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Rheumatology 2001; 40: 336-340
© 2001 British Society for Rheumatology

The role of hyaluronic acid in protecting surface-active phospholipids from lysis by exogenous phospholipase A2

D. W. Nitzan, U. Nitzan1, P. Dan1 and S. Yedgar1

Department of Oral and Maxillofacial Surgery, The Hebrew University–Hadassah School of Dental Medicine, Jerusalem,
1 Department of Biochemistry, Hadassah University Hospital, The Hebrew University–Hadassah Medical School, Jerusalem, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. This in vitro study aimed to elucidate the extent and kind of involvement of hyaluronic acid (HA) in the currently accepted view of synovial joint lubrication, in which surface-active phospholipids (SAPL) constitute the main boundary lubricant. The integrity of SAPL is apparently threatened by the lysing activity of phospholipase A2 (PLA2).

Methods. The effects of increasing concentrations of HA degraded by free radicals and non-degraded HA on the lysing activity of PLA2 were examined in vitro. Liposomes (lipid model membrane) containing phosphatidylcholine (PC) were used as the substrate, on the assumption that they are appropriate representatives of SAPL.

Results. HA adhered to the phospholipid membrane (liposomes), inhibiting their lysis by PLA2. However, in its degraded form, HA not only failed to inhibit PLA2-lysing activity, but accelerated it.

Conclusions. It is reasonable to assume that HA plays an important indirect role in the steady state of the boundary lubrication process of joints by protecting SAPL from being lysed by PLA2. However, as excessive loading generates free radicals within the joint (among other effects), the HA that is degraded in this way is incapable of protecting SAPL from lysis by PLA2. When the rate of degradation exceeds that of synthesis, there will be insufficient replacement of HA and/or SAPL, resulting in denudation of the articular surfaces. These are then exposed to increasing friction, and hence increased danger of degenerative joint changes.

KEY WORDS: Hyaluronic acid, Lubrication, Phospholipase A2, Surface-active phospholipids, Synovial joint, Degenerative joint changes.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Hyaluronic acid (HA) lends the normal human synovial fluid (SF) its remarkable rheological properties. Consequently, for many years HA was credited with the key role in the boundary lubrication system of joints, and as such was believed to occupy a primary place in the pathophysiology and therapy of joint disorders [1–6]. However, the role of HA within the framework of the joint's boundary lubrication system has been questioned. HA possesses a negligible load-bearing capacity and, moreover, degradation of the viscous HA by the use of hyaluronidase does not have a detrimental effect on the lubricating ability of the SF [7–9]. As a result, an array of other possible functions of HA in joint movement has been suggested, among which are those of space filler, wetting agent, flow barrier within the synovium, and protector of the cartilage surfaces [1–6]. Beside its mechanical role in joint function, HA has been found, inter alia, to support joint integrity by acting as a scavenger, inhibiting phagocytosis and chemotaxis, and by preventing scar tissue formation and angiogenesis [1–6, 10–15].

It seems, however, that the multifunctionality of HA is not the whole picture, as some aspects of the role of HA in synovial joint boundary lubrication are still obscure. A comprehensive understanding of its role requires clarification of the currently accepted view of the lubrication of synovial joints.

In the last decade it has been proposed that surface-active phospholipids (SAPL) serve as the major boundary lubricant, reducing the coefficient of kinetic friction to a very low value (0.001–0.006) and thus lessening wear of the articular surfaces, even under high loads [16–20]. The role of SAPL as a lubricant has been examined by removing them from joints by the use of lipid solvents [21] or, more specifically, of phospholipase A2 (PLA2) [22]. Their removal induces a significant increase in friction.

Lubricin, a glycoprotein that has been isolated from the load-bearing fraction of the synovial fluid and has been identified as a major boundary lubricant [23, 24], has been shown to be a water soluble-carrier of SAPL [22]. Proteolipid, another component of the SF, has been shown to facilitate the deposition of the oligolamellar (graphite-like) layer of SAPL, and so aids in boosting boundary lubrication of the articular surfaces [20].

When functioning under load, the boundary lubrication system adapts itself constantly by a process of remodelling. In this process, PLA2, which is secreted by synoviocytes, chondrocytes and osteoblasts into the synovial fluid [25], is probably responsible for the lysis of SAPL. It is therefore assumed that, when uncontrolled, its presence in the SF constitutes a threat to the continuity of the SAPL lining. Hence, it is of interest to establish the factor that governs the lysing activity of PLA2 and to identify the element that protects the continuity of SAPL in the steady-state remodelling process in such a way that the rate of lysis does not exceed the rate of synthesis.

This study, which was undertaken in order to elucidate the role of HA in preserving the integrity of the boundary lubricating system of synovial joints, explored two hypotheses. The first hypothesis was that high molecular weight HA inhibits PLA2 activity and thus protects the integrity of SAPL. In the event of excessive joint loading, or during an inflammatory process, reactive oxidative species are generated, which are known for their high potency in degrading HA and impeding its synthesis [14, 26]. The second hypothesis was that HA in its degraded form (dHA) loses its ability to inhibit PLA2 activity, leaving the SAPL vulnerable to lysis by PLA2.

