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The effect of synovial hyaluronate on the ingestion of monosodium urate crystals by leukocytes
307
Clinica Chimica Acta, 55 (1974) 307-315 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6560
THE EFFECT OF SYNOVIAL HYALURONATE ON THE INGESTION MONOSODIUM URATE CRYSTALS BY LEUKOCYTES
KENNETH
OF
D. BRANDT*
Robert Dawson Evans Department of Clinical Research, University Hospital, and the Department of Medicine, Boston University School of Medicine, Boston, Mass. 02118 (U.S.A.)
(Received
April 9, 1974)
Summary To extend previous studies which showed that large-sized hyaluronate molecules inpeded chemotactic movement of leukocytes, the effect of synovial hyaluronate on the rate at which crystals of monosodium urate were ingested by leukocytes was examined. Hyaluronates with viscometry-average molecular weights ranging from 1.13 * lo6 to 0.56 - 10’ were isolated and purified after treatment of synovial fluid with hyaluronidase for varying periods of time. Crystals of monosodium urate and normal peripheral blood leukocytes were suspended in buffer or in various concentrations of the hyaluronate preparations and the percentage of cells containing intracellular crystals was determined by polarization microscopy after incubation for 15 minutes. Results indicated that the rate of phagocytosis in all solutions of macromolecular hyaluronate was reduced in comparison with that in buffer. With increasing concentration of hyaluronate progressively greater impedence to the ingestion of crystals was observed. Moreover, at equimolar concentrations of uranic acid, the number of cells which ingested crystals was inversely proportional to the molecular weight of the hyaluronate. Thus, the physicochemical state of synovial hyaluronate may modify the response of leukocytes to monosodium urate crystals. The relatively small polymeric size of hyaluronate in gouty synovial fluid, therefore, may facilitate the phagocytosis of urate crystals and, through the consequent release of mediators of tissue injury from phagocytic cells, augment the inflammatory response within the joint.
* Reprint requests should be addressed Boston, Mass. 02118, U.S.A.
to
the author, University Hospital, 750 Harrison Avenue,
308
Introduction
The pathogenesis of the joint inflammation in gouty arthritis is related by considerable experimental evidence to the consequences attending phagocytosis of crystals of monosodium mate (MSU) in the synovial fluid [l].Recently we reported that large-sized hyaluronate (HA) from synovial fluid impeded the chemotactic movement of leukocytes [2]. It has not been previously demonstrated, however, that physicochemical properties of synovial fluid or of synovial HA significantly affect the ability of cells to dispose of biological material. In the present study HA samples of varying molecular weight are shown to retard the removal of MSU crystals from suspension by normal leukocytes. The degree to which removal of crystals was impaired, relative to controls, is related to both the concentration and the molecular weight of the HA in the preparations. Materials and Methods
Synovial fluid, obtained by aspiration from the knee of a patient with degenerative joint disease, was centrifuged for 30 minutes at 3500 rpm to remove cells and particulate material. Approximately 1 ml was accurately weighed [3] into a volumetric flask and diluted with 25 volumes of 0.05 M phosphate buffer, pH 7.0, containing 0.15 M NaCl. The relative viscosity*, qrel, was then determined at 37” in an Ostwald viscometer (bulb capacity 3 ml; buffer flow time 71 seconds). Isolation of hyaluronate. HA was isolated essentially as described by Castor et al. [4]. Four volumes of absolute ethanol were added to 2 ml aliquots of the diluted synovial fluid. After standing overnight at 4” the samples were centrifuged at 3000 rpm for 30 minutes and the precipitates recovered by decantation, washed in 85% ethanol, absolute ethanol and acetone, and dried in vacua. The material was then dissolved in 4 ml of 0.05 M Tris buffer, pH 8.0, containing 3 mg of pronase (Calbiochem, Los Angeles, CA), and incubated overnight at 60” under toluene. The small amount of insoluble material remaining after digestion was removed by centrifugation. The clear digest was then diluted with two volumes of double-distilled water and approximately 0.3 ml of a 5% solution of cetylpyridinium chloride (CPC) in 0.2 M Na, SO, was added. After standing overnight at 37” the precipitate which formed was collected by centrifugation at 18 000 rpm for 45 minutes and dissolved in 3 ml of 2 M sodium acetate, pH 7.0, following which the concentrations of uranic acid [ 51 and protein [ 61 were determined. Completeness of precipitation of the HA after addition of both ethanol and CPC (see above) was assured by the inability to detect uranic acid in the supernatants after concentration by rotary evaporation at 40”. Determination of molecular weight of the HA. The concentration of HA in the CPC precipitates was derived from the concentration of uranic acid,
* Relative
viscosity
of the buffer.
is defined
herein
as the ratio
of the flow
time
of the diluted
svnovial
fluid
to that
assuming a molecular weight of 400 for the disaccharide. concentration the intrinsic viscosity, [n] , was derived: [77]
=
From the q,,i and HA
qsp
c (1 + 0.16 rjsp)’ in which qsp is the specific viscosity (qsp = nre, - 1) and c the concentration of HA in g/100 ml [7]. Molecular weight, M, of the HA was then determined according to the relationship described by Laurent et al. [Q] = 0.036 X IvIO.~~. Preparation
of polymeric
HAS of various sizes.
