Cartilage-derived biomarkers and lipid mediators of inflammation in horses with osteochondritis dissecans of the distal intermediate ridge of the tibia

Janny C. de Grauw Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 12, 3584 CM, Utrecht, the Netherlands

Search for other papers by Janny C. de Grauw in
Current site
Google Scholar
PubMed
Close
 BVSc
,
Pieter A. Brama Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 12, 3584 CM, Utrecht, the Netherlands

Search for other papers by Pieter A. Brama in
Current site
Google Scholar
PubMed
Close
 DVM, PhD
,
Peter Wiemer Lingehoeve Equine Veterinary Clinic, Veldstraat 3a, 4033 AK, Lienden, the Netherlands

Search for other papers by Peter Wiemer in
Current site
Google Scholar
PubMed
Close
 DVM
,
Harold Brommer Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 12, 3584 CM, Utrecht, the Netherlands

Search for other papers by Harold Brommer in
Current site
Google Scholar
PubMed
Close
 DVM, PhD
,
Chris H. van de Lest Department of Biochemistry, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 12, 3584 CM, Utrecht, the Netherlands

Search for other papers by Chris H. van de Lest in
Current site
Google Scholar
PubMed
Close
 PhD
, and
P. Rene van Weeren Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 12, 3584 CM, Utrecht, the Netherlands

Search for other papers by P. Rene van Weeren in
Current site
Google Scholar
PubMed
Close
 DVM, PhD

Click on author name to view affiliation information

Abstract

Objective—To assess whether reported alterations in metabolism of cartilage matrix in young (0 to 24 months old) horses with osteochondritis dissecans (OCD) may also be found in older (24 to 48 months old) horses with clinical signs of OCD and to investigate the role of eicosanoids in initiating these clinical signs.

Sample Population—Synovial fluid was collected from 38 tarsocrural joints of 24 warmblood horses with (22 joints of 16 horses) or without (16 joints of 8 horses) clinical signs and a radiographic diagnosis of OCD of the distal intermediate ridge of the tibia.

Procedures—Turnover of type II collagen was investigated by use of specific immunoassays for synthesis (carboxypropeptide of type II collagen [CPII]) and degradation (collagenase-cleaved fragments of type II collagen [C2C]) products. Furthermore, glycosaminoglycan (GAG), leukotriene (LT) B4, cysteinyl LTs, and prostaglandin (PG) E2 concentrations were determined, and concentrations in joints with OCD were compared with those in joints without OCD.

Results—Concentrations of CPII, C2C, and GAG did not differ significantly between affected and nonaffected joints. Fluid from joints with OCD had significantly higher LTB4 and PGE2 concentrations than did fluids from nonaffected joints.

Conclusions and Clinical Relevance—Altered collagen or proteoglycan turnover was not detected in 24- to 48-month-old horses at the time they developed clinical signs of OCD of the distal intermediate ridge of the tibia. However, increased concentrations of LTB4 and PGE2 in fluid of joints with OCD implicate these mediators in the initiation of clinical signs of OCD.

Abstract

Objective—To assess whether reported alterations in metabolism of cartilage matrix in young (0 to 24 months old) horses with osteochondritis dissecans (OCD) may also be found in older (24 to 48 months old) horses with clinical signs of OCD and to investigate the role of eicosanoids in initiating these clinical signs.

Sample Population—Synovial fluid was collected from 38 tarsocrural joints of 24 warmblood horses with (22 joints of 16 horses) or without (16 joints of 8 horses) clinical signs and a radiographic diagnosis of OCD of the distal intermediate ridge of the tibia.

Procedures—Turnover of type II collagen was investigated by use of specific immunoassays for synthesis (carboxypropeptide of type II collagen [CPII]) and degradation (collagenase-cleaved fragments of type II collagen [C2C]) products. Furthermore, glycosaminoglycan (GAG), leukotriene (LT) B4, cysteinyl LTs, and prostaglandin (PG) E2 concentrations were determined, and concentrations in joints with OCD were compared with those in joints without OCD.

Results—Concentrations of CPII, C2C, and GAG did not differ significantly between affected and nonaffected joints. Fluid from joints with OCD had significantly higher LTB4 and PGE2 concentrations than did fluids from nonaffected joints.

Conclusions and Clinical Relevance—Altered collagen or proteoglycan turnover was not detected in 24- to 48-month-old horses at the time they developed clinical signs of OCD of the distal intermediate ridge of the tibia. However, increased concentrations of LTB4 and PGE2 in fluid of joints with OCD implicate these mediators in the initiation of clinical signs of OCD.

Osteochondrosis, which is also termed OCD when there are loose osteochondral fragments, is a major developmental joint disease in horses and can be defined as a disruption of the endochondral ossification process in the epiphyseal cartilage.1 Because this process is typically only active during the first year after birth, osteochondrotic lesions, by definition, will only develop during this period.2,3 The tarsocrural joint is one of the joints most commonly affected by osteochondrosis or OCD in warmblood horses,4,5 and lesions at this location often do not clinically manifest until later in life when training commences and young horses are first subjected to athletic challenges.6

In the tarsocrural joint, lesions characteristically develop at the cranial end of the intermediate ridge of the tibia as well as at the lateral trochlear ridge of the talus.7 In longitudinal experimental studies5,a conducted by our research group, the radiographic appearance of tarsocrural OCD lesions was highly dynamic in foals between 1 and 5 months of age, after which lesions remained permanent and radiographically stable until foals were 24 months old.

