• View in gallery
    Figure 1—

    Cytokine mRNA expression in SF cells obtained from stifle joints with CrCL rupture (CrCL-R) or MPL and normal control stifle joints (Con). In the ratio, the absolute amount of cytokine mRNA expression was calculated from the relative amount of cytokine and total amount of RNA in the sample by use of the following equation: absolute amount = (relative amount × total RNA)/0.3. Each symbol represents results for 1 joint; values < 0 represent no expression.

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    Figure 2—

    Mean ± SD IL-8 mRNA expression in SF cells obtained from the contralateral stifle joints of dogs with unilateral CrCL rupture at 6 and 12 months after reconstructive surgery to repair the initially affected stifle joint. Results are reported for contralateral stifle joints that sustained rupture of the CrCL by 12 months (diagonal-striped bars) and joints that did not sustain rupture of the CrCL of the contralateral stifle joint during the study (crosshatched bars).

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Cytokine mRNA expression in synovial fluid of affected and contralateral stifle joints and the left shoulder joint in dogs with unilateral disease of the stifle joint

Tanya de BruinDepartment of Diagnostic Imaging of Domestic Animals, Faculty of Veterinary Medicine, University of Ghent, Salisburylaan 133, 9820 Merelbeke, Belgium.

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Hilde de RoosterDepartment of Small Animal Medicine and Clinical Biology, Faculty of Veterinary Medicine, University of Ghent, Salisburylaan 133, 9820 Merelbeke, Belgium.

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Henri van BreeDepartment of Diagnostic Imaging of Domestic Animals, Faculty of Veterinary Medicine, University of Ghent, Salisburylaan 133, 9820 Merelbeke, Belgium.

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Luc DuchateauDepartment of Physiology and Biometrics, Faculty of Veterinary Medicine, University of Ghent, Salisburylaan 133, 9820 Merelbeke, Belgium.

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Eric CoxLaboratory of Veterinary Immunology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, University of Ghent, Salisburylaan 133, 9820 Merelbeke, Belgium.

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Abstract

Objective—To examine mRNA expression of cytokines in synovial fluid (SF) cells from dogs with cranial cruciate ligament (CrCL) rupture and medial patellar luxation (MPL) and determine mRNA expression for 3 joints (affected stifle, unaffected contralateral stifle, and left shoulder joints) in dogs with unilateral CrCL rupture.

Sample Population—29 stifle joints with CrCL rupture (29 dogs), 8 stifle joints with MPL (7 dogs), and 24 normal stifle joints (16 clinically normal dogs).

Procedures—Immediately before reconstructive surgery, SF was aspirated from the cruciate-deficient stifle joint or stifle joint with MPL. Fourteen of 29 dogs had unilateral CrCL rupture; SF was also aspirated from the unaffected contralateral stifle joint and left shoulder joint. Those 14 dogs were examined 6 and 12 months after reconstructive surgery. Total RNA was extracted from SF cells and reverse transcription–PCR assay was performed to obtain cDNA. Canine-specific cytokine mRNA expression was determined by use of a real-time PCR assay.

Results—Interleukin (IL)-8 and -10 and interferon-G expression differed significantly between dogs with arthropathies and dogs with normal stifle joints. For the 14 dogs with unilateral CrCL rupture, a significant difference was found for IL-8 expression. Before reconstructive surgery, IL-8 expression differed significantly between the affected stifle joint and left shoulder joint or contralateral stifle joint. Six months after surgery, IL-8 expression was significantly increased in the unaffected contralateral stifle joint, compared with the shoulder joint.

Conclusions and Clinical Relevance—No conclusions can be made regarding the role of the examined cytokines in initiation of CrCL disease.

Abstract

Objective—To examine mRNA expression of cytokines in synovial fluid (SF) cells from dogs with cranial cruciate ligament (CrCL) rupture and medial patellar luxation (MPL) and determine mRNA expression for 3 joints (affected stifle, unaffected contralateral stifle, and left shoulder joints) in dogs with unilateral CrCL rupture.

Sample Population—29 stifle joints with CrCL rupture (29 dogs), 8 stifle joints with MPL (7 dogs), and 24 normal stifle joints (16 clinically normal dogs).

Procedures—Immediately before reconstructive surgery, SF was aspirated from the cruciate-deficient stifle joint or stifle joint with MPL. Fourteen of 29 dogs had unilateral CrCL rupture; SF was also aspirated from the unaffected contralateral stifle joint and left shoulder joint. Those 14 dogs were examined 6 and 12 months after reconstructive surgery. Total RNA was extracted from SF cells and reverse transcription–PCR assay was performed to obtain cDNA. Canine-specific cytokine mRNA expression was determined by use of a real-time PCR assay.

