ADSORPTION OF POLYMORPHONUCLEAR LEUKOCYTE LYSOSOMAL ENZYMES TO MONOSODIUM URATE CRYSTALS MARK H. GINSBERG, FRANKLIN KOZIN. DONALD CHOW, JAMES MAY, and JOHN L. SKOSEY Monosodium urate ( M S U ) crystals induced prompt release of iysozyme, and slower release of pglucuronidase, a-mannosidase, and lactic dehydrogenase ( L D H ) from polymorphonuclear leukocytes. At increasing crystal concentrations, an increasing delay in the apparent onset of p-glucuronidase release was detected which appears to be due to selective'adsorption of enzyme activities to the M S U crystals: P-glucuronidase > LDH > a-mannosidase > lysozyme. Lysosomal enzyme adsorption by MSU crystals may then contribute to experimental error or may modulate gouty inflammation.
The hypothesis that monosodium urate (MSU) crystal-induced enzyme release from polymorphonuclear (PMN) leukocytes occurs by perforation of the cell from within ( I ) has gained widespread acceptance (2-7). The observation that release of the lysosomal enzyme P-glucuronidase lags behind that of cytoplasmic From the Departments of Medicine, The University of Chicago Pritzker School of Medicine, and the Franklin McLean Memorial Research Institute, Chicago, Illinois, 60637, and the Medical College of Wisconsin, Milwaukee. Wisconsin, 53200. These studies were supported in part by USPHS Training Grant (AM-05621) from the National Institute of Arthritis, Metabolic, and Digestive Diseases, and by the Louis Block Board o f the University of Chicago. Address reprint requests to Dr. Mark Ginsberg. Scripps Clinic and Research Foundation, Department of Immunopathology. 10666 North Torrey Pines Road, La Jolla. California 92037. Submitted for publication February 23. 1977: accepted July
5. 1977. Arthritis and Rheumatism, Vol. 20, No. 8 (November-December 1977)
lactic dehydrogenase (LDH) (2,6) has been thought to support this concept. In such studies, MSU crystals are incubated with PMNs, the PMNs and crystals separated from medium by centrifugation, and the medium is assayed for enzyme activity. In similar studies, we noted a paradoxical decrease in 0-glucuronidase activity in the medium of MSU crystal-treated PMN suspensions with increasing crystal concentrations. Weissmann et al. (2) have shown that MSU crystals do not interfere with the assay for this enzyme and that this enzyme is not adsorbed to cellular surfaces. I n this report, we demonstrate significant adsorption of p-glucuronidase to MSU crystals and show that MSU-induced release of the granule enzymes, lysozyme, and a-mannosidase, which are less avidly adsorbed, does not lag behind LDH release. Further, lysozyme release precedes LDH release, a result supporting the recent suggestion (6) that perforation from within is not the sole mechanism of MSU crystal-induced lysosomal enzyme release.
MATERIALS AND METHODS Monosodium urate (MSU)crystals were prepared by neutralization of uric acid with I N NaOH (8) to form negatively birefringent needle-shaped 1-40 p long crystals (primarily 10-30 p ) . Crystals were dried, heated at 200°C for 2 hours to destroy possible adsorbed pyrogens, and suspended at the appropriate concentrations in Krebs-Ringer phosphate (KRP) buffer (9), pH 7.4. Washed human peripheral blood leukocytes (92-95% polyrnorphonuclear) in K R P at 5-10 X 10' cells/ml were
PMN LEUKOCYTE LYSOSOMAL ENZYMES
prepared as previously described (10). Three hundred pl of cell suspensions in K R P were incubated with 2 0 0 4 crystal suspensions in polyethylene tubes at 37°C with continuous shaking. Three ml of ice-cold K R P were added at appropriate times and the crystals plus cell pellet separated from the supernatant by centrifugation at 300 g for 10 minutes at 4°C. A sample of the suspension was lysed by addition of 3.0 ml of K R P containing Triton X-100 (final concentration 0.1% vol/vol) followed by sonication. Enzyme activities were assayed in supernatants and lysed cell suspensions. Results were expressed as percentage of total enzyme activity. In adsorption studies, Triton X-100was added to cell suspensions prepared as above to a concentration of 0.196, following which the suspensions were sonicated four times in a Sonifier cell disruptor (#1850) at “7” for 4 seconds per time. This material was then centrifuged 14,000 g in a Sorvall RCZB centrifuge at 0°C for 30 minutes to sediment cell ghosts and unruptured lysosomes ( 1 1 ). In some experiments, cell sap was prepared as above in the absence of Triton X-100 and behaved identically with regard to adsorption to crystals. Three hundred pl of this cell sap were added to 200 pl of crystal suspension at 37°C and incubated with constant shaking in a Dubnoff Metabolic Incubator for 10 minutes, at which time 3 ml of ice-cold K R P was added. The resultant suspension was centrifuged at 300 g for 10 minutes at 0°C. Enzyme determinations on the supernatant and crystal pellet were done a s outlined below. Lactic dehydrogenase (LDH) was assayed by the oxidation of N A D H recorded at 340 mp in a Beckman Acta I I I spectrophotometer and expressed as 1,000. Wroblewski units
a decrease of 1 optical density unit/minute/ml of sample
( 1 2).