The underlying molecular changes in osteochondrotic cartilage have been the subject of many investigations. Studies8–13 involving markers of cartilage turnover in synovial fluid or serum have revealed significant differences in metabolism of collagen and proteoglycan between OCD-affected foals and healthy, age-matched control foals. Evidence11,13 points toward an early involvement of the collagen network in the pathogenesis of lesions (ie, during the first year after birth). However, the eventual clinical expression of OCD is believed to be the outcome of 2 consecutive events: the initial onset of lesions and the ensuing repair process.13 Little is known about cartilage metabolism in established OCD lesions after horses exceed 1 year of age. The fact that tarsocrural OCD lesions stabilize radiographically after horses are 5 months old, whereas clinical disease often does not become apparent until after horses are 24 months old, raises the issue as to whether affected joints may remain metabolically aberrant despite being radiographically stable and whether such metabolic aberrations may contribute to clinical manifestation of lesions.

Another poorly understood aspect of clinical expression of tarsocrural OCD is the pathophysiologic background of its most common signs (ie, prominent joint effusion with minimal disturbance of motion).7,14 Synovial membranes release vasoactive mediators, notably E-series PGs, when irritated by cartilage detritus and osteochondral fragments within the joint cavity,15–17 and this may partially account for the effusion. However, PGs are also believed to be intimately linked with joint pain and lameness in dogs and horses.18,19 Because lameness is an inconsistent finding in horses with OCD of the distal intermediate ridge of the tibia,7,14 we hypothesized that in clinically affected animals with joint effusion but no notable lameness, other inflammatory mediators may dominate to initiate the clinical signs. The LTs, which are also members of the eicosanoid family of inflammatory mediators, are potent vasoactive mediators that may be released by neutrophils and resident articular cells, such as synoviocytes, chondrocytes, and osteoblasts,20–22 but bear no direct relation to joint nociception. Leukotriene B4 and CysLTs are prime mediators of plasma extravasation and joint swelling in the stifle joints of guinea pigs23 and rats.24 So far, little is known about the involvement of the various LTs in joint disorders of horses, although LTB4 was considered a good indicator of experimentally induced synovitis in 1 study.25

In the study reported here, the underlying molecular mechanisms governing clinical signs of disease were examined in 24- to 48-month-old horses with OCD of the distal intermediate ridge of the tibia. The study was intended to serve 2 purposes. First, we investigated whether changes in metabolism of articular cartilage matrix, which have been reported in other studies8–13 in OCD-affected foals during the first year after birth, are also detectable in 24- to 48-month-old horses by the time that clinical signs become evident. Second, we tested the hypothesis that LTs (rather than PGs) may be involved in horses with joint effusion without associated lameness, which is typical of horses with OCD of the distal intermediate ridge of the tibia.

Materials and Methods

Sample population—Synovial fluid was collected from 38 tarsocrural joints of 24 warmblood horses. Samples of synovial fluid were obtained from 16 horses with joint effusion and OCD of the distal intermediate ridge of the tibia and from 8 clinically normal control horses. The study was approved by the Utrecht University Ethical Committee.

Samples from joints with OCD were obtained from 22 joints of 16 client-owned warmblood horses evaluated to determine the cause of prominent joint effusion of 1 or both tarsocrural joints. Because these were samples that would otherwise have been discarded during surgery, informed consent of the owners was not obtained. All selected horses were between 24 and 48 months of age at the time of examination. All horses were subjected to a full lameness examination, but lameness was not detected in any horse. Diagnosis of OCD of the tarsocrural joint was confirmed by standard radiographic examination. Only fluids from joints in which the OCD lesion was limited to the distal intermediate ridge of the tibia and that had no additional signs of degenerative changes at arthroscopy were included in the study.

Eight control horses, maintained as part of a university research herd, were matched on the basis of age with the OCD-affected horses. Each control horse underwent a full clinical lameness examination, and all were deemed free of musculoskeletal disease. Tarsocrural joints of control horses were considered free of OCD on the basis of examination of 3 standard radiographic views (dorsomedial-plantarolateral oblique, lateral-medial, and cranial-caudal) of each tarsocrural joint.

Sample collection—Horses with OCD of the distal intermediate ridge of the tibia were admitted for arthroscopic removal of fragments. Before surgical exploration of a joint, synovial fluid was aspirated into a sterile 20-mL syringe. Within 1 hour after collection, part of the sample was submitted for a cell count, whereas the remainder was transferred to polypropylene tubes and centrifuged at 10,000 Xg for 10 minutes. Synovial fluid was then divided into aliquots and stored at −80°C until further analysis.

For control horses, aseptic arthrocentesis of both tarsocrural joints was performed via a dorsomedial approach by use of an 18-gauge, 1.5-inch needle connected to a sterile 20-mL sterile syringe. When needed, a nose twitch or mild sedation achieved by the administration of detomidineb (0.01 mg/kg, IV) were used to ensure proper immobilization of horses during the procedure. Processing and storage of synovial fluid were conducted in accordance with the same protocol described for the samples from joints with OCD.

CPII assay—A commercially available competitive ELISAc for CPII was used. In the course of fibril formation, CPII is released into synovial fluid, where its concentration reflects synthesis of type II collagen.26 The assay included a bovine CPII standard and rabbit polyclonal capture antibody. Although this antibody was raised against human CPII, studies11,27 have established cross-reactivity as well as parallelism and effective spike recovery in samples obtained from horses.