Results—Interleukin (IL)-8 and -10 and interferon-G expression differed significantly between dogs with arthropathies and dogs with normal stifle joints. For the 14 dogs with unilateral CrCL rupture, a significant difference was found for IL-8 expression. Before reconstructive surgery, IL-8 expression differed significantly between the affected stifle joint and left shoulder joint or contralateral stifle joint. Six months after surgery, IL-8 expression was significantly increased in the unaffected contralateral stifle joint, compared with the shoulder joint.

Conclusions and Clinical Relevance—No conclusions can be made regarding the role of the examined cytokines in initiation of CrCL disease.

Rupture of the CrCL is one of the most common causes of hind limb lameness in dogs.1 Most dogs damage the CrCL during typical daily physical activities as a result of ligament deterioration.2–4 The exact etiopathogenesis of this CrCL rupture remains unknown despite extensive research. Degenerative changes within the microstructure of the ligament have been associated with loss of elasticity that result from increasing age, lack of exercise, chronic abnormal loading as a result of skeletal abnormalities, breed predisposition, obesity, sex predisposition, and immune-mediated inflammation.2,5–12

Rupture of the CrCL has been considered a noninflammatory joint disease caused by biomechanical instability because there is a lack of classical cytologic indicators of inflammation. However, studies13–16 have revealed an inflammatory process in the pathogenesis of CrCL rupture. Evidence of an inflammatory process is reflected in many of the clinical signs of cruciate disease, including swelling of the affected stifle joint, an increase in joint effusion, a slight increase in total cell numbers in the SF, and joint stiffness.17,18 Tearing of the CrCL could initiate an inflammatory process with the production of inflammatory cytokines, chemokines, nitric oxide, and prostaglandins as well as destructive enzymes. These factors and enzymes can further degrade the torn ligament.19 During this process, reexposed collagen particles could stimulate the immune system, which could result in an immune response that would increase inflammation and cause a self-perpetuating cycle of chronic inflammation and loss of joint integrity.20,21

The synovial tissue of cruciate-ligament–deficient stifle joints has signs of chronic inflammation, including hyper-plasia of the synovial lining with moderate infiltration of inflammatory cells into the sublining tissue.13,15,16,22 The accumulation and activity of the inflammatory cell populations within the inflamed synovium are likely to depend on the secretion of proinflammatory cytokines. Cytokines are chemical cellular messengers that can be produced by all cell types and are able to influence the cell of origin, adjacent cells, and cells remote from the cell of origin. Researchers are clarifying the roles of certain cytokines whose activities promote inflammation; are chondrodestructive; or, alternatively, are anti-inflammatory, chondroprotective, or chondroreparative. In studies23–25 in which investigators examined the role of cytokines in chronic joint inflammation and cartilage destruction in humans with rupture of the anterior cruciate ligament, an imbalance was detected between anabolic and catabolic cytokines.

Cytokines IL-1 and TNF-α have been identified as the major effectors of cartilage catabolism26 and suppressors of synthesis of new cartilage matrix.27 Interleukin-1 and TNF-α strongly induce IL-8 production in a broad range of cells.28–32 The chemokine IL-8 is chemotactic for neutrophils and T lymphocytes29,33 and stimulates the activation of neutrophils.34,35 Cytokines IL-4 and IL-10 have been described as anti-inflammatory cytokines.36 Interleukin-4 inhibits the production (natural and induced) of IL-1, IL-6, IL-8, TNF-α, and prostaglandin E2 in monocyte and synovial cell cultures.37–39 In addition, IL-4 can enhance the antigenpresenting capacity of B cells toward T-helper cells, thereby enhancing interactions between T and B cells.38 Interleukin-10 inhibits expression of major histocompatibility complex class II on antigen-presenting cells, proliferation of T cells,40,41 and production of IL-8 in SF and synovial tissue cell cultures.36,39 Interleukin-10 also inhibits the production of IL-1β and TNF-α in activated synovial tissue cultures, stimulates the production of IL-1 receptor antagonist and soluble TNF receptors, and downregulates the surface expression of the membrane receptor for TNF-α.41,42 In addition, IL-10 suppresses the release of reactive oxygen intermediates and nitric oxide in macrophages, thereby limiting tissue damage.43,44

The role of cytokines in naturally occurring joint diseases in dogs is still largely unknown. Therefore, in the study reported here, our objective was to evaluate gene expression for a chosen set of cytokines in stifle joints of dogs with CrCL rupture and MPL (at the time of reconstructive surgery) and in unaffected control stifle joints. In addition, a longitudinal study was initiated to monitor mRNA expression of cytokines before and up to 12 months after CrCL rupture in dogs with initial unilateral CrCL rupture.