Aliquots (10 ml) of the 1:25 dilution of the synovial fluid were incubated at 37” for 5 and 15 minutes, respectively, with 50 units of testicular hyaluronidase (Worthington Biochemical Corp., Freehold, N.J.). Four volumes of absolute ethanol were then added to each aliquot and to undigested controls to precipitate the remaining macromolecular HA which, after standing overnight, was recovered by centrifugation, washed, dried, digested with pronase and purified by precipitation with CPC (see above). Three ml of 2 M sodium acetate were added to solubilize the HA-CPC complexes and, after aliquots were removed for determination of uranic acid, the HA was precipitated from the remainder of the sample by addition of 4 volumes of ethanol. After standing overnight in the cold the precipitated HA was recovered by centrifugation in the cold and dissolved in phosphate-buffered saline at 4” for use in studies of phagocytosis and determinations of molecular weight. Isolation of oligosaccharides. The oligosaccharides of HA which had resulted from digestion of the synovial fluid with hyaluronidase for 15 minutes (see above) were isolated as follows: After addition of absolute ethanol to the digests the supernatants were dried in vacua, dissolved in 2 ml of distilled water and applied to a column (0.8 cm X 7.0 cm) of Dowex-l-formate [9]. The column was washed with distilled water and the oligosaccharides were then eluted with 0.8 M formic acid, lyophilized, dissolved in phosphate buffer and analyzed for uranic acid content. Preparation of crystals. Crystals of MSU were obtained by centrifugation of synovial fluid aspirated from a patient during an attack of gouty arthritis, washed several times with distilled water to lyse associated cells, and lyophilized. Before use they were resuspended (10 mg/ml) in the phosphate buffer. Phagocytosis studies. The rate at which MSU crystals were ingested by leukocytes was studied in siliconized tubes containing 0.2 ml of the crystal suspension in buffer. Leukocytes, obtained from the peripheral blood of a normal individual by dextran sedimentation, were resuspended in heparinized plasma and 0.3 ml was added to each tube, giving a concentration of approxibuffer without HA, or of HA mately 6000 cells/mm3 . 0.5 ml of phosphate diluted with buffer, was then pipetted into each tube. In the tubes which contained HA a sufficient quantity was added to give final concentrations of uranic acid of either 500 or 100 pg/ml. The tubes were tightly stoppered and rotated constantly at low speed at
310
37” for 15 minutes. A drop of each sample was then stained directly with 1% eosin to assess cell viability and the remainder centrifuged for 5 minutes at 1000 rpm. The cells were washed once in phosphate-buffered saline and smears prepared on cover slips, stained for 10 minutes with ethanolic methylene blue, washed in ethanol and dried. Two hundred cells on each slide were examined by polarization microscopy and the number containing intracellular crystals determined. Each experiment was performed in triplicate and the results expressed as the percentage of cells * 1 standard deviation (SD.) containing intracytoplasmic crystals. No indication was obtained of any effect on the cells which could be attributed to CPC remaining associated with the HA preparations. Thus, when results of studies with undigested HA purified by CPC precipitation were compared with those obtained with the same HA treated as above with pronase and ethanol but not with CPC, results of eosin exclusion and crystal ingestion studies were essentially identical. Indeed, since the HAS were solubilized at 4” (i.e., below the solubility temperature of the cetylpyridinium) after precipitation with ethanol, the bulk of the CPC was removed from the polyanion in this step. The effect of the oligosaccharides resulting from hyaluronidase digestion of the HA was similarly tested. The oligosaccharides (50 or 250 pg uranic acid/ml) in 0.5 ml of buffer were employed in lieu of the macromolecular HA solution or, to examine their effect in the presence of macromolecular HA, 50 or 250 pg of the lyophilized oligosaccharides were added to tubes containing the undigested HA (see above) (Table II). Samples were incubated and smears prepared and examined as described. Results
Digestion with hyaluronidase for increasing periods of time resulted in increasing degradation of the synovial HA, as indicated by progressive fall in the intrinsic viscosity from 40 to 11 dl/g. The molecular weight of the undigested HA, derived from the viscometric data, was 2.94 * 106. After only 5 minutes of hyaluronidase treatment the molecular weight of the recovered HA had fallen to 1.13 * 106. Further digestion, for a total of 15 minutes, resulted in a degraded HA with molecular weight of 0.56 * lo6 (Table I). No evidence was obtained that leukocyte viability, as measured by eosin exclusion, was affected by either the purified HA preparation, which contained TABLE PARTIAL
I DEGRADATION
OF
SYNOVIAL
HYALURONATE
HYALURONIDASE
Digestion
Intrinsic
time
vicosity
(minutes)
(dlkm)
Mol.