A preliminary study was conducted to assess dilution and digestion of samples for optimal assay performance. The preliminary study revealed that digestion of synovial fluid with testicular hyaluronidased (0.05 mg/mL) for 30 minutes at 37°C, followed by a 1:1 dilution of the digested synovial fluid, resulted in values within the optimal concentration range for the assay (50 to 2,000 ng/mL). Absorbance was measured at 450 nm.

C2C assay—A commercially available competitive ELISAe was used for the detection of C2C. This assay included a mouse primary C2C antibody (formerly referred to as col2-3/4Clong) that detects the neoepitope located at the C terminus of the three-fourths length cleavage product of type II collagen fibrils. This antibody was raised against a synthetic peptide representing the neoepitope (used as the assay standard) and can successfully detect cleavage fragments carrying this neoepitope in synovial fluid obtained from dogs and humans.28–30 Because the structure of collagen type II and collagenase cleavage sites are highly conserved,30,31 there was ample reason to assume that the assay would be cross-reactive in samples obtained from horses.

Proper assay performance was confirmed in a preliminary study of equine synovial fluid. Linearity was assessed by assay of serial dilution of samples with known concentrations of C2C, which yielded a correlation coefficient of 0.99 between the measured and expected concentration at each dilution. In addition, effective spike recovery in synovial fluid was observed (mean recovery, 95.3%; range, 92.6% to 105.8%). This preliminary study further revealed that after digestion with testicular hyaluronidased (0.05 mg/mL) for 30 minutes at 37°C, no dilution of synovial fluid samples was necessary to yield values within the dynamic range of the assay (1 μg/mL to 10 ng/mL). Absorbance was measured at 450 nm.

GAG assay—Proteoglycan content of synovial fluid was estimated by measuring sulfated GAG concentrations by use of the 1,9-dimethylmethylene blue metachromatic dyeassay,32 which was modified for use in microtiter plates. Eighty microliters of buffered hyaluronidase (500 μg of testicular hyaluronidased/mL in 50mM sodium acetate [pH, 5.2]) was added to 20 μL of synovial fluid; the mixture was allowed to digest for 30 minutes at 37°C. Then, 40 μL of the mixture was transferred to a well of a microtiter plate, and 200 μL of Farndale reagent (46μM 1,9-dimethylmethylene blue,f 40mM glycine, and 42mM NaCl, adjusted to pH 3.0 by the addition of hydrogen chloride) was added. The plate was incubated for 15 minutes, and absorbance was then measured at 525 nm. Shark cartilage chondroitin sulfateg served as the assay standard.

Eicosanoid immunoassays—Commercial competitive ELISA kits were used to determine synovial fluid concentrations of LTB4,h CysLTs,i and PGE2,j. Each kit was used in accordance with the manufacturer's instructions. The kits used rabbit polyclonal antibodies raised against the synthetic eicosanoids. Although these kits were intended for use in human samples, there are no species differences in the structure of arachidonic acid derivatives among species, so the assays can also be applied to samples obtained from horses.33

Samples were extracted before analysis by use of the immunoassays. In brief, 200 μL of synovial fluid and 10 μL of testicular hyaluronidased (10 mg/mL) were allowed to digest for 30 minutes at 37°C. Then, 100 μL of 0.1% formic acid was added, and the solution was vortexed, after which samples were centrifuged for 10 minutes at 13,000 Xg. Supernatant was harvested and applied to an RP-18 extraction columnk that had been conditioned with 1 mL of acetone followed by 1 mL of water. The column was then flushed with 1 mL of water, 1 mL of 5% ethanol, and 2 applications of hexane (1 mL of hexane/application). Eicosanoids were eluted from the column by use of 500 μL of ethylacetate, dried under a stream of nitrogen gas, and stored under nitrogen at −80°C until analysis.

All samples were analyzed within 1 week after sample extraction. At the time of assay, samples were reconstituted by the addition of 250 μL of assay buffer and analyzed in accordance with the manufacturer's instructions. Absorbance was measured at 405 nm, with a reference wavelength of 595 nm. Lower limit of detection of the assay was 19.4 pg/mL for LTB4, 26.6 pg/mL for CysLTs, and 8.25 pg/mL for PGE2.

Statistical analysis—Joints were grouped on the basis of lesions into OCD (22 joints) and control (16 joints) groups. Data were assessed for normality of distribution, and differences in synovial fluid variables between groups were tested by use of an unpaired t test. When data from the 2 groups failed the Levene test for equality of variance, unequal variances were assumed in statistical testing (Welch correction). Computer softwarel was used, and a value of P < 0.05 was considered significant for all statistical analyses.

Results

Animals—Mean age of horses for the OCD and control groups was 37.6 months (range, 24 to 47.4 months) and 37.8 months (range, 25 to 47 months), respectively. All horses in the OCD group had grade 4 radiographic OCD lesions5 on the distal intermediate ridge of the tibia.

Analysis of synovial fluid—All data for the variables were normally distributed. Hence, no data transformations were performed.

WBC counts—Mean ± SEM WBC counts did not differ significantly between synovial fluid obtained from joints with OCD (0.73 × 109 ± 0.03 × 109 cells/L) and normal joints (0.71 × 109 ± 0.05 × 109 cells/L; Table 1).

Table 1—

Mean ± SEM concentrations of cartilage-derived markers and eicosanoids in synovial fluid obtained from 22 OCD-affected and 16 normal (control) tarsocrural joints of horses.

