, joint μ values, cartilage sulfated GAG content, and expression of mRNA for target genes in the synovial tissue as the mean ± SEM. Student's unpaired 2-sample t-test was used to test for differences in measurements between the right and left knee joints. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Effects of cathepsin B on human lubricin.

Incubation with 0.5 units/ml of cathepsin B resulted in a time-dependent degradation of lubricin, as illustrated by diminishing band intensities at 2, 4, 6, 12, and 24 hours postincubation (Figure 1). PNA-positive bands were still detectable up to 6 hours after incubation of cathepsin B with human lubricin. However, at 12 hours and 24 hours, PNA reactivity was lost, indicating that β(1-3)Gal-GalNAc that was O-linked to the central exon 6 was completely degraded.

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Figure 1. Western blot analysis of purified human lubricin digested with cathepsin B (CB). Lubricin (5 μg/well) was incubated with cathepsin B at a final concentration of 0.5 units/ml reconstituted in 0.25M sodium acetate buffer (pH 5.5) at 37°C, and sampled after 2, 4, 6, 12, and 24 hours of incubation. The enzymatic reaction was stopped by addition of E-64, at a final concentration of 100 μM. Blots were probed with peroxidase-conjugated peanut agglutinin (PNA).

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Effects of cathepsin B on in vitro lubricating ability (Δμ) of human lubricin.

The Δμ values of human lubricin following time-dependent digestion with 0.5 units/ml of cathepsin B are reported in Table 1. The boundary-lubricating ability of human lubricin progressively deteriorated following cathepsin B digestion, as evidenced by positive Δμ values following 12 hours and 24 hours of incubation. In contrast, undigested control human lubricin exhibited a consistently negative Δμ value throughout the 24-hour period. Digestion of human lubricin with cathepsin B resulted in a significant (P < 0.001) increase in Δμ at 4, 6, 12, and 24 hours, compared with values obtained with undigested human lubricin.

Table 1. Coefficient of friction (Δμ) of purified human lubricin with and without digestion with 0.5 units/ml of cathepsin B*
Time of incubationWithout cathepsin B digestionWith cathepsin B digestion
  • *

    Negative Δμ values indicate lubrication; positive values indicate friction. Values are the mean ± SD of 2 experiments, each with 4 distinct Δμ measurements.

  • P < 0.001 versus the value obtained with undigested lubricin.

0 hours−0.068 ± 0.005−0.068 ± 0.005
2 hours−0.061 ± 0.004−0.048 ± 0.006
4 hours−0.059 ± 0.004−0.025 ± 0.005
6 hours−0.059 ± 0.002−0.012 ± 0.003
12 hours−0.061 ± 0.0090.008 ± 0.001
24 hours−0.060 ± 0.0040.051 ± 0.004

Progression of inflammation.

The progression of inflammation in the joints with AIA compared with control joints, determined by mean joint diameter, is illustrated in Figure 2A. One week following induction of arthritis, the mean diameter of the right joints was significantly (P < 0.001) higher than that of the left joints. The increase in the mean diameter of right joints compared with left joints remained significant (P < 0.001) 21, 24, and 28 days following arthritis induction.

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Figure 2. Diameter and ex vivo coefficient of friction (μ) of the right and left knee joints (n = 6 rats per group) 0, 7, 21, 24, and 28 days following arthritis induction. Arthritis was induced in the right femorotibial joints of male Lewis rats by subcutaneous injection of methylated bovine serum albumin (BSA) and Freund's complete adjuvant on days 0 and 14. Methylated BSA was subsequently injected intraarticularly on day 20. A, Joint diameter. The diameters of the right joints were significantly (P < 0.001) higher than those of the left joints on days 7, 21, 24, and 28. B, Ex vivo coefficient of friction. The ex vivo μ values in the right joints were significantly (P < 0.001) higher than those in the left joints on days 7, 21, and 24. Values are the mean and SEM.

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Ex vivo μ values in control joints and joints with AIA.

The temporal pattern of change in ex vivo μ values in rat joints following arthritis induction is illustrated in Figure 2B. The μ values in the excised right joints were significantly (P < 0.001) higher than those in excised left joints at 1 week following arthritis induction. Similarly, the μ values in excised right joints were significantly (P < 0.001) higher than those in excised left joints 21 and 24 days following arthritis induction. On day 28, there was no significant difference between ex vivo μ values in the right and left joints.