0
40
5
19
1.13
11
0.56
15
2.94
wt X lo+
BY
LIMITED
DIGESTION
WITH
Fig. 1. Smear of leukocytes following incubation for 15 minutes at 37’ with monosodium urate crystals. Examination by polarization microscopy. Intracytoplasmic crystals (arrows) could be readily differentiated from those which were extracellular.
8% protein, the partially degraded macromolecular HAS, the oligosaccharides resulting from hyaluronidase digestion, or the crystals. At least 90% of cells excluded eosin after incubation for 1 hour in solutions containing each of the above reagents, a result comparable to that obtained after incubation in buffer. The suspensions of MSU were sufficiently thin at the final concentrations employed so that intra- and extracellular crystals could be differentiated with little difficulty (Fig. 1). The percentage of cells which had ingested crystals was thus readily determined. Standard deviations of the triplicate samples of HA or of buffer were in all cases less than 14%. When MSU crystals were suspended in buffer in the absence of HA, approximately 40% of cells ingested crystals after 15 minutes. (Fig. 2). The rate of phagocytosis in all solutions of macromolecular HA was reduced in comparison with that in buffer and varied with both (a) the molecular weight and (b) the concentration of the HA, as follows: (a) At equimolar concentrations of uranic acid the number of cells which ingested crystals was inversely proportional to the molecular weight of the HA. Thus, at concentrations of uranic acid of 500 pg/ml, 1% of cells ingested
q -HA, n =HA. 0
500
pg
100 fq
uranic uranic
acid/ml acid/ml
=Eluffer
Fig. 2. Rate at which monosodium urate crystals were ingested by leukocytes suspended in phosphatebuffered saline or in solutions of synovial HA. Cells were incubated for 15 minutes at 37’ and the percentage containing intracytoplasmic crystals was determined by polarization microscopy. Results represent the mean of tripIicate determinations. S.D. was f 14% or less in each case.
crystals in solutions containing undigested HA, 13% in solutions of HA of intermediate size and 28% in the smallest HA tested. (b) Regardless of the polymeric size of the HA, phagocytosis was greater in solutions containing 100 c(g uranic acid/ml than in those containing 500 pg uranic acid/ml. At the lower concentration, the proportion of cells ingesting crystals rose progressively from 13% in undigested HA to 30% in HA of intermediate size and to 50% in the HA of lowest molecular weight. Oligosaccharides of HA, alone or in the presence of undigested HA, at concentrations of the latter of either 100 or 500 pg uranic acid/ml, had no appreciable effect on the rate of removal of MSU crystals by cells (Table II).
TABLE II EFFECT OF OLIGOSACCHARIDES TALS BY LEUKOCYTES
OF HYALURONIC
ACID ON PHAGOCYTOSIS
OF URATE CRYS-
Results represent the mean of triplicate experiments, with incubation for 15 minutes at 37’. Undigested HA (/.a uranic acid/ml)
Oligosaccharides (/xg uranic acid) 0
0
100
500
50 250 0 50 250 0 50 250
Cells containing crystals (% k 1 S.D.) 40.5 36.1 42.7 34.0 30.3 34.8 5.1 5.0 7.2
+ + f f * + * ? ?
3.2 3.9 4.4 2.9 4.0 3.3 0.5 0.3 0.8
313
Discussion The importance of phagocytic leukocytes in the genesis of tissue injury in a variety of joint diseases [lO,ll] , including gout [l] , has been delineated. Indeed, in the absence of polymorphonuclear leukocytes the synovitis which follows intra-articular injection of MSU crystals does not develop [ 121. After injection of MSU crystals into canine joints chemotactic activity has been detected in the synovial fluid [13]. By facilitating crystal phagocytosis, synovial fluid chemotactic factors may augment the inflammatory response in gouty arthritis, insofar as the inflammation is related to the release from phagocytes of lysosomal hydrolases or other mediators of tissue injury [l] . It has previously been shown that HA may impede the response of cells to defined chemotactic agents. For example, the directional migration of normal leukocytes induced by an Escherichia coli preparation has been found to be suppressed by synovial HA [ 21. In other circumstances HA may modify the chemotactic response by a different mechanism, i.e., by suppressing the stimulation of lymphocytes induced by mitogens [ 141. Since the stimulated lymphocyte elaborates chemotactic factors, synovial HA may affect release of the latter in cell-mediated hypersensitivity reactions within the joint. Thus, previous data suggested that HA might modify the ability of leukocytes to dispose of particulate material, such as crystals, in synovial fluid. The present study extends these observations and shows that when leukocytes and MSU crystals are suspended in synovial HA, the avidity with which the crystals are ingested by the cells is inversely related to both the concentration and molecular weight of the HA. The determinations of uranic acid employed in the present study to calculate intrinsic viscosities were presumably accurate, since precipitation of the various macromolecular HAS with CPC and ethanol was complete and volumes were determined gravimetrically to eliminate the inaccuracies inherent in pipetting viscous solutions of HA [ 31. Phagocytosis in all HA solutions in the present study was less than that in buffer. However, since phagocytosis in HA of lowest polymeric size was greater than that in HA which had been digested less extensively by hyaluronidase, the possibility was considered that the greater phagocytosis rate observed in low polymeric HA might have been due to an opsonizing effect of oligosaccharides The methods employed, which arising from the hyaluronidase digestions. involved precipitation with ethanol and cetylpyridinium chloride, should have excluded contamination of the partially digested HAS with oligosaccharides. Moreover, when oligosaccharides were added to the test system there was no evidence that they facilitated removal of crystals by the cells. Studies in Boyden chambers, described elsewhere [15], excluded the presence of chemotactic activity in the HA and the crystal preparations, although the possibility that a chemotactic factor was generated following interaction of crystals and leukocytes [ 131 was not excluded. This is significant in interpreting the progressive reductions observed in crystal phagocytosis in HAS of increasing size; if a chemotactic factor had been present the results could have been attributed to its selective adsorption or inactivation by HA of larger molecular size.