VariableControl jointsOCD-affected joints
CPII (ng/mL)528.0 ± 29.5740.2 ± 135.5
C2C (ng/mL)170.9 ± 8.5192.8 ± 10.2
GAG (μg/mL)13.9 ± 2.112.8 ± 1.5
CysLT (pg/mL)416.9 ± 38.3400.5 ± 20.6
LTB4 (pg/mL)568.9 ± 35.9934.5 ± 60.0*
PGE2 (pg/mL)127.3 ± 33.6362.3 ± 93.6
WBCs (× 109 cells/L)0.71 ± 0.050.73 ± 0.03

Means differed significantly (P < 0.001) between groups.

Means and variances differed significantly (means, P < 0.001; variances, P < 0.05) between groups.

Markers of metabolism of cartilage matrix—No significant differences in CPII (type II collagen synthesis marker), C2C (type II collagen degradation marker), and GAG (estimate of proteoglycan degradation) concentrations were found between synovial fluid obtained from OCD-affected joints and normal tarsocrural joints (Table 1).

Eicosanoid concentrations—A significant (P < 0.001) increase in the LTB4 concentration was found in synovial fluid obtained from OCD-affected joints, compared with concentrations in synovial fluid obtained from control joints of age-matched horses (Figure 1). Mean ± SEM value for the OCD-affected horses (934.5 ± 60.0 pg/mL) was nearly twice that for the control horses (568.9 ± 35.9 pg/mL). Similarly, mean PGE2 concentration for the OCD-affected horses (362.3 ± 93.6 pg/mL) was significantly higher than the concentration for the control horses (127.3 ± 33.6 pg/mL; Figure 2). Concentrations of LTB4 and PGE2 had significantly (P = 0.005 and P < 0.001, respectively) greater variance for the OCD-affected horses than for the control horses. Concentrations of CysLTs were detectable in all synovial fluid samples, but we did not detect significant differences between mean concentrations for OCD-affected (400.4 ± 20.6 pg/mL) and control (416.9 ± 38.3 pg/mL) joints.

Figure 1—
Figure 1—

Mean ± SEM LTB4 concentrations in synovial fluid obtained from 22 OCD-affected and 16 normal (control) tarsocrural joints of horses. The mean value differed significantly (P < 0.001) between the groups.

Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1156

Figure 2—
Figure 2—

Mean ± SEM PGE2 concentrations in synovial fluid obtained from 22 OCD-affected and 16 normal (control) tarsocrural joints of horses. The mean value differed significantly (P < 0.05) between the groups.

Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1156

Discussion

Osteochondrosis and OCD affect the young of numerous species, including humans,34 and the underlying molecular mechanisms of OCD have been the subject of many scientific investigations. Alterations in turnover of articular cartilage matrix have been reported8–13 for foals with OCD, and these changes have been associated with pathogenesis of the disease condition.

It has been asserted13 that because lesions only develop within the first year after birth, studies of OCD in older animals may reflect only the secondary reparative processes. However, evidence5,a has revealed that most radiographic lesions in foals heal uneventfully within the first year after birth. Hence, it would be of value to know whether cartilage metabolism in horses that do not have proper healing of defects and subsequently develop clinical disease differ from those of healthy age-matched control horses.

Alterations in the collagen network and, in particular, changes in metabolism of type II collagen have been implicated in the OCD disease process.11–13 In the young horses reported here, we found no differences in synthesis of type II collagen, on the basis of synovial fluid concentrations of CPII, between OCD-affected and normal joints. Absolute concentrations of CPII in the synovial fluid reported here are 10-fold higher than those for horses with OCD reported elsewhere,11,27 which may reflect differences in methods used in the various studies. Samples from OCD-affected and control horses were treated identically, so any such discrepancies in methods would have affected both groups and would therefore not have influenced the final outcome of our study.

The lack of differences in CPII concentrations between synovial fluid obtained from horses with OCD and healthy control horses in our study is in close agreement with results from another study.11 In that study, investigators found increased concentrations of CPII in synovial fluid obtained from young (9 to 18 months old) horses with OCD, compared with concentrations in synovial fluid of control horses, but they found no such difference between similar groups of older (24 to 48 months old) horses. Analysis of results from that study and the study reported here suggests that the enhanced synthesis of type II collagen seen in young OCD-affected horses subsides as the horses mature, with the rate of synthesis then being similar in OCD-affected and normal tarsocrural joints. This phenomenon may be related to the typical age-related decrease in metabolic activity of chondrocytes as horses mature35,36 and indicates that, in this respect, osteochondrotic joints in older horses behave similarly to normal joints.

Enhanced degradation of type II collagen has been reported12 in a younger (7 to 12 months old) group of OCD-affected horses, compared with results for healthy, age-matched control foals. In the older horses in the study reported here, C2C concentrations in synovial fluid of OCD-affected and normal joints did not differ significantly, with type II collagen apparently being degraded at comparable rates in both groups of tarsocrural joints. This suggests that the enhancement of collagen degradation in joints with OCD during the development of lesions subsides at a later age.

