Investigation of synovial fluid lubricants and inflammatory cytokines in the horse: a comparison of recombinant equine interleukin 1 beta-induced synovitis and joint lavage models

Abstract

Background: Lameness is a debilitating condition in equine athletes that leads to more performance limitation and loss of use than any other medical condition. There are a limited number of non-terminal experimental models that can be used to study early inflammatory and synovial fluid biophysical changes that occur in the equine joint. Here, we compare the well-established carpal IL-1β-induced synovitis model to a tarsal intra-articular lavage model, focusing on serial changes in synovial fluid inflammatory cytokines/chemokines and the synovial fluid lubricating molecules lubricin/proteoglycan 4 and hyaluronic acid. The objectives of this study were to evaluate clinical signs; synovial membrane and synovial fluid inflammation; and synovial fluid lubricants and biophysical properties in response to carpal IL-1β synovitis and tarsal intra-articular lavage.

Results: Hyaluronic acid (HA) concentrations, especially high molecular weight HA, and synovial fluid viscosity decreased after both synovitis and lavage interventions. Synovial fluid lubricin concentrations increased 17-20-fold for both synovitis and lavage models, with similar changes in both affected and contralateral joints, suggesting that repeated arthrocentesis alone resulted in elevated synovial fluid lubricin concentrations. Synovitis resulted in a more severe inflammatory response based on clinical signs (temperature, heart rate, respiratory rate, lameness and joint effusion) and clinicopathological and biochemical parameters (white blood cell count, total protein, prostaglandin E₂, sulfated glycosaminoglycans, tumor necrosis factor-α and CC chemokine ligands - 2, - 3, - 5 and - 11) as compared to lavage.

Conclusions: Synovial fluid lubricin increased in response to IL-1β synovitis and joint lavage but also as a result of repeated arthrocentesis. Frequent repeated arthrocentesis is associated with inflammatory changes, including increased sulfated glycosaminoglycan concentrations and decreased hyaluronic acid concentrations. Synovitis results in more significant inflammatory changes than joint lavage. Our data suggests that synovial fluid lubricin, TNF-α, CCL2, CCL3, CCL5, CCL11 and sGAG may be useful biomarkers for synovitis and post-lavage joint inflammation. Caution should be exercised when performing repeated arthrocentesis clinically or in experimental studies due to the inflammatory response and loss of HA and synovial fluid viscosity.

Keywords: Chemokine; Hyaluronic acid; Lubrication; Lubricin; Osteoarthritis; Repeated arthrocentesis; Rheology.

Fig. 1

Lameness scores and total protein (TP), white blood cell (WBC), and prostaglandin E₂ (PGE₂) concentrations in the synovial fluid of middle carpal joints (MCJ) and tarsocrural joints (TCJ) following IL-1β-induced synovitis or intra-articular lavage. Forelimb lameness, as measured by mean vector sum using an inertial motion sensor system, increased markedly from baseline for 12 h post-induction of synovitis on the synovitis limb (a). Hind limb lameness as measured by pushoff factor showed that horses started the study with a trend toward mild lameness of the contralateral hindlimb (negative values) in the first week but became sound (− 2 to + 2) in the last 4 weeks of the study (b). TP increased from baseline for 72 h following synovitis induction and was greater in synovitis MCJ as compared to contralateral MCJ for 4 weeks post-synovitis induction. TP decreased from baseline at 5 weeks post-induction (c). TP increased from baseline for 72 h following lavage with no differences from contralateral TCJ. TP decreased from baseline at 3 and 4 weeks post-lavage (d). WBC counts increased from baseline and were greater in synovitis MCJ as compared to contralateral MCJ for 1 week post-synovitis induction (e). WBC counts increased from baseline for 72 h following lavage with no differences from contralateral TCJ (f). PGE₂ increased from baseline for 24 h following synovitis and was greater in synovitis MCJ as compared to contralateral MCJ for the majority of 2 weeks post-synovitis induction (g). Following lavage, PGE₂ increased from baseline at 6 h and was greater in lavage TCJ as compared to contralateral TCJ at 6 and 24 h post-lavage (h). An asterisk indicates a difference from baseline, and a triangle indicates a difference from the contralateral limb at the same time point. Graphed values are marginal means +/− SEM