314
Although the above experiments show clearly that HA suppressed phagocytosis of MSU crystals, they do not contribute to an understanding of the mechanisms involved. However, it has been shown that chemotactic responsiveness of leukocytes may be impaired following phagocytosis of macromolecular material [ 16,171. If, by analogy, higher molecular weight HA had been selectively phagocytized, diminished chemotaxis in solutions containing larger-sized HA might have occurred. On the other hand, differences in phagocytosis rates in HAS of varying polymeric size may have been due to mechanical factors, i.e., the movement of cells may have been progressively impeded by increasingly viscous HA solutions. To further probe these possible mechanisms studies are currently in progress of HA phagocytosis by leukocytes and of chemotaxis in synthetic materials having viscosities similar to that of synovial fluid. The significance of the present study lies in the fact that it indicates that synovial fluid HA itself may modify the biological activity of phagocytes in the joint space. In the context of the above results it may be relevant that phagocytosis of staphylococci has been found to be diminished in synovial fluid in comparison with that in serum of patients with rheumatoid arthritis
[W. Although measurements of HA size in gouty synovial fluid are lacking, intrinsic viscosities and concentrations of the HAS utilized in the present study are similar to those encountered in synovial fluid of patients with other types of inflammatory joint disease [19]. On the basis of the mucin test [20], 86% of 75 synovial fluids from patients with proven gouty arthritis examined in this unit have demonstrated an abnormally low degree of HA polymerization. The above results suggest that the smaller polymeric size of HA in gouty synovial fluids may facilitate removal of MSU crystals by leukocytes. It could thereby contribute to the intensity of the inflammation in the joint. Acknowledgements These investigations were supported by grants from the United States Public Health Service, National Institute of Arthritis and Metabolic Diseases (AM-04599 and TI-AM-5285), from the General Clinical Research Centers Branch of the Division of Research Sources, National Institutes of Health (RR-533), from the Massachusetts Chapter of the Arthritis Foundation and from the Arthritis Foundation. The author is the recipient of a Special Research Fellowship from the National Institutes of Health. References 1
G. Weissman,
2
K. Brandt.
Advan. Arthritis
3
D. Hamerman
4
C.W.
5
T. Bitter
6
O.H.
7
L. Sundblad,
Castor. and
Lowry,
8
T.C.
9
B. Weissmann.
10
J.L.
fiaurent, Holkmder,
Int.
Med.,
Rheum.,
and
H. Schuster,
D. Wright
and
H. Muir,
An&t.
N.J.
Sm.
M. Ryan Myer,
D.J.
A.L.
37
4 (1962)
Farrand 58
(1958)
Arthritis
J. Biol.
G. Astorga.
57 Rheum.,
11
(1968)
652
330
R.J.
(1953)
Randall,
J. Biol.
Chem.,
193
(1951)
113
Pietruszkiewicz.
P. Sampson.
McCarty.
Invest.,
Buckingham,
Ups., A.
239 308
Biochem.,
Med. and
(1974)
(1970) J. Clin.
R.V.
Rosebrough,
Acta K.
19
13
Biochim. Chem.,
Ann.
Int.
Biophys.
208
(1954)
Med..
62
Acta. 417
(1965)
271
42
(1960)
476
265
315 11 12 13 14 15 16 17 18 19 20
G. Weissmann, Arthritis Rheum., 9 (1966) 834 P. Phelps and D.J. McCarty, J. Exp. Med., 124 (1966) 115 P. Phelps, J. Lab. Clin. Med., 76 (1970) 622 Z. Darzynkiewicz and E.A. B&m, Expt. Cell Res., 66 (1971) 113 K.D. Brandt and A.S. Cohen. In preparation H.U. Keller and E. Sorkin, Int. Arch. Allergy, 34 (1968) 513 A.G. Mowat and J. Baum, J. CIin. Invest. 50 (1971) 2541 P.T. Bode1 and J.W. HoIlingsworth. J. Clin. Invest., 45 (580) 1966 A.J. BoUet. J. Lab. Clin. Med. 48 (1954) 721 AS. Cohen. In AS. Cohen (Ed.), Laboratory Diagnostic Procedures Brown and Co. Boston. 1967
in the Rheumatic
Diseases, Little,
Clinica Chimica Acta, 55 (1974) 307-315 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6560
THE EFFECT OF SYNOVIAL HYALURONATE ON THE INGESTION MONOSODIUM URATE CRYSTALS BY LEUKOCYTES
KENNETH
OF
D. BRANDT*
Robert Dawson Evans Department of Clinical Research, University Hospital, and the Department of Medicine, Boston University School of Medicine, Boston, Mass. 02118 (U.S.A.)