Similar to our findings for collagen metabolism, synovial fluid concentrations of GAG in the study reported here were independent of OCD status of the joint from which the sample was collected and corresponded closely with concentrations for normal tarsocrural joints obtained in other studies.37,38 There are contradictory results with regard to aberrations in proteoglycan metabolism in osteochondrotic horses. Investigators in 1 study8 found low GAG contents and altered proteoglycan composition of OCD-affected cartilage of the distal intermediate ridge of the tibia, and investigators in an in vivo study39 found significant increases in synovial fluid concentrations of GAGs in OCD-affected horses. In 2 other studies,10,12 investigators found no difference in GAG release from osteochondrotic or normal cartilage explants. Because the aforementioned in vivo study39 does not provide information about the age of the horses or whether there were degenerative changes in the osteochondrotic joints, it is hard to compare results from that study and the study reported here. Investigators in yet another study11 found a decrease in synthesis of proteoglycans in 9- to 18-month-old horses with OCD of the tarsocrural joint but not in 24- to 48-month-old horses with OCD of the tarsocrural joint, which suggests that this aspect of proteoglycan metabolism was no longer aberrant by the time horses were > 2 years old, and this is in agreement with data for proteoglycan degradation reported here.

Analysis of our results for concentrations of metabolic markers in synovial fluid points toward a stabilization in turnover of type II collagen as well as proteoglycan degradation in OCD-affected horses at 24 to 48 months of age. This supports the idea that OCD lesions in the tarsocrural joint become both radiographically5,a and metabolically stable over time.

Lack of alterations in concentrations of markers of cartilage matrix metabolism at the time of clinical manifestation reinforces the idea that other factors are involved, with synovitis appearing to be a likely candidate. However, synovial inflammation as a cause of clinical signs of OCD has received relatively little attention from researchers.19

In 1 study19 on the involvement of PGE2 in joint disease of horses, the authors included a subgroup of 8 horses with OCD. In that study, no increase in PGE2 concentrations was detected in synovial fluid from OCD-affected joints, compared with concentrations in normal joints. The authors conjectured that because OCD is commonly associated with minimal lameness, it could be expected that PGE2 concentrations would be low in OCD-affected joints. Unfortunately, no information was supplied in their report on the type of joints from which samples were obtained or the exact location of lesions within affected joints. The study reported here involved 22 narrowly defined clinical cases of OCD of the distal intermediate ridge of the tibia. We detected a significant increase in PGE2 concentration in synovial fluid obtained from OCD-affected joints, despite the fact that none of the horses were lame. In accordance with our findings, investigators in another study40 also detected higher concentrations of PGE2 in synovial fluid collected from all 7 horses affected by OCD in their study; however, they partially explained this by reference to the more clinically active nature of the disease in their horses, all of which were lame at the time of sample collection.

Our finding of increased PGE2 concentrations in synovial fluid of OCD-affected joints despite the fact that horses were not lame raises questions about the supposed intimate link between joint pain and concentrations of PGE2 in synovial fluid.18,41,42 Although the high PGE2 concentrations in OCD-affected joints are perhaps unexpected with regard to the lack of lameness, they are not illogical. Bone fragments cause release of PGE2 from equine synoviocytes,16 and PGE2 can influence the diameter and permeability of synovial vessels, thus contributing to joint effusion.42 Because joint effusion was the primary clinical sign in the horses of our report, it may have been expected that we would detect increased PGE2 concentrations.

The actions of LTs, another member of the eicosanoid group of inflammatory mediators, have been relatively overlooked in joint disease.20,24 Synoviocytes and subchondral osteoblasts are both capable of production and release of LTs in response to various stimuli,20,43 whereas chondrocytes are believed to release LTs following interactions with granulocytes.22 Increased concentrations of LTB4 in synovial fluid have been associated with arthritic conditions42,44,45 and can be significantly correlated with severity of synovitis in equids.26 In the study reported here, a significant (P < 0.001) increase in LTB4 concentrations was found in synovial fluid obtained from osteochondrotic joints, compared with concentrations in control joints.

Although increases in LTB4 concentrations in synovial fluid reportedly correlate with increases in WBCs,25,33 this was not the situation in our study. The WBC counts were comparably low for both the OCD-affected and control joints. It is hard to explain the reason that increases in LTB4 concentrations in synovial fluid were not accompanied by increased infiltration by polymorphonuclear neutrophils in these joints. Possibly, the powerful chemotactic actions of LTB4 in joints with infectious, rheumatoid, or chemically induced arthritis (conditions in which many more mediators are liberated that may contribute to leukocyte attraction into the joint46) may not be as pronounced in the relatively calm OCD-affected joints. Alternatively, the LTB4 concentrations found in our study may have been too low to cause a substantial influx of leukocytes into the joint cavity, given that picomolar or nanomolar concentrations of LTB4 are needed in humans to induce an influx, and equine leukocytes seem less responsive to LTB4 than their human counterparts.47,48 Even so, an in vivo study49 of humans with rheumatoid arthritis also did not yield consistent correlations between LTB4 concentrations and WBC counts,49 despite the known chemotactic properties of LTB4 and leukocyte involvement in this disease.

Because leukocytes are apparently not the major source of LTB4 in OCD, 2 other likely candidates remain (ie, the synovial membrane and subchondral bone). The synovial membrane is likely to be irritated by cartilage and bone detritus in animals with OCD, and synoviocytes are believed to be major contributors to eicosanoid concentrations in synovial fluid.50 Interestingly, osteoblasts in subchondral bone can also be apt producers of LTB4 in vivo,50–52 and in the subchondral bone compartment, LTB4 is an important regulator of the extracellular matrix.51 Because in osteochondrosis, especially the OCD form, the subchondral bone is exposed and may communicate with the synovial cavity, we speculate that the increased concentrations of LTB4 in synovial fluid may partially originate from subchondral bone. The finding of increased LTB4 concentrations in osteochondrotic joints of older horses raises the issue of whether LTB4 is also involved in the earlier stages of the disease. In this respect, it is interesting to mention that LTB4 can stimulate osteoclast differentiation and bone resorption51 and affect bone remodeling during endochondral ossification.14,53 Moreover, LTB4 can cause upregulation of cartilage catabolic factors, such as matrix metalloproteinases,20 and could thus hypothetically play a role in the disturbance of normal chondrocyte maturation in animals with OCD. Additional investigation of the involvement of LTB4 in osteochondrosis in horses, especially young horses in which lesions are developing, would seem warranted.