Materials and Methods

Animals—Thirty-six client-owned dogs admitted to the Ghent University Veterinary School with lameness of the stifle joint along with 16 clinically normal dogs that were euthanatized for reasons unrelated to the stifle joints were included in the study. Twenty-nine of 36 affected dogs were admitted for surgical stabilization of stifle joints with CrCL rupture, whereas 7 dogs were admitted for surgical correction of MPL (1 dog had bilateral MPL). In the clinically normal dogs, lack of signs of osteoarthritis and lack of other pathologic lesions in the joints were confirmed by visual inspection during necropsy.

Owners of all client-owned dogs provided written consent to acquire SF from the affected stifle joint for research purposes. In addition, owners of 14 of those dogs provided written consent to have their dogs examined 6 and 12 months after surgery. The study was approved by the ethical commission of the Faculty of Veterinary Medicine, Ghent University.

The diagnosis of CrCL rupture was confirmed in all affected dogs at the time of corrective surgery. Breed, age at time of hospital admission, body weight, duration of lameness, and findings during surgical correction of the affected stifle joint were recorded for all affected dogs. Corrective surgery was performed by 1 surgeon (TdB). During surgery, dogs were further categorized with regard to the degree of CrCL rupture (partial or complete). Partial rupture was characterized as rupture of the craniomedial or caudolateral band of the CrCL, whereas complete rupture was characterized as total rupture of the CrCL. Menisci were evaluated during surgery, and the damaged portions were removed.

Collection and processing of samples—Immediately before the stab incision for the arthrotomy during corrective surgery, SF was aspirated from the affected stifle joints (29 samples from dogs with unilateral CrCL rupture, 8 samples from dogs with MPL) of the clientowned dogs. Samples of SF were obtained by percutaneous arthrocentesis from 24 stifle joints of the 16 clinically normal control dogs immediately after they were euthanatized by administration of a combination producta that contained embutramide (200 mg/mL), mebezonium iodide (50 mg/mL) and tetracainehydrochloride (5 mg/mL).

Samples of SF were also collected from the contralateral stifle joint and left shoulder joint of the 14 dogs that were monitored longitudinally. During subsequent examinations, SF was aspirated from both stifle joints and the left shoulder joint; dogs were heavily sedated or anesthetized for joint aspiration.

When < 0.2 mL of SF was aspirated from a joint, a modified washing method that used vitamin B12 as an internal standard was performed as described elsewhere.45 Briefly, a solution containing 25% vitamin B12b (1 mg/mL):75% saline (0.9% NaCl) solutionc (vol:vol) was injected in the joint, the joint was passively moved, and the solution was aspirated. The volume and character of aspirates were recorded. Samples with visible blood contamination were discarded. Direct smears of SF were made of all remaining samples for differential cell counts; total leukocyte count was not performed because all cells were used for RNA extraction. Direct smears were stained with H&E stain.d Samples of SF were kept on ice until processed; all SF samples were processed within 6 hours after collection.

RNA extraction—Samples of SF were centrifuged (9,390 × g for 10 minutes at 4°C) to separate cells and debris. Purified SF was stored at −20°C. The SF cell pellet was used for total RNA extraction. Total RNA was isolated by use of a phenol–guanidine isothiocyanate mixture,e as described elsewhere.46

Reverse transcription–PCR and real-time PCR assays—Reverse transcription–PCR assay was performed as described elsewhere.46 Resulting cDNA served as a template for a real-time PCR assay. An essential expressed housekeeping gene, GAPDH, was used as a control sample for the uniformity of the reverse transcription reaction and as a reference for quantification of cytokine mRNA.47 The oligonucleotide primers used for detection of canine GAPDH, TNF-α, IFN-γ, IL-4, IL-8, and IL-10 were obtained from published reports48,49 or derived from known canine cytokine sequences by use of commercially available softwaref (Appendix).

Cytokine cDNA was amplified and quantified with real-time PCR techniques by use of a 2.0 real-time PCR systemg and a DNA kit.h The reaction mixture consisted of a master mix containing Taq DNA polymerase, deoxynucleoside triphosphate mixture, and fluorescent dyei; 3mM MgCl2; 0.3μM of each primer; and 2 μL of template cDNA in a total volume of 20 μL. After samples were heated at 95°C for 10 minutes, cycling (35 to 40 cycles) consisted of denaturation at 94°C for 15 seconds; annealing at 60°C (TNF-α), 64°C (GAPDH, IL-4, and IL-8), or 65°C (IFN-γ and IL-10) for 5 seconds; and extension at 72°C for 12 seconds. Fluorescence acquisition was measured at 81°C (IFN-γ), 82°C (IL-8), 84°C (IL-4), or 86°C (GAPDH, TNF-G, and IL-10) in a single mode. Melting curve analysis was conducted at 65° to 98°C with continuous fluorescence acquisition for the confirmation of the specificity of PCR products. As an additional control of specificity, PCR products were subjected to agarose gel electrophoresis, when necessary. Quantification of cytokines was achieved by use of external standards of GAPDH, TNF-α, IFN-γ, IL-4, IL-8, and IL-10 cDNA. Calculation was performed with analysis software.j