(Received
April 9, 1974)
Summary To extend previous studies which showed that large-sized hyaluronate molecules inpeded chemotactic movement of leukocytes, the effect of synovial hyaluronate on the rate at which crystals of monosodium urate were ingested by leukocytes was examined. Hyaluronates with viscometry-average molecular weights ranging from 1.13 * lo6 to 0.56 - 10’ were isolated and purified after treatment of synovial fluid with hyaluronidase for varying periods of time. Crystals of monosodium urate and normal peripheral blood leukocytes were suspended in buffer or in various concentrations of the hyaluronate preparations and the percentage of cells containing intracellular crystals was determined by polarization microscopy after incubation for 15 minutes. Results indicated that the rate of phagocytosis in all solutions of macromolecular hyaluronate was reduced in comparison with that in buffer. With increasing concentration of hyaluronate progressively greater impedence to the ingestion of crystals was observed. Moreover, at equimolar concentrations of uranic acid, the number of cells which ingested crystals was inversely proportional to the molecular weight of the hyaluronate. Thus, the physicochemical state of synovial hyaluronate may modify the response of leukocytes to monosodium urate crystals. The relatively small polymeric size of hyaluronate in gouty synovial fluid, therefore, may facilitate the phagocytosis of urate crystals and, through the consequent release of mediators of tissue injury from phagocytic cells, augment the inflammatory response within the joint.
* Reprint requests should be addressed Boston, Mass. 02118, U.S.A.
to
the author, University Hospital, 750 Harrison Avenue,
308
Introduction
The pathogenesis of the joint inflammation in gouty arthritis is related by considerable experimental evidence to the consequences attending phagocytosis of crystals of monosodium mate (MSU) in the synovial fluid [l].Recently we reported that large-sized hyaluronate (HA) from synovial fluid impeded the chemotactic movement of leukocytes [2]. It has not been previously demonstrated, however, that physicochemical properties of synovial fluid or of synovial HA significantly affect the ability of cells to dispose of biological material. In the present study HA samples of varying molecular weight are shown to retard the removal of MSU crystals from suspension by normal leukocytes. The degree to which removal of crystals was impaired, relative to controls, is related to both the concentration and the molecular weight of the HA in the preparations. Materials and Methods
Synovial fluid, obtained by aspiration from the knee of a patient with degenerative joint disease, was centrifuged for 30 minutes at 3500 rpm to remove cells and particulate material. Approximately 1 ml was accurately weighed [3] into a volumetric flask and diluted with 25 volumes of 0.05 M phosphate buffer, pH 7.0, containing 0.15 M NaCl. The relative viscosity*, qrel, was then determined at 37” in an Ostwald viscometer (bulb capacity 3 ml; buffer flow time 71 seconds). Isolation of hyaluronate. HA was isolated essentially as described by Castor et al. [4]. Four volumes of absolute ethanol were added to 2 ml aliquots of the diluted synovial fluid. After standing overnight at 4” the samples were centrifuged at 3000 rpm for 30 minutes and the precipitates recovered by decantation, washed in 85% ethanol, absolute ethanol and acetone, and dried in vacua. The material was then dissolved in 4 ml of 0.05 M Tris buffer, pH 8.0, containing 3 mg of pronase (Calbiochem, Los Angeles, CA), and incubated overnight at 60” under toluene. The small amount of insoluble material remaining after digestion was removed by centrifugation. The clear digest was then diluted with two volumes of double-distilled water and approximately 0.3 ml of a 5% solution of cetylpyridinium chloride (CPC) in 0.2 M Na, SO, was added. After standing overnight at 37” the precipitate which formed was collected by centrifugation at 18 000 rpm for 45 minutes and dissolved in 3 ml of 2 M sodium acetate, pH 7.0, following which the concentrations of uranic acid [ 51 and protein [ 61 were determined. Completeness of precipitation of the HA after addition of both ethanol and CPC (see above) was assured by the inability to detect uranic acid in the supernatants after concentration by rotary evaporation at 40”. Determination of molecular weight of the HA. The concentration of HA in the CPC precipitates was derived from the concentration of uranic acid,
* Relative
viscosity
of the buffer.
is defined
herein
as the ratio
of the flow
time
of the diluted
svnovial
fluid
to that
assuming a molecular weight of 400 for the disaccharide. concentration the intrinsic viscosity, [n] , was derived: [77]
=
From the q,,i and HA
qsp
c (1 + 0.16 rjsp)’ in which qsp is the specific viscosity (qsp = nre, - 1) and c the concentration of HA in g/100 ml [7]. Molecular weight, M, of the HA was then determined according to the relationship described by Laurent et al. [Q] = 0.036 X IvIO.~~. Preparation
of polymeric
HAS of various sizes.