To our knowledge, the study reported here is the first to provide concentrations of CysLTs in synovial fluids obtained from horses. Although CysLTs were detectable in substantial amounts in samples from all osteochondrotic joints, there were no significant differences from concentrations detected in control joints. Hence, despite the fact that CysLTs are involved in synovial effusion in rats,24 it appears that they are unlikely to be involved in the synovial effusion seen in horses with OCD.

The usual caveats associated with analysis of mediator or marker concentrations in synovial fluid should be considered when interpreting results from the study reported here. Most importantly, the difficulty in comparing concentrations of analytes between joints with and without prominent joint effusion should be appreciated. In addition, concentrations of metabolic markers only allow indirect investigation of tissue metabolism. Many factors other than those involved with actual rate of synthesis or degradation of matrix components may affect synovial clearance rates and thus marker concentrations in synovial fluid. Among these, the amount of active inflammation may be an important determinant,54 which could not be corrected for.

Analysis of our findings supports the idea that alterations in cartilage metabolism cannot account for the observed clinical signs in horses between 24 and 48 months of age with OCD of the distal intermediate ridge of the tibia. This study extends the findings from radiographic studies,5,a which revealed that tarsocrural OCD lesions remained radiographically stable in affected horses from 5 to 24 months of age. We can conclude that in addition to being radiographically stable, these lesions also appear to be metabolically stable. Furthermore, we established the local involvement of various eicosanoids in joints of horses with OCD of the distal intermediate ridge of the tibia and found that PGE2 and LTB4, but not CysLTs, are likely to play a role in the prominent joint effusion that is the most common clinical sign.

ABBREVIATIONS

OCD

Osteochondritis dissecans

PG

Prostaglandin

LT

Leukotriene

CysLT

Cysteinyl LT

CPII

Carboxypropeptide of type II collagen

C2C

Collagenase-cleaved fragments of type II collagen

GAG

Glycosaminoglycan

a.

Enzerink E, Dik KJ, Knaap J, et al. Radiographic development of lesions in hock and stifle in a group of Dutch Warmblood horses from 1 – 24 months of age (abstr), in Proceedings. Br Equine Vet Assoc 2000;39:195.

b.

Domosedan, Orion Corp, Espoo, Finland.

c.

CPII immunoassay, IBEX, Montreal, QC, Canada.

d.

Testicular hyaluronidase, Sigma Chemical Co, St Louis, Mo.

e.

C2C immunoassay, IBEX, Montreal, QC, Canada.

f.

DMMB, Sigma Chemical Co, St Louis, Mo.

g.

Chondroitin-6-Sulfate, Sigma Chemical Co, St Louis, Mo.

h.

LTB4 ELISA, RnDsystems, Minneapolis, Minn.

i.

Cysteinyl leukotriene ELISA, RnDsystems, Minneapolis, Minn.

j.

PGE2 high sensitivity ELISA, RnDsystems, Minneapolis, Minn.

k.

LiChrolut RP-18, Merck, Darmstadt, Germany.

l.

SPSS, version 11.0.1, SPSS Inc, Chicago, Ill.

References

  • 1

    Rejnö S, Strömberg B. Osteochondrosis in the horse II. Pathology. Acta Radiol Suppl 1978;358:153178.

  • 2

    Carlson CS, Cullins LD, Meuten DJ. Osteochondrosis of the articular-epiphyseal cartilage complex in young horses: evidence for a defect in cartilage canal blood supply. Vet Pathol 1995;32:641647.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3

    McIlwraith CW, ed. AQHA developmental orthopaedic disease symposium. Amarillo, Tex: American Quarter Horse Association, 1986;177.

  • 4

    van Weeren PR, Barneveld A. The effect of exercise on the distribution and manifestation of osteochondrotic lesions in the Warmblood foal. Equine Vet J Suppl 1999;31:1625.

    • Search Google Scholar
    • Export Citation
  • 5

    Dik KJ, Enzerink E, van Weeren PR. Radiographic development of osteochondral abnormalities, in the hock and stifle of Dutch Warmblood foals, from age 1 to 11 months. Equine Vet J Suppl 1999;31:915.

    • Search Google Scholar
    • Export Citation
  • 6

    McIlwraith CW. Clinical aspects of osteochondritis dissecans. In: McIlwraith CW, Trotter GW, eds. Joint disease in the horse. Philadelphia: WB Saunders Co, 1996;369374.

    • Search Google Scholar
    • Export Citation
  • 7

    McIlwraith CW, Foerner JJ, Davis DM. Osteochondritis dissecans of the tarsocrural joint: results of treatment with arthroscopic surgery. Equine Vet J 1991;23:155162.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8

    Lillich JD, Bertone AL & Malemud CJ, et al. Biochemical, histochemical, and immunohistochemical characterization of distal tibial osteochondrosis in horses. Am J Vet Res 1997;58:8998.