Sulfated GAG content of articular patellar cartilage from control joints and joints with AIA.

There was no significant difference in sulfated GAG content between the right and left joints 1 week following arthritis induction, as illustrated in Figure 3. Twenty-one, 24, and 28 days after arthritis induction, the sulfated GAG content in the right joint patellar cartilage was significantly (P < 0.001) lower than that in corresponding left joint patellar cartilage.

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Figure 3. Sulfated glycosaminoglycan (GAG) content of patellar cartilage from the right and left knee joints (n = 6 rats per group) 0, 7, 21, 24, and 28 days following arthritis induction. The sulfated GAG content in the patella was significantly (P < 0.001) lower in the right joints compared with the left joints on days 21, 24, and 28. Values are the mean and SEM.

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Lubricin, IL-1β, and cathepsin B expression in synovial tissue from control joints and joints with AIA.

Lubricin, IL-1β, and cathepsin B expression in synovial tissue from the right and left knee joints of control and arthritic rats is illustrated in Figure 4. The mRNA isolated from each synovial tissue specimen was sufficient to conduct the quantitative PCR experiments. Using primers spanning exons 6 and 7, lubricin mRNA expression levels in synovial tissue from the right and left joints of untreated control rats were shown to be identical. In rats with AIA, lubricin mRNA expression in synovial tissue from the right joints relative to that from the left joints was significantly (P < 0.001) lower on days 4 and 7 compared with control. On days 14 and 28 following arthritis induction, there was no longer a significant difference between relative lubricin mRNA expression in the right and left joints.

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Figure 4. Relative levels of mRNA for interleukin-1β (IL-1β), lubricin, and cathepsin B in synovial tissue from the right and left knee joints of untreated control rats and of rats with antigen-induced arthritis (AIA) 4, 7, 14, and 28 days following arthritis induction (n = 6 rats per group). Relative expression of the target genes in synovial tissue was calculated as the ratio of mRNA expression in the right knees to that in the left knees. Compared with untreated controls, IL-1β expression in the right joints relative to the left joints of rats with AIA was significantly higher 4, 7, and 14 days following arthritis induction, lubricin expression in the right joints relative to the left joints of rats with AIA was significantly lower 4 and 7 days following arthritis induction, and cathepsin B expression in the right joints relative to the left joints of rats with AIA was significantly higher 4, 7, 14, and 28 days following arthritis induction (all P < 0.001). Values are the mean and SEM.

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Cathepsin B mRNA expression in synovial tissue from the right joints relative to the left joints was highest 4 days following arthritis induction, and it progressively declined on days 7, 14, and 28. On days 4, 7, 14, and 28, cathepsin B mRNA expression in synovial tissue from the right joints relative to the left joints was significantly (P < 0.001) higher than the corresponding relative expression in control rats.

IL-1β mRNA expression in synovial tissue from the right joints relative to the left joints was also highest 4 days following arthritis induction and progressively declined on days 7, 14, and 28. The expression of IL-1β mRNA in synovial tissue from the right joints relative to the left joints was significantly (P < 0.001) higher than the corresponding relative expression in control rats on days 4, 7, and 14.

Histologic staining of rat cartilage specimens using mAb S6.89 and hematoxylin and eosin.

Lubricin staining on the surface and the superficial zone of representative articular cartilage specimens, 1 week following the initiation of arthritis, is illustrated in Figures 5A and B. The right knee exhibited no lubricin staining in the superficial zone cartilage. The contralateral left knee exhibited lubricin staining on the surface of articular cartilage and in the superficial zone, indicating that down-regulation of lubricin expression in the superficial layer was confined to arthritic knees at the initial stages of arthritis.