Alpha-mannosidase and P-glucuronidase were measured simultaneously by a modification of the technique of Talalay et al. (13) in which 1.1 ml of acetate buffer, pH 4.5, containing 1 mM phenolphthalein 0-glucuronide and 5.5 mM p-nitrophenyl a - D - mannoside (Sigma Chemical, St. Louis, Missouri) was incubated with 300 pi of sample for 17 hours. Color was developed by addition of 2 ml of 0.4 M glycine buffer pH 10.5 and absorbance read at 550 mp for P-glucuronidase and 405 mp for a-mannosidase. Results are expressed as nanomoles of phenolphthalein and nanomoles of nitrophenol released per hours of incubation per milliliter of sample. Lysozyme was assayed turbidometrically ( 14) using a suspension of M lysodeikticus (Difco). Results were expressed as 1,000 units = a decline of 1 optical density unit/minute/ml of sample.
RESULTS When whole cells were incubated with MSU crystals (Figure l ) , initial release of lysozyme was more rapid than that of a-mannosidase o r 0-glucuronidase at all crystal concentrations employed. Lysozyme release preceded LDH release as well, a result suggesting that this enzyme is released before cell death occurs. At 2 mg/ml, MSU crystals, LDH, and p-glucuronidase release were nearly parallel. As the crystal concentration increased, an increasing lag in the appearance of p-
1 c 0
.-,>” 4 0 .c u
40 80 Time [min)
Fig 1. Time course of enzyme release from human PMN incubated in the presence of M S U crystals. Two hundred microliters of MSU crystals were added to a 300-pl cell suspension and incubated at 37°C. Reactions were stopped by addition o f 3 ml ice cold KRP and centrifugation at 4°C. A-A percent of total lysozyme activity (100% = 321 unitslnrl 1.vsare): x-x percent of total LDH activity (100% = 778 Wroblewski unitslml lysate); 0-0 percent of total a-mannosidase activity (100% = 7.7 nmofes nitrophenol releasedlhrlml lysate): 0-0 percent of total 8glucuronidase activity (100% = 33 nmoles phenolphthalein releasedlhrlml lysate). Values are mean f S E M of at least triplicate determinations in this and succeedingjigures. Where no error bars appear the standard error is within the point. Data shown represent one of two experiments measuring all four enzymes and six experiments measuring only lysozyme, L D H , and P-glucuronidase.