    • Search Google Scholar
    • Export Citation
  • 9

    Billinghurst RC, Brama PA & van Weeren PR, et al. Evaluation of serum concentrations of biomarkers of skeletal metabolism and results of radiography as indicators of severity of osteochondrosis in foals. Am J Vet Res 2004;65:143150.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10

    van den Hoogen BM, van de Lest CH & van Weeren PR, et al. Changes in proteoglycan metabolism in osteochondrotic articular cartilage of growing foals. Equine Vet J Suppl 1999;31:3844.

    • Search Google Scholar
    • Export Citation
  • 11

    Laverty S, Ionescu M & Marcoux M, et al. Alterations in cartilage type-II procollagen and aggrecan contents in synovial fluid in equine osteochondrosis. J Orthop Res 2000;18:399405.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12

    Laverty S, Okouneff S & Ionescu M, et al. Excessive degradation of type II collagen in articular cartilage in equine osteochondrosis. J Orthop Res 2002;20:12821289.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13

    van de Lest CH, Brama PA & van El B, et al. Extracellular matrix changes in early osteochondrotic defects in foals: a key role for collagen? Biochim Biophys Acta 2004;1690:5462.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14

    Hurtig MB, Pool RR. Pathogenesis of equine osteochondrosis. In: McIlwraith CW, Trotter GW, eds. Joint disease in the horse. Philadelphia: WB Saunders Co, 1996;362383.

    • Search Google Scholar
    • Export Citation
  • 15

    Kidd JA, Fuller CJ, Barr ARS. Osteoarthritis in the horse. Equine Vet Educ 2001;13:160168.

  • 16

    May SA, Hooke RE, Lees P. Bone fragments stimulate equine synovial lining cells to produce the inflammatory mediator prostaglandin E2. Equine Vet J Suppl 1988;6:131132.

    • Search Google Scholar
    • Export Citation
  • 17

    May SA, Hooke RE, Lees P. Identity of the E-series prostaglandin produced by equine chondrocytes and synovial cells in response to a variety of stimuli. Res Vet Sci 1989;46:5457.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18

    Trumble TN, Billinghurst RC, McIlwraith CW. Correlation of prostaglandin E2 concentrations in synovial fluid with ground reaction forces and clinical variables for pain or inflammation in dogs with osteoarthritis induced by transection of the cranial cruciate ligament. Am J Vet Res 2004;65:12691275.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19

    May SA, Hooke RE, Lees P. Prostaglandin E2 in equine joint disease. Vlaamsch Diergeneeskundig Tijdschrift 1994;63:187191.

  • 20

    Martel-Pelletier J, Mineau F & Fahmi H, et al. Regulation of the expression of 5-lipoxygenase-activating protein/5-lipoxygenase and the synthesis of leukotriene B(4) in osteoarthritic chondrocytes: role of transforming growth factor beta and eicosanoids. Arthritis Rheum 2004;50:39253933.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21

    Fermor B, Haribabu B & Weinberg JB, et al. Mechanical stress and nitric oxide influence leukotriene production in cartilage. Biochem Biophys Res Commun 2001;285:806810.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22

    Amat M, Diaz C, Vila L. Leukotriene A4 hydrolase and leukotriene C4 synthase activities in human chondrocytes: transcellular biosynthesis of leukotrienes during granulocyte-chondrocyte interaction. Arthritis Rheum 1998;41:16451651.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23

    Kuwabara K, Jyoyama H & Fleisch JH, et al. Inhibition of antigen-induced arthritis in guinea pigs by a selective LTB4 receptor antagonist LY293111Na. Inflamm Res 2002;51:541550.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24

    Wang Y, Mitchell J & Sharma M, et al. Leukotrienes mediate 5-hydroxytryptamine-induced plasma extravasation in the rat knee joint via CysLT-type receptors. Inflamm Res 2004;53:6671.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25

    Bertone AL, Palmer JL, Jones J. Synovial fluid inflammatory mediators as markers of equine synovitis. Vet Surg 1993;22:372373.

  • 26

    Nelson F, Dahlberg L & Laverty S, et al. Evidence for altered synthesis of type II collagen in patients with osteoarthritis. J Clin Invest 1998;102:21152125.

  • 27

    Frisbie DD, Ray CS & Ionescu M, et al. Measurement of synovial fluid and serum concentrations of the 846 epitope of chondroitin sulfate and of carboxy propeptides of type II procollagen for diagnosis of osteochondral fragmentation in horses. Am J Vet Res 1999;60:306309.

    • Search Google Scholar
    • Export Citation
  • 28

    Matyas JR, Atley L & Ionescu M, et al. Analysis of cartilage biomarkers in the early phases of canine experimental osteoarthritis. Arthritis Rheum 2004;50:543552.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29

    Fraser A, Fearon U & Billinghurst RC, et al. Turnover of type II collagen and aggrecan in cartilage matrix at the onset of inflammatory arthritis in humans: relationship to mediators of systemic and local inflammation. Arthritis Rheum 2003;48:30853095.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30

    Chu Q, Lopez M & Hayashi K, et al. Elevation of a collagenase generated type II collagen neoepitope and proteoglycan epitopes in synovial fluid following induction of joint instability in the dog. Osteoarthritis Cartilage 2002;10:662669.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31

    Bramono DS, Richmond JC & Weitzel PP, et al. Matrix metalloproteinases and their clinical applications in orthopaedics. Clin Orthop 2004;428:272285.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32

    Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 1986;883:173177.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33

    Bertone AL, Palmer JL, Jones J. Synovial fluid cytokines and eicosanoids as markers of joint disease in horses. Vet Surg 2001;30:528538.