The relative amount of cytokine expression was plotted as a ratio of the copy numbers of cytokine per the copy numbers of GAPDH, with the quotient multiplied by 1,000. The absolute amount of cytokine mRNA expression in a sample was determined by use of the following equation:

article image

Statistical analysis—Wilcoxon-Mann-Whitney rank sum tests were used to compare age, body weight, and duration of clinical signs between affected dogs with rupture of the CrCL and dogs with MPL. The Fisher exact test was used to compare the frequency of concomitant medial meniscus tear and the frequency of history of severe trauma.

The Fisher exact test was used to compare prevalence for a specific cytokine in affected versus control stifle joints. The absolute amount of cytokine mRNA expression in the SF samples was compared among dogs with CrCL rupture, dogs with MPL, and control dogs by use of the Kruskal-Wallis test, and pairwise comparisons were made by use of the Wilcoxon-Mann-Whitney rank sum test.

The absolute amount of cytokine mRNA expression in the various joints (left shoulder joint, affected stifle joint, and contralateral stifle joint) within dogs with CrCL rupture was compared by use of the Friedman test, with stratification by dog for all time points except at the first assessment (ie, operation for surgical repair). Pairwise comparisons were based on the Wilcoxon signed rank test. At the first assessment time point, which was immediately before corrective surgery, a substantial number of dogs had an assessment of only the affected stifle joint. Therefore, a Kruskall-Wallis test was performed so that all information could be considered, and a Wilcoxon rank sum test was used for the pairwise comparisons.

All analyses were performed by use of commercially available software.k Values were considered significant at P ≤ 0.05.

Results

Animals—Several breeds comprised the 3 groups of dogs. Dogs with rupture of the CrCL included 7 Golden Retrievers, 5 Labrador Retrievers, 3 Bernese Mountain Dogs, 3 Boxers, 2 Rottweilers, 2 American Staffordshire Terriers, 2 mixed-breed dogs, 1 Flat-Coated Retriever, 1 Chow Chow, 1 Great Dane, 1 German Shepherd Dog, and 1 Dogue de Bordeaux. Dogs with MPL included 3 Labrador Retrievers, 3 American Staffordshire Terriers, and 1 Bull Terrier. Clinically normal control dogs included 8 American Staffordshire Terriers, 3 Labrador Retrievers, 2 Boxers, 1 Doberman Pinscher, 1 Rottweiler, and 1 Belgian Shepherd Dog.

Characteristics of the 3 groups of dogs—Mean age, body weight, and duration of lameness and whether there was a meniscal tear or a history of trauma were determined for the various groups (Table 1). Thirteen of 29 (44.8%) dogs had partial CrCL rupture; 4 of the 13 also had a concomitant medial meniscus tear. The remaining 16 (55.2%) dogs had complete rupture of the CrCL; 13 of the 16 also had medial meniscus tear. The prevalence for complete rupture was significantly (P = 0.008) higher than that for dogs with partial rupture. Nineteen of 29 (65.5%) dogs with a ruptured CrCL did not have a history of severe trauma; there was no significant (P = 0.83; post hoc power = 0.040) difference between dogs with partial rupture and dogs with complete rupture. No significant difference was found for age (P = 0.98; post hoc power = 0.050) or body weight (P = 0.20; post hoc power = 0.054) between dogs with complete or partial CrCL rupture. Dogs with partial CrCL rupture were lame for a significantly (P = 0.01) longer time than were dogs with complete CrCL rupture.

Table 1—

Distribution and mean ± SD values for variables of stifle joints in 3 groups of dogs on the basis of various characteristics.

GroupNo. of stifle jointsNo. with MMTNo. associated with traumaAge (y)Body weight (kg)Duration of lameness (mo)
Rupture of CrCL2917104.5 ± 2.534.4 ± 8.42.6 ± 2.9
Complete161364.5 ± 2.432.8 ± 8.41.9 ± 2.8
Partial13444.4 ± 2.536.4 ± 8.33.7 ± 2.9
Patellar luxation8002.1 ± 2.427.0 ± 7.34.9 ± 3.5
Control dogs240NA3.8 ± 3.323.1 ± 8.7NA

MMT = Medial meniscus tear. NA = Not applicable.

