Department of Veterinary Basic Sciences, The Royal Veterinary College, London NW1 0TU,
1 Department of Biology, University of York, York YO1 5YW and
2 Rheumatology Unit, University College London, London W1P 9PG, UK
Correspondence to:
A. A. Pitsillides, Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London NW1 0TU, UK.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. Hock joints of five adult sheep were immobilized by external fixation. Twelve weeks later, SF and synovium samples were collected. The HA concentration in SF was assayed using an ELISA-based method. Non-specific esterase (NSE) and uridine diphosphoglucose dehydrogenase (UDPGD) activities were assessed in cryostat sections of snap-chilled synovial samples using cytochemical techniques, and UDPGD activity per cell was measured in synovial lining cells by scanning and integrating microdensitometry.
Results. We found that the SF HA concentration was decreased from 1.65±0.25 mg/ml in control joints to 0.68±0.16 mg/ml in immobilized joints. Synovial intimal cell UDPGD activity decreased from 18.0±2.7 U/cell in control joints to 12.2±1.5 after immobilization. There was also a decrease in UDPGD-positive intimal cell numbers. Intimal surfaces in controls contained numerous NSE-positive cells, which were rarely observed in intima from immobilized joints.
Conclusions. These results suggest that immobilization decreases SF HA levels and that this is associated with reduced intimal cell UDPGD activity (essential for HA formation). Immobilization also decreased the prevalence of (NSE-positive) intimal macrophages. These findings suggest that mechanosensitive homeostatic mechanisms exist within the synovial intima.
KEY WORDS: Synovial lining cells, Hyaluronan, Immobilization, Joint, Uridine diphosphoglucose dehydrogenase, Type B cells, Synovial fluid
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The SF of normal diarthrodial joints contains high concentrations of HA [1, 2], suggesting that high levels are required for joint function to be preserved. Experimental findings support the notion that these high HA concentrations may contribute to the joint's functional competence. For example, the functional improvement observed in rheumatoid arthritis (RA) joints (where HA concentrations are low whilst the total amount of HA may be high; [23]) after intra-articular corticosteroid treatment is associated with a restoration in these SF HA concentrations [4]. Nevertheless, the mechanisms by which joint HA concentrations are controlled, and how they are influenced by articulation and loading, are not fully elucidated.
Whilst the changes in SF HA content in disease are well established, their relationship with the cellular changes occurring in the intimal lining of synovium remains largely unaddressed [5]. Synovial intima contains two major cell populations: macrophages (type A cell; [6, 7]) and specialized fibroblasts (type B cell). One specialized feature of intimal fibroblasts relates to HA synthesis. We have shown that uridine diphosphoglucose dehydrogenase (UDPGD) activity, which is essential for HA production and rate limiting in UDP-glucuronate (one of HA's monosaccharide subunits) formation, is 6-fold higher in normal synovial intimal fibroblasts than other fibroblasts, and that this decreases in chronically inflamed rabbit and human RA joints [810]. Cells with high UDPGD activity also appear in tissue lining a cavity containing a moving implant, but not in an air-filled cavity [5], suggesting that mechanical stimuli control cellular HA-synthetic characteristics.
Synovial intima appears to have a low rate of cell replacement and, although direct evidence is lacking, intimal fibroblasts are probably derived locally from subintimal fibroblasts, whilst intimal macrophages are likely to arrive in the joint as circulating monocytes, undergoing their macrophagic transition in the subintimal perivascular stroma. However, the effect of normal joint function on the local differentiation of these apparently distinct cell types has not been established. The aim of this study was to determine the effect of an extended period of external fixator-induced immobilization on SF HA levels and to establish whether immobilization is also associated with alterations in the cellular characteristics of the synovial intima.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vivo strain gauge recordings in other sheep showed that in addition to reducing the peak strain magnitude in the calcaneus during walking, the fixator also abolished the high-magnitude transient strains experienced when the sheep made sudden movements. Thus, application of the fixator protects the hock joint from loading and also prevented hock joint articulation [11].