Aliquots (10 ml) of the 1:25 dilution of the synovial fluid were incubated at 37” for 5 and 15 minutes, respectively, with 50 units of testicular hyaluronidase (Worthington Biochemical Corp., Freehold, N.J.). Four volumes of absolute ethanol were then added to each aliquot and to undigested controls to precipitate the remaining macromolecular HA which, after standing overnight, was recovered by centrifugation, washed, dried, digested with pronase and purified by precipitation with CPC (see above). Three ml of 2 M sodium acetate were added to solubilize the HA-CPC complexes and, after aliquots were removed for determination of uranic acid, the HA was precipitated from the remainder of the sample by addition of 4 volumes of ethanol. After standing overnight in the cold the precipitated HA was recovered by centrifugation in the cold and dissolved in phosphate-buffered saline at 4” for use in studies of phagocytosis and determinations of molecular weight. Isolation of oligosaccharides. The oligosaccharides of HA which had resulted from digestion of the synovial fluid with hyaluronidase for 15 minutes (see above) were isolated as follows: After addition of absolute ethanol to the digests the supernatants were dried in vacua, dissolved in 2 ml of distilled water and applied to a column (0.8 cm X 7.0 cm) of Dowex-l-formate [9]. The column was washed with distilled water and the oligosaccharides were then eluted with 0.8 M formic acid, lyophilized, dissolved in phosphate buffer and analyzed for uranic acid content. Preparation of crystals. Crystals of MSU were obtained by centrifugation of synovial fluid aspirated from a patient during an attack of gouty arthritis, washed several times with distilled water to lyse associated cells, and lyophilized. Before use they were resuspended (10 mg/ml) in the phosphate buffer. Phagocytosis studies. The rate at which MSU crystals were ingested by leukocytes was studied in siliconized tubes containing 0.2 ml of the crystal suspension in buffer. Leukocytes, obtained from the peripheral blood of a normal individual by dextran sedimentation, were resuspended in heparinized plasma and 0.3 ml was added to each tube, giving a concentration of approxibuffer without HA, or of HA mately 6000 cells/mm3 . 0.5 ml of phosphate diluted with buffer, was then pipetted into each tube. In the tubes which contained HA a sufficient quantity was added to give final concentrations of uranic acid of either 500 or 100 pg/ml. The tubes were tightly stoppered and rotated constantly at low speed at
310
37” for 15 minutes. A drop of each sample was then stained directly with 1% eosin to assess cell viability and the remainder centrifuged for 5 minutes at 1000 rpm. The cells were washed once in phosphate-buffered saline and smears prepared on cover slips, stained for 10 minutes with ethanolic methylene blue, washed in ethanol and dried. Two hundred cells on each slide were examined by polarization microscopy and the number containing intracellular crystals determined. Each experiment was performed in triplicate and the results expressed as the percentage of cells * 1 standard deviation (SD.) containing intracytoplasmic crystals. No indication was obtained of any effect on the cells which could be attributed to CPC remaining associated with the HA preparations. Thus, when results of studies with undigested HA purified by CPC precipitation were compared with those obtained with the same HA treated as above with pronase and ethanol but not with CPC, results of eosin exclusion and crystal ingestion studies were essentially identical. Indeed, since the HAS were solubilized at 4” (i.e., below the solubility temperature of the cetylpyridinium) after precipitation with ethanol, the bulk of the CPC was removed from the polyanion in this step. The effect of the oligosaccharides resulting from hyaluronidase digestion of the HA was similarly tested. The oligosaccharides (50 or 250 pg uranic acid/ml) in 0.5 ml of buffer were employed in lieu of the macromolecular HA solution or, to examine their effect in the presence of macromolecular HA, 50 or 250 pg of the lyophilized oligosaccharides were added to tubes containing the undigested HA (see above) (Table II). Samples were incubated and smears prepared and examined as described. Results
Digestion with hyaluronidase for increasing periods of time resulted in increasing degradation of the synovial HA, as indicated by progressive fall in the intrinsic viscosity from 40 to 11 dl/g. The molecular weight of the undigested HA, derived from the viscometric data, was 2.94 * 106. After only 5 minutes of hyaluronidase treatment the molecular weight of the recovered HA had fallen to 1.13 * 106. Further digestion, for a total of 15 minutes, resulted in a degraded HA with molecular weight of 0.56 * lo6 (Table I). No evidence was obtained that leukocyte viability, as measured by eosin exclusion, was affected by either the purified HA preparation, which contained TABLE PARTIAL
I DEGRADATION
OF
SYNOVIAL
HYALURONATE
HYALURONIDASE
Digestion
Intrinsic
time
vicosity
(minutes)
(dlkm)
Mol.