Dogs with MPL were significantly younger (P = 0.009) and weighed less (P = 0.03) than dogs with CrCL rupture. In addition, dogs with MPL were lame for a significantly (P = 0.03) longer time than dogs with CrCL rupture.

Cytokine mRNA expression in stifle joints with CrCL deficiency or MPL at the time of surgery and in normal stifle joints—Expression of TNF-α was detected in 4 of 29 (13.8%) stifle joints with a ruptured CrCL, whereas expression was not detected in the 8 stifle joints with MPL or 24 normal stifle joints. However, prevalence for TNF-α expression did not differ significantly (P = 0.082; post hoc power = 0.500) among the 3 groups of dogs.

Expression of IL-8 was detected in 15 of 29 (51.7%) stifle joints with a ruptured CrCL but only in 1 of 24 (4.2%) normal stifle joints. This difference in prevalence was significant (P < 0.001). In addition, IL-8 expression was detected in all 8 (100%) stifle joints with MPL. This prevalence was significantly different, compared with prevalence for the joints with CrCL rupture (P = 0.015) and the normal stifle joints (P < 0.001).

Expression of IL-10 was detected in 7 of 29 (24.1%) stifle joints with CrCL rupture and in 5 of 8 (62.5%) stifle joints with MPL, whereas expression was not detected in the 24 normal stifle joints. Prevalence differed significantly between joints with CrCL rupture and normal stifle joints (P = 0.012) and between joints with MPL and normal stifle joints (P < 0.001), whereas prevalence did not differ significantly (P = 0.08; post hoc power = 0.530) between the stifle joints with CrCL rupture and stifle joints with MPL.

Expression of IFN-γ was detected in 1 of 29 (3.4%) stifle joints with CrCL rupture, 2 of 8 (25.0%) stifle joints with MPL, and none of the normal stifle joints. Prevalence did not differ significantly among the 3 groups. In addition, IL-4 expression was not detected in any of the stifle joints of the 3 groups.

Significant differences in the absolute amount of cytokine mRNA expression were found for IL-8 (P < 0.001), IL-10 (P < 0.001), and IFN-γ (P = 0.023) but not for TNF-α (P = 0.082) and IL-4 (P = 1.000; Figure 1). Pairwise comparisons of the absolute amount of mRNA expression between samples obtained immediately before surgery from dogs with a ruptured CrCL, dogs with an MPL, and clinically normal control dogs revealed a significant (P < 0.001) difference for IL-8 expression between CrCL-deficient stifle joints and normal stifle joints and between stifle joints with MPL and normal stifle joints. In addition, a significant difference was found for IL-10 expression between CrCL-deficient stifle joints and normal stifle joints (P = 0.008) and between stifle joints with MPL and normal stifle joints (P < 0.001). A significant (P = 0.013) difference was also found for IFN-γ expression between joints with MPL and normal stifle joints. Expression of IL-4 and TNF-α did not differ significantly among the various groups.

Figure 1—
Figure 1—

Cytokine mRNA expression in SF cells obtained from stifle joints with CrCL rupture (CrCL-R) or MPL and normal control stifle joints (Con). In the ratio, the absolute amount of cytokine mRNA expression was calculated from the relative amount of cytokine and total amount of RNA in the sample by use of the following equation: absolute amount = (relative amount × total RNA)/0.3. Each symbol represents results for 1 joint; values < 0 represent no expression.

Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.953

Longitudinal expression of cytokine mRNA in dogs with unilateral CrCL rupture—Cytokine mRNA expression for the 14 dogs monitored during the longitudinal portion of the study was summarized (Table 2). Expression of IL-4 and IFN-γ was not detected in any of the samples.

Table 2—

Cytokine mRNA expression in SF cells obtained from the left shoulder joint (LS), stifle joint with the initially ruptured CrCL (Init Rup), and contralateral stifle joint (Contra) of 14 dogs with unilateral rupture that were monitored longitudinally for 12 months after reconstructive surgery to repair the initially ruptured CrCL.