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Figure 5. Representative rat articular cartilage specimens stained with monoclonal antibody (mAb) S6.89 (A and B) and with hematoxylin and eosin (C and D). Histologic staining of cartilage from a right knee joint with mAb S6.89 1 week following induction of arthritis revealed little lubricin staining in superficial zone articular chondrocytes (A). Similar staining of cartilage from a sham-injected left knee joint 1 week following arthritis induction showed lubricin staining in superficial zone articular chondrocytes and on the cartilage surface (B). Hematoxylin and eosin staining of cartilage from a right knee joint 3 weeks following induction of arthritis revealed synovial hyperplasia, pannus formation, and articular cartilage destruction (C). Similar staining of a sham-injected left knee joint 3 weeks following arthritis induction showed little synovial hyperplasia and no significant cartilage invasion (D). Bars in A and B = 150 μm; bars in C and D = 100 μm.

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Hematoxylin and eosin staining of representative cartilage specimens, 3 weeks following the initiation of arthritis, is shown in Figures 5C and D. The right knee exhibited synovial hyperplasia, pannus overgrowth, and infiltration of the articular cartilage. In contrast, the left knee showed minimal synovial hyperplasia with no pannus formation or cartilage infiltration.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Proteolytic degradation of lubricin may increase friction between articular cartilage surfaces, which leads to damage and wear, thereby accelerating the progression of degenerative joint diseases. This supposition is strengthened by the observation that patients with camptodactyly-arthropathy–coxa vara–pericarditis syndrome developed precocious joint failure as a result of a lack of lubricin expression (22). RA is a chronic inflammatory condition characterized by infiltration of polymorphonuclear cells (23) and accumulation, in SF, of proteases, e.g., cathepsin B (24), which plays an important role in RA pathogenesis (25). Cathepsin B also has an important role in the pathogenesis of osteoarthritis; elevation of cathepsin B expression has been demonstrated in a model of early osteoarthritis (26). Cathepsin B activity has been linked to the progressive loss of boundary lubrication of lubricin-supplemented pooled RA SF, and inhibition of cysteine protease activity, and more specifically that of cathepsin B, using enzyme inhibitors resulted in significant inhibition of lubricin degradation by RA SF (14). This indicates that cathepsin B is active in RA and contributes to the loss of SF boundary lubrication evidenced in RA SF.

Cathepsin B proteolytically degrades lubricin in a time-dependent manner, as shown by diminishing intensity of the ∼240-kd lubricin band intensity when probed with peroxidase-linked PNA. Following 6 hours of incubation with cathepsin B, the mucin domain was still detectable with PNA, although to a reduced extent compared with control. Following 12 and 24 hours of incubation, PNA reactivity was completely lost, indicating loss of mucin domain integrity as a result of cathepsin B proteolytic activity. The boundary-lubricating ability of human lubricin, measured in a latex:glass system, continuously declined with cathepsin B treatment, and was completely abolished by 12 hours. After 6 hours of cathepsin B treatment, the ability of lubricin to lubricate was still evidenced by a negative Δμ value, albeit to a diminished extent. It appears, therefore, that loss of the boundary-lubricating ability of lubricin is more likely associated with loss of central mucin domain integrity following cathepsin B treatment. This is consistent with previously reported results indicating that lubricating ability was attributable to O-linked β(1-3)Gal-GalNAc moieties to threonine residues in the mucin domain (12).

The rat AIA model simulates key pathogenic events in human RA, e.g., articular joint destruction, persistent inflammation, and joint effusion. Models of monarticular disease, similar to the one used in our study, allow the initiation and progression of pathology to be confined to a specific articular joint, with minimal involvement of other joints (17). The progression of inflammation in our model, as determined by mean joint diameter, indicates that large fluid effusions accumulated in the arthritic joints and persisted for the duration of the study. These effusions accumulated in the right joints and not in the left, sham-injected joints.

The inflammatory nature of the disease in our model was reinforced by the determination of the synovial tissue expression level of a key inflammation mediator, IL-1β. In response to an inflammatory insult, IL-1β was markedly induced in synovial tissue from the right joints as opposed to the left joints at an early stage and persisted for a considerable period during the progression of arthritis, before IL-1β mRNA expression in the right joints ultimately declined. This pattern of induction was observed by measuring relative IL-1β mRNA expression levels by real-time PCR using IL-1β–specific cDNA amplification primers. The focus on IL-1β as an inflammation mediator in this research does not exclude a possible role of other inflammation mediators, e.g., tumor necrosis factor and oncostatin M, in the changes in joint lubrication observed in this model.