0
40
5
19
1.13
11
0.56
15
2.94
wt X lo+
BY
LIMITED
DIGESTION
WITH
Fig. 1. Smear of leukocytes following incubation for 15 minutes at 37’ with monosodium urate crystals. Examination by polarization microscopy. Intracytoplasmic crystals (arrows) could be readily differentiated from those which were extracellular.
8% protein, the partially degraded macromolecular HAS, the oligosaccharides resulting from hyaluronidase digestion, or the crystals. At least 90% of cells excluded eosin after incubation for 1 hour in solutions containing each of the above reagents, a result comparable to that obtained after incubation in buffer. The suspensions of MSU were sufficiently thin at the final concentrations employed so that intra- and extracellular crystals could be differentiated with little difficulty (Fig. 1). The percentage of cells which had ingested crystals was thus readily determined. Standard deviations of the triplicate samples of HA or of buffer were in all cases less than 14%. When MSU crystals were suspended in buffer in the absence of HA, approximately 40% of cells ingested crystals after 15 minutes. (Fig. 2). The rate of phagocytosis in all solutions of macromolecular HA was reduced in comparison with that in buffer and varied with both (a) the molecular weight and (b) the concentration of the HA, as follows: (a) At equimolar concentrations of uranic acid the number of cells which ingested crystals was inversely proportional to the molecular weight of the HA. Thus, at concentrations of uranic acid of 500 pg/ml, 1% of cells ingested
q -HA, n =HA. 0
500
pg
100 fq
uranic uranic
acid/ml acid/ml
=Eluffer
Fig. 2. Rate at which monosodium urate crystals were ingested by leukocytes suspended in phosphatebuffered saline or in solutions of synovial HA. Cells were incubated for 15 minutes at 37’ and the percentage containing intracytoplasmic crystals was determined by polarization microscopy. Results represent the mean of tripIicate determinations. S.D. was f 14% or less in each case.
crystals in solutions containing undigested HA, 13% in solutions of HA of intermediate size and 28% in the smallest HA tested. (b) Regardless of the polymeric size of the HA, phagocytosis was greater in solutions containing 100 c(g uranic acid/ml than in those containing 500 pg uranic acid/ml. At the lower concentration, the proportion of cells ingesting crystals rose progressively from 13% in undigested HA to 30% in HA of intermediate size and to 50% in the HA of lowest molecular weight. Oligosaccharides of HA, alone or in the presence of undigested HA, at concentrations of the latter of either 100 or 500 pg uranic acid/ml, had no appreciable effect on the rate of removal of MSU crystals by cells (Table II).
TABLE II EFFECT OF OLIGOSACCHARIDES TALS BY LEUKOCYTES
OF HYALURONIC
ACID ON PHAGOCYTOSIS
OF URATE CRYS-
Results represent the mean of triplicate experiments, with incubation for 15 minutes at 37’. Undigested HA (/.a uranic acid/ml)
Oligosaccharides (/xg uranic acid) 0
0
100
500
50 250 0 50 250 0 50 250
Cells containing crystals (% k 1 S.D.) 40.5 36.1 42.7 34.0 30.3 34.8 5.1 5.0 7.2
+ + f f * + * ? ?
3.2 3.9 4.4 2.9 4.0 3.3 0.5 0.3 0.8
313
Discussion The importance of phagocytic leukocytes in the genesis of tissue injury in a variety of joint diseases [lO,ll] , including gout [l] , has been delineated. Indeed, in the absence of polymorphonuclear leukocytes the synovitis which follows intra-articular injection of MSU crystals does not develop [ 121. After injection of MSU crystals into canine joints chemotactic activity has been detected in the synovial fluid [13]. By facilitating crystal phagocytosis, synovial fluid chemotactic factors may augment the inflammatory response in gouty arthritis, insofar as the inflammation is related to the release from phagocytes of lysosomal hydrolases or other mediators of tissue injury [l] . It has previously been shown that HA may impede the response of cells to defined chemotactic agents. For example, the directional migration of normal leukocytes induced by an Escherichia coli preparation has been found to be suppressed by synovial HA [ 21. In other circumstances HA may modify the chemotactic response by a different mechanism, i.e., by suppressing the stimulation of lymphocytes induced by mitogens [ 141. Since the stimulated lymphocyte elaborates chemotactic factors, synovial HA may affect release of the latter in cell-mediated hypersensitivity reactions within the joint. Thus, previous data suggested that HA might modify the ability of leukocytes to dispose of particulate material, such as crystals, in synovial fluid. The present study extends these observations and shows that when leukocytes and MSU crystals are suspended in synovial HA, the avidity with which the crystals are ingested by the cells is inversely related to both the concentration and molecular weight of the HA. The determinations of uranic acid employed in the present study to calculate intrinsic viscosities were presumably accurate, since precipitation of the various macromolecular HAS with CPC and ethanol was complete and volumes were determined gravimetrically to eliminate the inaccuracies inherent in pipetting viscous solutions of HA [ 31. Phagocytosis in all HA solutions in the present study was less than that in buffer. However, since phagocytosis in HA of lowest polymeric size was greater than that in HA which had been digested less extensively by hyaluronidase, the possibility was considered that the greater phagocytosis rate observed in low polymeric HA might have been due to an opsonizing effect of oligosaccharides The methods employed, which arising from the hyaluronidase digestions. involved precipitation with ethanol and cetylpyridinium chloride, should have excluded contamination of the partially digested HAS with oligosaccharides. Moreover, when oligosaccharides were added to the test system there was no evidence that they facilitated removal of crystals by the cells. Studies in Boyden chambers, described elsewhere [15], excluded the presence of chemotactic activity in the HA and the crystal preparations, although the possibility that a chemotactic factor was generated following interaction of crystals and leukocytes [ 131 was not excluded. This is significant in interpreting the progressive reductions observed in crystal phagocytosis in HAS of increasing size; if a chemotactic factor had been present the results could have been attributed to its selective adsorption or inactivation by HA of larger molecular size.