Dog No.JointCrCL ruptureIL-8IL-10TNF-α
   Day 0*6 mo12 moDay 0*6 mo12 moDay 0*6 mo12 mo
1LSNA0.002NDND0.090NDND0.420NDND
Init RupPartialND30.100ND0.380NDND
ContraPartial4.6809.200ND0.5300.0620.4704.5904.480 ND 
2LSNANDND162.000NDNDND
Init RupPartial14.60063.40013.9002.950NDNDNDNDND
ContraCompleteNDND176.00014.200NDND
3LSNA0.008NDND2.220NDNDNDNDND
Init RupPartial0.4000.170ND31.10013.000NDNDNDND
ContraNAND147.000ND15.300NDNDNDNDND
4LSNANDNDNDNDNDNDNDNDND
Init RupCompleteNDNDNDNDNDNDNDNDND
ContraComplete0.44052.600NDNDNDNDNDNDND
5LSNANDNDNDNDND4.780NDNDND
Init RupCompleteND74.300NDNDND0.330NDND2.020
ContraNANDND5.520NDND1.010NDND4.130
6LSNANDNDNDNDND1.640NDNDND
Init RupPartialNDND12.700NDND0.870NDND0.710
ContraNAND2.87018.000NDND1.090NDNDND
7LSNANDNDNDNDNDND
Init RupComplete4,230.0004.4202,400.000NDNDND2,110.000NDND
ContraNANDNDNDND0.060NDNDNDND
8LSNAND14.200NDND9.760NDNDNDND
Init RupPartialNDNDNDNDNDNDNDNDND
ContraUnknownND21.600759.000ND10.70059.700NDND55.300
9LSNA17.4009.4501,610.000ND2.8701.170NDNDND
Init RupPartialND10.4001,160.000ND0.360NDNDNDND
ContraNAND6.750ND0.047NDNDNDNDND
10LSNAND1.980NDNDNDND
Init RupCompleteND8.260NDND
ContraNANDND0.110NDNDND
11LSNANDNDNDNDNDND
Init RupComplete30.100ND0.1700NDND
ContraUnknown25.700ND0.350ND0.070ND
12LSNANDND12.400NDND1.700NDND2.410
Init RupPartial49.300NDNDNDNDND
ContraUnknown10.900ND10.600ND1.30056.700NDND0.790
13LSNAND36.8005.490NDNDNDNDNDND
Init RupPartialND64.50013.900NDND0.024NDNDND
ContraNAND55.3003.030NDNDNDNDNDND
14LSNAND7.090ND2.710ND1.740
Init RupCompleteNDND29.200NDND2.610NDND10.500
ContraNANDND5.840NDND2.380NDND2.320

Values reported are the ratio of the absolute amount of cytokine mRNA expression to the amount of GAPDH expression. In the ratio, the absolute amount of cytokine mRNA expression was calculated from the relative amount of cytokine and total amount of RNA in the sample by use of the following equation: absolute amount = (relative amount × total RNA)/0.3.

Day 0 was defined as the day of reconstructive surgery on the stifle joint with the initially ruptured CrCL.

Represents dogs that subsequently sustained CrCL rupture in the contralateral stifle joint.

Represents mRNA expression at the time point for the CrCL rupture of the contralateral stifle joint. NA = Not applicable. — = Not determined. ND = Not detectable.

The absolute amount of cytokine mRNA expression was compared among the 3 joints (initially affected stifle joint, contralateral stifle joint, and left shoulder joint) in dogs initially examined because of unilateral CrCL rupture. Comparisons were made for the same assessment time points (ie, at the time of surgery and 6 and 12 months after reconstructive surgery). A significant difference was found only for IL-8. At the time of surgery of the initially affected stifle joint, the IL-8 expression of the 3 joints differed significantly (P = 0.01), with a significant (P = 0.007) difference between the initially affected stifle joint and left shoulder joint. Six months after surgery of the initially affected stifle joint, a significant (P = 0.039) difference was found only between the contralateral stifle joint and left shoulder joint because of an increase in IL-8 expression in the contralateral stifle joint.

Six months after initial surgery, absolute amounts of cytokine mRNA expression were compared between contralateral stifle joints that would sustain a CrCL rupture at 12 months after surgery of the initially affected stifle joint and those that did not (Figure 2). No significant differences were found for any cytokines, although IL-8 expression was slightly higher in the contralateral stifle joints that sustained a CrCL rupture by 12 months after initial surgery.

Figure 2—
Figure 2—

Mean ± SD IL-8 mRNA expression in SF cells obtained from the contralateral stifle joints of dogs with unilateral CrCL rupture at 6 and 12 months after reconstructive surgery to repair the initially affected stifle joint. Results are reported for contralateral stifle joints that sustained rupture of the CrCL by 12 months (diagonal-striped bars) and joints that did not sustain rupture of the CrCL of the contralateral stifle joint during the study (crosshatched bars).

Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.953

The absolute amount of cytokine mRNA expression in the contralateral stifle joint at the time of rupture was compared with that of the dogs in which the contralateral stifle joint was still intact. No significant differences were found. The absolute amount of cytokine mRNA expression in a specific joint was also compared among the various assessment time points, but no significant differences were found for any of the joints examined.