Histologic examination of representative rat cartilage specimens indicated that cartilage destruction was evident 3 weeks following the initiation of arthritis. At 1 week after arthritis induction, lubricin expression was significantly decreased in the affected joints compared with the contralateral joints, as demonstrated by immunohistochemistry analysis. This indicates that lack of lubricin expression by superficial zone chondrocytes precedes any significant cartilage damage in this model.

The frictional properties of arthritic knee joints were determined by measuring whole joint μ values with the modified Stanton pendulum technique. The early and persistent elevation of μ values in arthritic joints correlated with the extent of inflammation as determined by joint diameter and synovial tissue IL-1β expression. Although interstitial fluid pressurization contributes to joint lubrication in mammals (27), lubricin has been established as a very important factor in maintaining low μ values in diarthrodial joints. Previous research using Prg4-null mice indicated that joints from homozygotes had significantly higher μ values compared with those from either heterozygotes or normal mice (28). An interesting observation in the present study was that μ values in arthritic joints returned to control levels at 28 days following the initiation of arthritis. The reduction of μ values in arthritic joints in the later stages of arthritis can be partly explained by the return of synovial tissue lubricin expression in the arthritic joints to levels comparable with those in contralateral joints.

Lubricin expression following arthritis induction was significantly diminished in synovial tissue from the right joints compared with corresponding levels in synovial tissue from the left joints. This effect was transient and occurred during the early stages of the inflammatory response. In the later stages, it appeared that lubricin expression in synovial tissue from the right joints was restored to levels comparable with those in the left joints. Given the early elevation in IL-1β expression in synovial tissue from the right joints, it is plausible that the decrease in lubricin expression is caused by excessive IL-1β secreted by synovial tissue. This hypothesis is supported by earlier research showing that SZP, a lubricin-homologous protein secreted by superficial zone articular chondrocytes, is down-regulated by IL-1α (29).

Cathepsin B expression in synovial tissue from the right joints of rats with AIA exhibited an early and persistent pattern of elevation when compared with expression in synovial tissue from the left joints. The persistent elevation of cathepsin B indicates that even if normal lubricin expression is restored in arthritic joints, the secreted lubricin is still susceptible to proteolytic degradation by cathepsin B. This may explain why a high μ value was still evident in arthritic joints after lubricin expression in synovial tissue from these joints returned to control levels.

Cartilage damage was demonstrated by a significant decrease in the sulfated GAG content of patellar cartilage from right joints compared with left joints. The cartilage damage appeared to have been restricted to the arthritic right joints since histologic examination revealed extensive pannus formation 3 weeks after arthritis induction, while contralateral joints exhibited normal synovial lining. The association between loss of joint lubrication and articular cartilage damage was also previously demonstrated in a surgically induced joint injury model (30).

Loss of lubricin and the subsequent failure of fundamental joint lubrication mechanisms is a characteristic feature of inflammatory arthritic diseases. This conclusion is supported by previous work demonstrating loss of SF boundary-lubricating ability in RA and milder forms of synovitis (13). In the present study, we have demonstrated that cathepsin B can proteolytically degrade lubricin, resulting in the loss of lubricin's boundary-lubricating properties. Furthermore, in the early stages of arthritis, the presence of lubricin on the surface of articular cartilage is diminished, contributing to the elevation of μ values in arthritic joints. The loss of lubricin at the early stages of arthritis is likely a result of down-regulation of lubricin expression by synovial fibroblasts and increased proteolytic degradation by cathepsin B and other proteases. Restoring normal joint lubrication at an early stage following extensive joint inflammation may prove to be beneficial in limiting wear-induced cartilage damage.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Dr. Chichester had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Drs. Elsaid, Jay and Chichester.

Acquisition of data. Drs. Elsaid, Jay and Chichester.

Analysis and interpretation of data. Drs. Elsaid, Jay and Chichester.

Manuscript preparation. Drs. Elsaid, Jay and Chichester.

Statistical analysis. Dr. Elsaid.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

The authors would like to thank Dr. Tom Schmid of Rush Medical College for providing mAb S6.89.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
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