To examine these hypotheses, we used liposomes (a lipid model membrane), which contain phosphatidylcholine (PC), as a substrate. We assumed that liposomes are optimal simulators of SAPL.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Liposomes made from dioleoylphosphatidylcholine (DOPC) and 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole) aminocaproylphosphatidylcholine (C6-NBD-PC) were purchased from Avanti Biochemicals (Alabaster, AL, USA). Thin-layer chromatography plates were purchased from Whatman (LK6; Whatman, Maidstone, UK). Naja mocambique phospholipase A2, hyaluronic acid and all other laboratory chemicals and buffers were obtained from Sigma (St Louis, MO, USA).

Degradation of HA
HA (10 mg/ml of 100 mM Ca2+-free Tris buffer, pH 8.0) was exposed to free radicals by exposure to H2O2 (4 mM) in the presence of traces of Cu2+ and Fe2+ (10 µM) for 30 min. The reaction was terminated by the addition of catalase (200 µg) for 10 min followed by dialysis against Ca2+-free Tris buffer (pH 8.0). Degradation of HA was monitored by the reduction in viscosity of its solution, using an Oswald capillary viscometer (Cannon Instruments Co., State College, PA, USA).

Effect of HA and of dHA on lysis of phospholipids by PLA2
Liposomes were used as a substrate to study their lysis by PLA2 in the presence of either HA or dHA. A mixture of the liposomes DOPC and the fluorescent C6-NBD-PC (3:2 molar ratio) was evaporated under a stream of nitrogen. The lipid extract was then resuspended in Ca2+-free Tris buffer (100 mM, pH 8.0) and filtered successively through polycarbonate Millipore filters (0.2 and 0.4 µM). The purified liposome substrates were preincubated with HA (final concentration 0.0–6.88 mg/ml) and with dHA (final concentration 0.0–2.5 mg/ml) for 30 min in a water bath at 37°C. Naja mocambique PLA2 (30 µl; 0.014 U/ml) in Tris buffer (100 mm, pH 8.0) supplemented with Ca2+ (10 mM) was then added to the mixture to a final volume of 800 µl. Alternatively, PLA2 was preincubated with HA or dHA for 30 min in a 37°C water bath, after which liposomes in Tris buffer (100 mM, pH 8.0) supplemented with Ca2+ (10 mM) were added to each of the mixtures to a final volume of 800 µl per mixture. The reactions were allowed to continue for an additional 40 min in a shaking water bath at 37°C, after which they were stopped by the addition of 3 ml of termination solution consisting of chloroform:methanol:2 N hydrochloric acid at a ratio of 166:133:5 in the presence of 1 ml acid saline. The lipids were extracted into an organic phase, dried under a stream of nitrogen, resuspended in 40 µl chloroform:methanol (1:1), chromatographed on silica thin-layer plates and eluted in chloroform:methanol: water (65:35:5) [27]. The band corresponding to the NBD caproic acid (C6-NBD-FA) was scraped off and extracted into chloroform:methanol (1:1). Its fluorescence intensity was measured by excitation at 470 nm and emission at 535 nm [27].

Interaction between HA and liposomes
The potassium bromide density test was applied to determine whether a complex of HA with phospholipid (HA–PL complex) is formed during incubation of the lysosomes with HA. Samples of HA (2 ml, 5 mg/ml), liposomes and their mixture were placed at the bottom of three SW-41 centrifugation tubes and potassium bromide gradients (1.005–1.21 g/ml) were layered on top [28]. The tubes were then centrifuged at 40 000 r.p.m. for 21 h at 4°C, after which they were examined for the locations of HA, phospholipid and their mixture. HA was detected by the use of the stain-all reagent, a blue colour being formed by their interaction [29]. The liposomes were detected by C6-NBD-PC fluorescence. The high-density HA band was expected to be located at the bottom of the gradient, while the band of low-density liposomes was expected to be at the top of the gradient. The nature of the interaction between HA and liposomes determines the location of the components of the mixture in the gradient.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The inhibitory effect of HA on the hydrolysis of the phospholipid substrate by exogenous PLA2 is shown in Fig. 1Go. Dose-dependent inhibition of PLA2 activity occurred in the presence of HA (final concentration 0–6.88 mg/ml). The rate of inhibition showed a decline: a dramatic reduction in PLA2 activity (by 66%) occurred in the presence of 1.22 mg/ml HA. Increasing concentration of HA was associated with a decreasing rate of inhibition, to the point that PLA2 activity was almost entirely blocked at physiological HA concentrations. dHA (0.6–2.5 mg/ml) failed to inhibit hydrolysis of phospholipid by PLA2 (Fig. 2Go). In fact, there was a slight degree of acceleration rather than inhibition in PLA2 activity in the presence of increasing concentrations of dHA. It is known that HA, an antioxidant, may act as a proactivator when degraded by free radicals, as is frequently the case with oxidation products [30, 31]. The same results were obtained (i) when HA or dHA was incubated with liposomes to which PLA2 was later added, and (ii) when HA or dHA was incubated with PLA2 and phospholipid was added subsequently.