Discussion

In the study reported here, TNF-α expression was only detected in 4 stifle joints with pathologic changes of the CrCL and none of the joints with MPL or normal stifle joints. Tumor necrosis factor-A is considered a proinflammatory cytokine that plays an important role in the acute stage of inflammation.24,50,51 In another study,52 investigators found moderate TNF-α expression in dogs with CrCL rupture. Unfortunately, they did not state the number of dogs that had TNF-α expression, and mean expression can be influenced by a small number of dogs with high expression. Bioactivity of TNF-α in the SF of dogs has been examined by use of bioassays.14,53–55 Bioactivity of TNF-α varies from being undetectable to increased in dogs with rheumatoid arthritis, spontaneous CrCL rupture, and experimentally transected CrCL.14,53–55 It remains difficult to conclude from these studies the role that TNF-α plays in dogs with pathologic changes of the stifle joints.

In the study reported here, a significant difference in prevalence of IL-8 expression was found between stifle joints with CrCL rupture or MPL and normal joints. In addition, the prevalence differed significantly between joints with MPL (100%) and joints with CrCL rupture (51.7%). In another study46 conducted by our laboratory group, analysis of IL-8 mRNA expression in joints with osteoarthritis failed to reveal a significant difference in prevalence between joints with MPL or CrCL rupture. However, in the study reported here, we examined fewer samples with CrCL disease and slightly more with MPL, which probably resulted in this significant difference. The absolute amount of IL-8 expression did not differ significantly between joints with CrCL rupture or MPL, although it was slightly higher in dogs with cruciate disease. There is more evidence for the role of chemokines as a second-step mediator of inflammation in arthropathies in humans.28–30,32 Authors46,56 have suggested that IL-8 could relate to the ongoing inflammation within a joint. High IL-8 expression has also been detected in dogs with immune-mediated arthritis in a slightly higher amount than in dogs with cruciate disease.52 This is consistent with studies29,56,57 in humans in which joints with rheumatoid arthritis have significantly higher IL-8 concentrations and amounts of joint inflammation than for joints with osteoarthritis.

The prevalence and absolute amount of IL-10 expression was significantly different in joints with CrCL rupture or MPL, compared with values for normal joints; however, no difference was found between dogs with CrCL rupture and MPL. Investigators in another study52 also found that IL-10 mRNA expression was upregulated in SF cells of dogs with immune-mediated arthritis and CrCL rupture. Humans with rheumatoid arthritis also have high expression of IL-10 in affected joints58; however, the amounts are insufficient to control the inflammatory response.59 Results for the study reported here are consistent with results for the human studies because IL-8 expression was often higher than IL-10 expression in the stifle joints of dogs with arthropathies.

In the study reported here, a significant difference in IFN-γ expression was found between joints with MPL and normal stifle joints, which was surprising because only 2 of 8 joints with MPL had IFN-γ expression. It is possible that this was merely a coincidence attributable to the small number of dogs with MPL. Expression of IFN-γ was detected in only 1 of 29 stifle joints with CrCL rupture. In another study52 in which investigators examined dogs with joint diseases, low expression was detected; however, the expression was slightly higher in stifle joints with CrCL rupture than in joints with immune-mediated arthritis.

Results for the control joints of the study are worthy of mention. Lack of expression of the selected cytokines in these samples could have been attributable to small numbers of cells. However, this was unlikely because all samples included in the study had typical amounts of expression for the housekeeping gene. Samples with low amounts of expression for the housekeeping gene were discarded from the study.

To our knowledge, the study reported here is the first in which gene expression of several cytokines has been examined in both stifle joints and the left shoulder joint of dogs with unilateral CrCL rupture during a period of 12 months. A significant difference in cytokine expression between the 3 examined joints was found only for IL-8. At the time of surgery of the initially affected stifle joint, highest IL-8 expression was found in the stifle joints with CrCL rupture. Six months later, expression was higher in the contralateral stifle joints. As stated earlier, IL-8 expression could relate to ongoing inflammation within a joint.46,56 Therefore, the higher expression found in the contralateral stifle joints 6 months after surgery of the initially affected stifle joint may indicate preclinical inflammation in dogs that would subsequently sustain a rupture of the CrCL in the contralateral joint within the next 6 months. However, if this is the situation, significantly higher IL-8 mRNA expression would be expected at the time of rupture of the contralateral CrCL (ie, by 12 months after surgery to repair the initially affected stifle joint), yet no difference in IL-8 expression was found in the contralateral joint between dogs that sustained a subsequent CrCL rupture and dogs that did not. Protein expression may not have the same pattern as for mRNA expression, or additional factors are needed to lead to CrCL rupture. It remains possible that the dogs that did not sustain CrCL rupture of the contralateral stifle joint during this study could still have a rupture of that CrCL in the future because these dogs were only investigated for 12 months. Therefore, future studies should examine the total protein content of IL-8 and other mediators of inflammation for a longer period to clarify this issue.