  • 34

    Ekman S, Carlson CS. The pathophysiology of osteochondrosis. Vet Clin North Am Small Anim Pract 1998;28:1732.

  • 35

    Brama PA, TeKoppele JM & Beekman B, et al. Matrix metalloproteinase activity in equine synovial fluid: influence of age, osteoarthritis, and osteochondrosis. Ann Rheum Dis 1998;57:697699.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36

    Brama PA, van den Boom R & DeGroott J, et al. Collagenase1 (MMP-1) activity in equine synovial fluid: influence of age, joint pathology, exercise and repeated arthrocentesis. Equine Vet J 2004;36:3440.

    • Search Google Scholar
    • Export Citation
  • 37

    van den Boom R, Brama PA & Kiers GH, et al. Assessment of the effects of age and joint disease on hydroxyproline and glycosaminoglycan concentrations in synovial fluid from the metacarpophalangeal joint of horses. Am J Vet Res 2004;65:296302.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38

    Palmer JL, Bertone AL, McClain H. Assessment of glycosaminoglycan concentration in equine synovial fluid as a marker of joint disease. Can J Vet Res 1995;59:205212.

    • Search Google Scholar
    • Export Citation
  • 39

    Alwan WH, Carter SD & Bennett D, et al. Glycosaminoglycans in horses with osteoarthritis. Equine Vet J 1991;23:4447.

  • 40

    Kirker-Head CA, Chandna VK & Agarwal RK, et al. Concentrations of substance P and prostaglandin E2 in synovial fluid of normal and abnormal joints of horses. Am J Vet Res 2000;61:714718.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41

    Moses VS, Hardy J & Bertone AL, et al. Effects of anti-inflammatory drugs on lipopolysaccharide-challenged and -unchallenged equine synovial explants. Am J Vet Res 2001;62:5460.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42

    Owens JG, Kamerling SG & Stanton SR, et al. Effects of pretreatment with ketoprofen and phenylbutazone on experimentally induced synovitis in horses. Am J Vet Res 1996;57:866874.

    • Search Google Scholar
    • Export Citation
  • 43

    Tawara T, Shingu M & Nobunaga M, et al. Effects of recombinant human IL-1 beta on production of prostaglandin E2, leukotriene B4, NAG, and superoxide by human synovial cells and chondrocytes. Inflammation 1991;15:145157.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44

    Nishimura M, Segami N & Kaneyama K, et al. Relationships between pain-related mediators and both synovitis and joint pain in patients with internal derangements and osteoarthritis of the temporomandibular joint. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:328332.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45

    Tsuji F, Oki K & Fujisawa K, et al. Involvement of leukotriene B4 in arthritis models. Life Sci 1999;64:PL51PL56.

  • 46

    Leirisalo-Repo M. The present knowledge of the inflammatory process and the inflammatory mediators. Pharmacol Toxicol 1994;75(suppl 2):13.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 47

    Tarlowe MH, Kannan KB & Itagaki K, et al. Inflammatory chemoreceptor cross-talk suppresses leukotriene B4 receptor 1-mediated neutrophil calcium mobilization and chemotaxis after trauma. J Immunol 2003;171:20662073.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48

    Potter KA, Leid RW & Kolattukudy PE, et al. Stimulation of equine eosinophil migration by hydroxyacid metabolites of arachidonic acid. Am J Pathol 1985;121:361368.

    • Search Google Scholar
    • Export Citation
  • 49

    Seppala E, Nissila M & Isomaki H, et al. Effects of nonsteroidal anti-inflammatory drugs and prednisolone on synovial fluid white cells, prostaglandin E2, leukotriene B4 and cyclic AMP in patients with rheumatoid arthritis. Scand J Rheumatol 1990;19:7175.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 50

    Wittenberg RH, Willburger RE & Kleemeyer KS, et al. In vitro release of prostaglandins and leukotrienes from synovial tissue, cartilage, and bone in degenerative joint diseases. Arthritis Rheum 1993;36:14441450.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51

    Lajeunesse D, Reboul P. Subchondral bone in osteoarthritis: a biologic link with articular cartilage leading to abnormal remodeling. Curr Opin Rheumatol 2003;15:628633.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 52

    Paredes Y, Massicotte F & Pelletier JP, et al. Study of the role of leukotriene B4 in abnormal function of human subchondral osteoarthritis osteoblasts: effects of cyclooxygenase and/or 5-lipoxygenase inhibition. Arthritis Rheum 2002;46:18041812.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 53

    Meghji S, Sandy JR & Harvey W, et al. Stimulation of bone collagen and non-collagenous protein synthesis by products of 5and 12-lipoxygenase: determination by use of a simple quantitative assay. Bone Miner 1992;18:119132.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 54

    Lohmander LS, Atley LM & Pietka TA, et al. The release of crosslinked peptides from type II collagen into human synovial fluid is increased soon after joint injury and in osteoarthritis. Arthritis Rheum 2003;48:31303139.

    • Crossref
    • Search Google Scholar
    • Export Citation
All Time Past Year Past 30 Days
Abstract Views 73 0 0
Full Text Views 614 445 135
PDF Downloads 240 102 13
Advertisement