314
Although the above experiments show clearly that HA suppressed phagocytosis of MSU crystals, they do not contribute to an understanding of the mechanisms involved. However, it has been shown that chemotactic responsiveness of leukocytes may be impaired following phagocytosis of macromolecular material [ 16,171. If, by analogy, higher molecular weight HA had been selectively phagocytized, diminished chemotaxis in solutions containing larger-sized HA might have occurred. On the other hand, differences in phagocytosis rates in HAS of varying polymeric size may have been due to mechanical factors, i.e., the movement of cells may have been progressively impeded by increasingly viscous HA solutions. To further probe these possible mechanisms studies are currently in progress of HA phagocytosis by leukocytes and of chemotaxis in synthetic materials having viscosities similar to that of synovial fluid. The significance of the present study lies in the fact that it indicates that synovial fluid HA itself may modify the biological activity of phagocytes in the joint space. In the context of the above results it may be relevant that phagocytosis of staphylococci has been found to be diminished in synovial fluid in comparison with that in serum of patients with rheumatoid arthritis
[W. Although measurements of HA size in gouty synovial fluid are lacking, intrinsic viscosities and concentrations of the HAS utilized in the present study are similar to those encountered in synovial fluid of patients with other types of inflammatory joint disease [19]. On the basis of the mucin test [20], 86% of 75 synovial fluids from patients with proven gouty arthritis examined in this unit have demonstrated an abnormally low degree of HA polymerization. The above results suggest that the smaller polymeric size of HA in gouty synovial fluids may facilitate removal of MSU crystals by leukocytes. It could thereby contribute to the intensity of the inflammation in the joint. Acknowledgements These investigations were supported by grants from the United States Public Health Service, National Institute of Arthritis and Metabolic Diseases (AM-04599 and TI-AM-5285), from the General Clinical Research Centers Branch of the Division of Research Sources, National Institutes of Health (RR-533), from the Massachusetts Chapter of the Arthritis Foundation and from the Arthritis Foundation. The author is the recipient of a Special Research Fellowship from the National Institutes of Health. References 1
G. Weissman,
2
K. Brandt.
Advan. Arthritis
3
D. Hamerman
4
C.W.
5
T. Bitter
6
O.H.
7
L. Sundblad,
Castor. and
Lowry,
8
T.C.
9
B. Weissmann.
10
J.L.
fiaurent, Holkmder,
Int.
Med.,
Rheum.,
and
H. Schuster,
D. Wright
and
H. Muir,
An&t.
N.J.
Sm.
M. Ryan Myer,
D.J.
A.L.
37
4 (1962)
Farrand 58
(1958)
Arthritis
J. Biol.
G. Astorga.
57 Rheum.,
11
(1968)
652
330
R.J.
(1953)
Randall,
J. Biol.
Chem.,
193
(1951)
113
Pietruszkiewicz.
P. Sampson.
McCarty.
Invest.,
Buckingham,
Ups., A.
239 308
Biochem.,
Med. and
(1974)
(1970) J. Clin.
R.V.
Rosebrough,
Acta K.
19
13
Biochim. Chem.,
Ann.
Int.
Biophys.
208
(1954)
Med..
62
Acta. 417
(1965)
271
42
(1960)
476
265
315 11 12 13 14 15 16 17 18 19 20
G. Weissmann, Arthritis Rheum., 9 (1966) 834 P. Phelps and D.J. McCarty, J. Exp. Med., 124 (1966) 115 P. Phelps, J. Lab. Clin. Med., 76 (1970) 622 Z. Darzynkiewicz and E.A. B&m, Expt. Cell Res., 66 (1971) 113 K.D. Brandt and A.S. Cohen. In preparation H.U. Keller and E. Sorkin, Int. Arch. Allergy, 34 (1968) 513 A.G. Mowat and J. Baum, J. CIin. Invest. 50 (1971) 2541 P.T. Bode1 and J.W. HoIlingsworth. J. Clin. Invest., 45 (580) 1966 A.J. BoUet. J. Lab. Clin. Med. 48 (1954) 721 AS. Cohen. In AS. Cohen (Ed.), Laboratory Diagnostic Procedures Brown and Co. Boston. 1967
in the Rheumatic
Diseases, Little,