glucuronidase activity in the supernatant developed. In contrast, the pattern of a-mannosidase release did not change as the crystal concentration increased from 2 to 8 mg/ml. Because a-mannosidase activity is contained in the same granule population as P-glucuronidase activity (15), the disparity in apparent release of these lysosomal enzymes must be artifactual. At the highest concentrations, a lag in appearance of L D H activity occurred as well. At 8 mg MSU/ml, as percent total enzyme, significantly less L D H than a-mannosidase was detected in supernatants at 10- and 20-minute incubation (0.001 > P , 0.01 > P , respectively two-tailed t test). In adsorption experiments, a dramatic decline in supernatant /3-glucuronidase activity occurred at higher concentrations of MSU crystals. A corresponding increment in activity associated with the crystal pellet (Figure 2 ) was noted. The slight decrease in a-mannosidase activity was not statistically significant. L D H activity was negligibly decreased at higher enzyme concentrations; however, at lower L D H concentrations, loss of supernatant enzyme activity occurred at the higher crystal concentrations. Supernatant lysozyme recoveries were not materially altered by the concentrations of MSU crystals used. Heating MSU crystals to 200°C may alter their surface properties. T o exclude artifact related to heating, unheated MSU crystals were tested and showed similar lysosomal enzyme adsorption properties to the heated crystals. In addition, three different batches of needle-shaped MSU crystals gave similar results. The concentration of uric acid in solution was assayed in these samples by the enzymatic spectrophotometric technique (16) and was 2.5 mg/100 in all samples to which crystals had been added, this corresponds well with the reported solubility of MSU in buffers at 4°C (17). Figure 3 shows the effect of addition of fresh autologous serum on enzyme adsorption to urate crystals. Addition of up to 20% serum inhibits but does not abrogate the reduction of supernatant 0-glucuronidase activity. In the presence of serum, adsorbed enzyme activities were quantitatively recovered from the crystals. This indicates that adsorption artifact may have occurred in experiments done by others in the presence of 0.03 mg/ml(2) human serum albumin or 10%human serum (7). The slight increases in L D H activity at low crystal concentrations were not statistically significant; the declines in the absence of serum (diluted cell sap) 4 and 8 mg/ml were significant (0.05 > P , 0.01 > P respectively, two-tailed t test).
GINSBERG ET AL
'I 1 .o
[ M U Crystals] Img/mlj Fig 2. The supernatant activities of (3-glucuronidase, cu-mannosidase, L D H . and lysozyme as functions of M S U crystal concentration. Two hundred microliters of M S U crystals were added to 300 pl of cell sap and incubated at 37'C /or 10 minutes. M S U crystal concentrations are those achieved in a 500-pl incubarion mixture. Three milliliters oJ cold K R P were added and the supernatants isolated by centrifugation. One of two experiments with four enzymes and four experiments with omission of amannosidase. 0-0 activity in cell sap; 0-0 activity in corresponding cr.vstal pellets (crystal pellets not done for a-mannosidase); J-L] cell sap diluted 1 : l O with K R P . Mean zk S E M .
PMN LEUKOCYTE LYSOSOMAL ENZYMES
c VI .e 3
[ MSU Crystals] [mg/ml] Fig 3. Effect of serum on adsorption of LDH and P-glucuronidase to MSU crystals. Two hundred microliters of M S U crystals were added to 100 p l of $resh human serum neat. 1 : 2 and 1 : 4 ; 200 mi of cell sap or cell sap diluted I : 5 was added and the 500 p l of mixture was incubated at 37”C$or I0 minutes and treated as in Figure 2. Crystal and serum concentrations are those achieved in the 500-pl incubation mixture. 0-0 enzyme activities remaining in supernatants no serum present during adsorption; *-• enzyme activities remaining in supernatants 5% serum; 0-0 enzyme activities remaining in supernatanrs 10% serum; B-¤ enzyme activities remaining in supernatant 20% serum. Dotted lines represent 1 : s dilutions of cell sap. Data Jrom one of’ two experiments are shown.