Collection of SF and lining samples
Twelve weeks after surgery, the sheep were killed by lethal pentobarbitone injection and synovial cavities of left and right hock joints were sampled by needle insertion between the digital extensor tendons on the cranial aspect. After collection of SF, the skin was removed and the tibiotarsal joint disarticulated. Samples of synovial tissue were collected from equivalent sites in the anterior and posterior compartments of all joints. The synovial tissue was cut into pieces not greater than 0.5 cm3 , immersed for 1 min in a 5% solution of polyvinyl alcohol containing 5% CaCl2 2H2O [8] and then chilled to -70°C in n-hexane (BDH; low in aromatic hydrocarbons). The tissue was stored in dry tubes at -70°C and examined within 2 weeks.
Assessment of HA concentration by ELISA
Biotinylated HA binding region (HABr) prepared from pig laryngeal cartilage was a kind gift from Professor M. T. Bayliss (The Royal Veterinary College, London). Aliquots (0.5 ml) of SF were incubated with 0.5 ml of 20 µl/ml papain (Boehringer Mannheim) in 0.2 M acetate buffer (pH 5.8) containing 10 mM EDTA and 10 mM cysteine (added just prior to use), at 60°C for 16 h. The papain was inactivated by addition of 5 mM iodoacetic acid. Experiments in which HA of different, precisely defined, molecular weights (a gift from Pharmacia LKB, Sweden) were treated as above indicated that assay sensitivity was unaffected by HA's molecular mass (not shown).
After centrifugation, HA concentration in the supernatant was assayed, in duplicate, using an ELISA-based method [12]. Each sample was assayed at least twice, and samples which did not yield a dilution profile gradient corresponding to that obtained for standard HA were discarded and the assay repeated (no sample was excluded more than once). This method relies on competition between unknown concentrations of sample HA and a fixed quantity of plate-bound HA, for binding to a fixed concentration of biotinylated HABr. The biotinylated HABr's capacity to bind plate-bound HA is inversely related to the HA concentration of that sample. Sample HA concentration (mg/ml) was determined by comparison with the competition produced by known concentrations of exogenous HA, and results were evaluated by paired t-test.
Assessment of UDPGD activity
To assay UDPGD activity, we used the method described by Mehdizadeh et al. [13]. Briefly, fresh 10 µm sections were incubated at 37°C in an atmosphere of nitrogen, in medium containing 5.3 mM UDP-glucose (3 mg/ml; Sigma), 0.45 mM NAD (0.3 mg/ml; Boehringer) in 30% (w/v) polyvinyl alcohol (Grade G04/140; Wacker Chemicals Ltd, Surrey) in 0.05 M glycylglycine buffer (pH 7.8). Just prior to use, medium was saturated with nitrogen, adjusted to pH 7.8 and 3.7 mM nitroblue tetrazolium (3 mg/ml; Sigma) was added. In all cases, reaction time was 20 min. Serial sections were incubated in medium lacking substrate (no UDP-glucose) or in full medium including 0.2 mM UDP-xylose (0.21 mg/ml; Sigma), a specific inhibitor of UDPGD activity.
After reaction, sections were washed in water, dried and mounted in Aquamount (BDH). The amount of precipitated formazan was measured using a Vickers M85A scanning and integrating microdensitometer [8], at a wavelength of 560 nm, in individual cells within the synovial lining, with a x40 objective and a scanning spot of 0.5 mm in the plane of the section. At least 20 cells were measured in histologically defined intima in each of two duplicate sections in each assay, and up to three samples were examined from each joint. Results, expressed as units of mean integrated extinction (MIE x100) per cell, for a 20 min reaction time were evaluated by paired t-test.