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  		FIG. 1. Dose-dependent inhibition of PLA2 activity by increasing concentrations of HA.

 


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  		FIG. 2. dHA accelerates rather than inhibits PLA2 activity.

 
To investigate whether the inhibition of PLA2 activity is due to protection of the liposomes by HA, the absorption of the latter to the lipid membrane was determined with the potassium bromide density test. The interaction between the two components, i.e. HA and phospholipids, yielded a band in the gradient far below the location of the low-density phospholipid alone, but close to the high-density HA band, confirming that an HA–PL complex membrane had been created (Fig. 3Go).



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  		FIG. 3. Potassium bromide density gradient. The liposomes represented by the fluorescent band are located at a low-density level (left tube). The liposome/HA mixture is located at a high-density level, below the band of liposomes alone. This confirms that HA binds to the liposomes, forming a complex (right tube).

 
The possibility that HA in the solution interacts with PLA2 was also considered, but separation between the HA and PLA2 using column chromatography was technically difficult because of the viscosity of HA. Inhibition consequent on interaction between the enzyme and the free polymer in the solution was found to be negligible compared with interaction with polymer adsorbed to the membrane surface [32].


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In the last decade, HA has gradually lost its status as the key component responsible for boundary lubrication. However, the results of the present in vitro study suggest that HA might have an indirect role in the boundary lubrication of synovial joint. A major role in lubrication has been assigned to SAPL [17–21], which constantly adapt themselves by means of remodelling, and within the framework of this process PLA2 is probably responsible for the lysis of SAPL. When the existing situation becomes unbalanced and the lysogenic activity of this enzyme becomes uncontrolled, there may be an actual threat to the integrity of the SAPL. The steady-state condition of the joint is indicative of the presence of an efficient defence mechanism for the SAPL.

It was demonstrated in the present in vitro study that HA interacts with liposomes, indicating that this lends it the capability of protecting phospholipids against lysis by exogenous PLA2. As may be inferred from the potassium bromide density test, HA adheres to the liposomes firmly, thus forming a barrier against lysis by PLA2. The degradation of HA by reactive oxidative species impairs this function, thereby exposing the liposomes to lysis by PLA2. These in vitro results seem to imply that HA plays an indirect role in joint lubrication in functioning synovial joints by protecting the integrity of SAPL (Fig. 4aGo)



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  		FIG. 4. Synovial joint lubrication system and its partial collapse. (a) SAPL cover the articular surfaces (arrows). High molecular weight HA is attached to the SAPL as fluid film. PLA2 is present in the joint. HA protects the SAPL from attack by PLA2. (b) After excessive overloading of the joint, free radicals may be produced, and thereby degrading the HA. In its degraded form HA does not inhibit PLA2 activity, thus enabling lysis of the SAPL layer. (c) On lysis of the SAPL, the articular surfaces are stripped of their lubricants, exposing them to unwanted effects. Because these surfaces are smooth and possess high surface energy, friction is generated between the ‘naked’ articular surfaces (arrows). Such friction is probably the prime mover in the degenerative changes in the joint.

 
It must be noted, however, that excessive joint loading produces free radicals, which are capable of degrading HA [26]. In its degraded form, HA is presumably unable to inhibit PLA2 activity, and without such protection SAPL become accessible to hydrolysis (Fig. 4bGo and cGo). It is warranted to assume that, in these circumstances, HA not only loses its role as a protector of phospholipids but also increases their hydrolysis by PLA2. It is well known that under certain circumstances antioxidants may act [30, 31] as pro-oxidants (oxidation mediators), accelerating the lysis of SAPL.

What is the importance of the integrity of lubrication with regard to joint function? How does the elimination of SAPL affect joint function [10, 11, 33–35]? The uncovered articular surfaces are elastic smooth planes [36] with high surface energy [22, 37]; as such they generate increased friction between the surfaces when lacking in lubrication. Increased friction plays an important role in a variety of joint disorders [12, 38–41; D. W. Nitzan, submitted for publication], which may all culminate in deteriorating degenerative joint disease.

In a similar fashion, the high molecular weight HA inhibits angiogenesis, which probably accounts for the avascular, white appearance of the healthy articular surface [13]. The acceleration of angiogenesis by dHA, on the other hand, explains the typical capillary-rich appearance of the inflamed articular surface [13].

There is a comparable model of protection at the cellular level that underscores our theory: PLA2 is secreted into the extracellular fluid, where it constitutes an active component in the inflammatory process, posing a threat to the cell membrane phospholipids of the target cells [42]. It has been shown [42] that a mechanism similar to that described for SAPL in this study acts at the cellular level: cell-surface proteoglycans protect the cell membrane from being lysed by PLA2, a condition that is abrogated in the presence of reactive oxidative species [42]. The latter degrade cell-surface proteoglycans, rendering the membrane phospholipids accessible to hydrolysis by PLA2.


   Notes
 
Correspondence to: D. W. Nitzan, Department of Oral and Maxillofacial Surgery, Faculty of Dental Medicine, P.O.B. 12272, Jerusalem 91120, Israel. Back


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 14 January 2000; Accepted 9 October 2000


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