DISCUSSION MSU crystals induce the release of P-glucuronidase and other PMN enzymes. The data presented here show that under the conditions of a typical experiment, a substantial artifactual reduction in supernatant 0glucuronidase occurs as a result of exposure to the MSU crystals. This reduction occurs despite the constant concentration of uric acid in solution produced by addition of from 1.2-8 mg/ml of MSU crystals. Further, corresponding increments in enzyme activity associated with the crystal pellets occurred. This result suggests that enzyme adsorption, rather than inhibition of enzyme activity by uric acid in solution or by some impurity in the crystals, is responsible for this effect. Selective protein adsorption has been described for silica crystals (18,19) and more recently for MSU crystals (20). The apparent increase in the delay of P-glucuronidase release from PMNs with increasing MSU crystal quantities can be explained by greater degrees of enzyme adsorption from the supernatant. The difference between the time course of P-glucuronidase and a-mannosidase release time is then due to selective /3-glucuronidase adsorption by crystals. Release of lysozyme
preceding that of L D H and a-mannosidase may be partially due to differential adsorption, but the constant difference between a-mannosidase and lysozyme at three crystal concentrations suggests that MSU crystals, like phorbolmyristate acetate (21 ), may induce selective release of specific neutrophil granules. There has been abundant morphologic (4-6) and biochemical (2,3,6) confirmation of the hypothesis that MSU crystals are phagocytosed and subsequently rupture the phagolysosomes from within, leading ultimately to cell death and to release of L D H and lysosomal enzymes. O u r data showing substantial LDH release are compatible with this hypothesis. Recent studies with cytochalasin B (6,22) have shown that inhibition of phagocytosis of MSU by PMNs is associated with a marked diminution in L D H release and a much less dramatic decline (6) or increase (22) in 6-glucuronidase release, results leading to the hypothesis that MSUinduced enzyme release proceeds by “reverse endocytosis” as well as “perforation from within” (6). Our data, which suggest early release of a granule enzyme, support this hypothesis. MSU crystals adsorb a variety of proteins (20) and protein adsorption has been shown to modulate the
GINSBERG ET AL
ability of t h e crystal t o release cellular constituents by either secretion or lysis (1,23). T h e observation t h a t lysosomal enzymes a r e a d s o r b e d by MSU crystals raises t h e possibility t h a t such enzymes in gouty j o i n t fluid bind t o MSU crystals a n d influence t h e course of g o u t y arthritis.
ACKNOWLEDGMENTS We gratefully acknowledge the expert secretarial assistance of Ms. Karen Kosakowski, Ms. Gen LaPinska, and Ms. Tomes Dearmont.
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10. Skosey JL, Chow D, Damgaard E, Sorenson LB: Effect of cytochalasin B on response of human polymorphonuclear leukocytes to zymosan. J Cell Biol 57:237-240, 1973 1 I . Beaufay H: Methods for the isolation of lysosomes, Lysosomes in Biology and Pathology. Vol 1 . Edited by J Dingle and H.B. Fell. New York, American Elsevier Publishing Company, pp 5 15-554, 1969 12. Bergmeyer HU, Bernt E, Hess B: Methods of Enzymatic Analysis. Edited by HU Bergmeyer. New York, Academic Press, pp 736-741, 1963 13. Talalay P, Fishman WH, Huggins C: Chromogenic substrates. 11. Phenolphthalein glucuronic acid as substrate for the assay of glucuronidase activity. J Biol Chem 166:757-772, 1946 14. Wright D G , Malawista SE: The mobilization and extracellular release of granular enzymes from human leukocytes during phagocytosis. J Cell Biol53:788-794, 1973 15. Bretz U , Baggiolini M: Biochemical and morphological characterization of azurophil and specific granules of human neutrophilic polymorphonuclear leukocytes. J Cell Biol 63:251-269(s), 1974 16. Praetorius E, Poulson H: Enzymatic determination of uric acid with detailed directions. Scand J Clin Lab Invest 5:273-280, 1953 17. Kippen I, Klinenberg J R , Weinberger A, Wilcox WR: Factors affecting urate solubility in vitro. Ann Rheum Dis 33:313-317, 1974 18. Jones BM, Edwards JH, Wagner JC: Absorption of serum proteins by inorganic dust. Br J lndustr Med 29:287-292, I972 19. Ishiyama H, Yasada J, Ito H: Selective adsorption of serum proteins to aerosil. Japan J Med Sci Biol 27:269-272, I974 20. Kozin F, McCarty DJ: Protein adsorption to monosodium urate, calcium pyrophosphate dihydrate, and silica crystals. Relationship to the pathogenesis of crystal induced inflammation. Arthritis Rheum 19:433-438, 1976 21. Estenson RD, White JG, Holmes: Specific degranulation of human polymorphonuclear leukocytes. Nature 258:347-348, 1974 22. Spilberg I, Gallacher A, Mendell B: Studies on crystal induced chemotactic factor. 11. Role of phagocytosis. J Lab Clin Med 85:631-635, 1975 23. Skosey JL, Kozin F, Ginsberg MH, et al: Protein adsorption t o monosodium urate crystals: Differential responses of human peripheral blood neutrophils. Second International Symposium on Purine metabolism in Man, in press 1976