Fig. 2

Hyaluronic acid (HA) concentration, relative concentration of HA with a molecular weight of > 6.1 MDa (HMW HA), and viscosity of the synovial fluid of middle carpal joints (MCJ) and tarsocrural joints (TCJ) following IL-1β-induced synovitis or intra-articular lavage. HA decreased from baseline at 6, 24, and 72 h and at 1, 3, 4, and 5 weeks following synovitis induction in both synovitis MCJ and contralateral MCJ (a). HA decreased from baseline from 6 to 12 h, 72 h to 3 weeks, and at 5 weeks post-lavage in both lavage TCJ and contralateral TCJ (b). HMW HA decreased from baseline in synovitis MCJ for 1 week post-synovitis induction and was less than control MCJ from 6 to 48 h. HMW HA increased above baseline at 5 weeks (c). HMW HA increased from baseline at 48 h and from 3 to 5 weeks post-lavage with no statistical differences from contralateral TCJ (d). Viscosity decreased from baseline for the duration of the study period (4 weeks) following synovitis induction and was less than the contralateral MCJ for the same period (except at 1 weeks) (e). Following lavage, viscosity was unchanged from baseline except for a decrease at 168 h and was less than the contralateral TCJ at 72 h (f). Baseline viscosity between the MCJ and TCJ were statistically different. An asterisk indicates a difference from baseline, and a triangle indicates a difference from the contralateral limb at the same time point. Graphed values are marginal means +/− SEM

Fig. 3

Lubricin and sulfated glycosaminoglycan (sGAG) concentrations in the synovial fluid of middle carpal joints (MCJ) and tarsocrural joints (TCJ) following IL-1β-induced synovitis or intra-articular lavage. Lubricin increased from baseline for 2 weeks following synovitis induction in both synovitis and contralateral MCJ (a). Lubricin increased from baseline for 72 h following lavage with the contralateral TCJ concentration being greater than synovitis TCJ at 72 and 168 h post-lavage (b). sGAG concentration increased from baseline for 72 h and was greater in synovitis MCJ as compared to contralateral MCJ during that time. sGAG concentration decreased from baseline at 3, 4 and 5 weeks post-induction of synovitis (c). sGAG concentration increased from baseline from 24 to 72 h following lavage and decreased from baseline 3 and 4 weeks with no differences from contralateral TCJ (d). An asterisk indicates a difference from baseline, and a triangle indicates a difference from the contralateral limb at the same time point. Graphed values are marginal means +/− SEM

Fig. 4

Interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) concentrations in the synovial fluid of middle carpal joints (MCJ) and tarsocrural joints (TCJ) following IL-1β-induced synovitis or intra-articular lavage. IL-1β remained unchanged following synovitis induction (a) or intra-articular lavage (b). TNF-α was increased from baseline and was greater in synovitis MCJ as compared to contralateral MCJ at 6 h post-synovitis induction (c). TNF-α was unchanged from baseline following lavage with no differences from contralateral TCJ (d). An asterisk indicates a difference from baseline, and a triangle indicates a difference from the contralateral limb at the same time point. Graphed values are marginal means +/− SEM

Fig. 5

Chemokine ligand 2 (CCL2), chemokine ligand 3 (CCL3), chemokine ligand 5 (CCL5) and chemokine ligand 11 (CCL11) concentrations in the synovial fluid of middle carpal joints (MCJ) and tarsocrural joints (TCJ) following IL-1β-induced synovitis or intra-articular lavage. CCL2 was increased from baseline from 6 to 24 h following synovitis and was greater in synovitis MCJ as compared to contralateral MCJ at 6 and 24 h post-synovitis induction (a). Following lavage, CCL2 was increased from baseline from 6 to 12 h but did not differ from contralateral TCJ (b). CCL3 was increased from baseline and was greater in synovitis MCJ compared to contralateral MCJ at 6 h post-synovitis induction (c). CCL3 was less in the lavage TCJ as compared to the contralateral TCJ at baseline but otherwise had no response to lavage (d). Following synovitis, CCL5 was increased from baseline at 6, 12, and 72 h and was greater compared to the contralateral MCJ at 6, 72, and 168 h (e). CCL5 did not change in response to lavage (f). CCL11 was increased from baseline from 6 to 72 h following synovitis and was greater in synovitis MCJ than contralateral MCJ from 6 to 48 h (g). CCL11 was increased from baseline at 6 and 12 h post-lavage and did not differ from contralateral TCJ at any time. An asterisk indicates a difference from baseline, and a triangle indicates a difference from the contralateral limb at the same time point. Graphed values are marginal means +/− SEM