Assessment of non-specific esterase (NSE) activity
NSE activity was assessed in fresh 10 µm serial sections using -naphthyl acetate as described previously [10]. In all instances, serial sections were also stained with 0.1% toluidine blue in 0.1 M acetate buffer (pH 6.1), washed, air-dried and mounted in DePeX (BDH).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In accord with previous findings, the majority of intimal cells in control synovium showed high UDPGD activity, whilst subintimal synovial cells showed little, if any, UDPGD activity (Fig. 2a and c). Intimal cells with high UDPGD activity formed an almost continuous band of cells which followed the contours of the intimal layer (Fig. 2c
). In immobilized joints, the thickness of the UDPGD-containing intimal layer was frequently reduced; in control joints, it was often 23 cells thick, whilst in immobilized joints it was rarely more than one cell thick (Fig. 2b and d
).
|
Effect of immobilization on NSE activity in synovial lining cells
It has previously been found that macrophages in synovial intima show consistently high NSE activity [10]. The intimal lining of synovium from control joints contained distinct regions in which a significant proportion of cells were NSE positive (Fig. 2j). Commonly, these NSE-positive cells were superficial to cells in the same region showing high UDPGD activity. In contrast, regions of NSE-positive rich intima were not evident in synovium from immobilized joints, and single NSE-positive cells were only rarely observed (Fig. 2k
).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The immobilization-induced reduction in SF HA concentration could be contributed to by decreased synthesis, increased clearance or an increase in volume of distribution. Decreases in intimal cell UDPGD activity indicate that immobilization may reduce the formation of one of HA's essential monosaccharides, and until the mechanisms regulating HA synthesis are determined, it is possible that this alone may decrease rates of HA synthesis. Since the volume of SF retained in a normal, mobile joint is probably determined by its HA content, a reduced rate of HA synthesis might be expected to give rise to a reduced SF volume with an unchanged concentration of HA. However, immobility may also be expected to reduce SF clearance and thereby increase the volume of distribution directly [14]. Although the precise estimation of SF volume was not possible in this study, no major change was apparent in immobilized joints. Thus, immobilization may have a dual action, such that the concentration of HA falls and SF volume is not increased because any tendency to retain fluid is offset by the diminished water-retaining capacity of a reduced intra-articular HA content.
It is possible that the immobilization-induced changes observed were mediated by alterations in fluid flux consequent on muscle wastage and change in blood flow. However, the level of use of the limb was not significantly reduced and there was neither significant muscle wastage detectable by palpation nor oedema formation observed. Although our study has not addressed the role of movement in controlling HA's molecular mass, the immobilization-induced decreases in HA's concentration are consistent with an adaptive change to the removal of the joint's normal functionally related stimuli, suggesting also that high SF HA levels are essential in this regard.
The mechanisms by which intimal cell numbers are regulated in the synovium of normal joints and how they are modified by inflammation, in which intimal cell number increases significantly, are ill-defined. As well as decreasing UDPGD activity, we have found that immobilization reduces the number of both UDPGD-positive cells and NSE-positive macrophages in the intima, suggesting that their recruitment is modulated by mobility. Movement may influence intimal cell numbers by modifying local cytokine release, may involve nerve endings or even a paracrine influence from resident intimal fibroblast-like cells; the latter may explain why macrophages preferentially accumulate in the intima, rather than subintima, but this remains to be addressed.
Intimal macrophage function in normal synovium is not established. Several lines of evidence suggest that these cells are employed in remodelling their extracellular matrix [15]. If this is the case, it is tempting to speculate that fibroblast-derived HA synthesis is decreased primarily by movement's withdrawal and that this has secondary effects on macrophage recruitment (see Fig. 3). By corollary, findings based on the analysis of SF after episodes of exercise are consistent with movement increasing local degradation [16, 17]. Further, Culty et al. [18] showed that alveolar macrophages are capable of uptake and lysosome-mediated degradation of HA, suggesting that decreases in macrophage numbers would point to a diminished HA-degradatory potential in the intima. This would be in harmony with coincident decreases in HA synthesis, and the possibility that changes in local (fibroblast-derived) HA concentration modify macrophage recruitment.
|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|