The amount and temporal patterns of gene expression of the selected cytokines appeared to vary widely within a patient, which has also been reported25 in humans with anterior cruciate ligament rupture. Such variation may be attributable to medications, modifications in exercise, or alterations in diet, which are difficult, if not impossible, to standardize in client-owned dogs.

Although the number of dogs that was longitudinally monitored in the study reported here was relatively small, the population was representative for dogs with naturally developing CrCL disease because no history of vigorous trauma was recorded in these 14 dogs but nearly half of the dogs sustained a CrCL rupture in the contralateral stifle joint during the study period. However, this group of dogs was not homogeneous and included dogs of various breeds, ages, and sexes. Etiopathogenesis of the CrCL rupture could have differed among these dogs, and it is therefore not surprising that the acquired results varied considerably. In addition, the chronicity and severity of the disease could not be standardized in these client-owned animals, which is in contrast to the conditions possible for experimental animals. It also would be difficult to control medications administered to treat client-owned dogs with osteoarthritis prior to sample collection.

Another limitation of our study was the use of SF cells for the detection of mRNA expression of cytokines. Detection of mRNA does not necessarily indicate biologically active proteins.60 However, because of the lack of reagents for the detection of most canine cytokine proteins, we chose to evaluate mRNA expression as an indicator of cytokine pattern. Numerous cytokine-producing sources are found within a joint, including articular cartilage, synovium, and subchondral bone.61–63 Our objective was to analyze SF cells rather than synovial tissue because of the way in which dogs were recruited for the study and a desire to obtain consecutive samples. In addition, marked variation in degree of synovitis can be found at various sites in the same stifle joint,13 which means multiple biopsy specimens from several sites of the same joint should have been obtained to provide accurate results. This is an extremely tedious process and could result in an increased risk for initiating joint damage, whereas arthrocentesis is simple and acquires SF cells, which are exudative cells that consist of mononuclear cells, macrophages, neutrophils, synoviocytes, and a few chondrocytes.

Although the cause of naturally occurring CrCL rupture is still unknown, studies14,20,21,46,52,53,55 have revealed that cytokines could play a major role in the maintenance of the inflammatory process and induction of intra-articular tissue destruction. Results from our study provided no inferences about the role of these mediators in the initiation of CrCL disease. Gene expression of IL-8 was the most prominent, together with expression of IL-10, in the joints with CrCL rupture or MPL. It is possible that other cytokines play a more important role in CrCL rupture than the cytokines selected for examination in this study. In addition, cytokine bioactivity is influenced by the absolute amount contained in the SF as well as by the number of receptors and specific inhibitors. Therefore, future attempts to define the inflammatory reaction in CrCL rupture should also involve studies on the regulatory functions of cytokines and growth factors. Only a complete understanding of the role of cytokines and their antagonists in cruciate disease will result in a therapeutic approach to alter the course of CrCL rupture and possibly help prevent rupture of the contralateral CrCL.

ABBREVIATIONS

CrCL

Cranial cruciate ligament

SF

Synovial fluid

IL

Interleukin

TNF

Tumor necrosis factor

MPL

Medial patellar luxation

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

IFN

Interferon

a.

T61, Intervet, Boxmeer, The Netherlands.

b.

Hydroxocobalamin, Sterop Laboratories, Brussels, Belgium.

c.

Plurule, Baxter, Lessines, Belgium.

d.

H&E, Merck & Co, Leuven, Belgium.

e.

Trizol, Invitrogen, Merelbeke, Belgium.

f.

Light Cycler probe design, Roche, Vilvoorde, Belgium.

g.

Light Cycler, Roche, Vilvoorde, Belgium.

h.

Light Cycler-faststart DNA Master SYBR Green I kit, catalogue No. 2239264, Roche, Vilvoorde, Belgium.

i.

SYBR Green I, Roche, Vilvoorde, Belgium.

j.

Light Cycler analysis software, Roche, Vilvoorde, Belgium.

k.

StatXact, Cytel Software Corp, Salt Lake City, Utah.

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Appendix

Primer sequences used in real-time PCR assays for the detection of cytokines in SF cells obtained from dogs.

table3

Contributor Notes

Supported by the Special Research Fund of the Ghent University, Belgium.

Address correspondence to Dr. de Bruin.