Urate crystals have been identified in virtually all gouty synovial fluids by compensated polarized light microscopy (1, 2). Phagocytosis of crystals by polymorphonuclear, and to a lesser degree, by mononuclear phagocytes is a constant feature of the acute attack (3). Microcrystalline calcium pyrophosphate and adrenocorticosteroid esters are phagocytosed during acute pseudogout (4) and during the "postinjection flare" (5) respectively. Injection of microcrystalllne sodium urate (6, 7), sodium orotate (6), calcium oxalate (7), calcium pyrophosphate (4)' and corticosteroid esters (5) all induce an acute inflammatory reaction. Phlogistic responses to crystals of different chemical composition are identical, leading to the use of the phrase "crystal-induced synovitis" as a generic term to emphasize the lack of specificity of the response (4).The response is also nonspecific with respect to host species (7-9). Therefore, an experimental model of crystal-induced inflammation was developed in the dog (10). The purpose of this work was to investigate the role of the polymorphonuclear leukocyte in crystal synovitis using this model. MethodIn a previous paper, we described a reproducible method for quantitating the intra-articular pressure, pH, and leukocyte (WBC) response to intrasynovial injection of monosodium urate crystals (10). In this study, 10 male mongrel dogs weighing between 12 and 25 kg were given 0.2 to 0.3 mg vinblastine 1 (VLB) per kg intravenously. Total and differential peripheral blood leukocyte counts were performed daily; crystals were injected 3 to 4 days later when the animals were lenkopenic.

The nearly constant finding of sodium urate crystals in aspirated gouty synovial fluid (1) led to the induction of acute arthritis in both human (2, 3) and canine (3) subiects by intrasynovial crystal injection. Calcium pyrophosphate crystals, identified in the synovial fluid from patients with a goutlike syndrome ("pseudogout"), and crystals of adrenocorticosteroid esters are also irritants when injected (4, 5). Crystal-induced inflammation is completely reversible, dose related, and nonspecific with regard to host or to the chemical composition of the injected crystal. These observations confirmed and extended findings recorded by His and Freudweiler at the end of the nineteenth century (6)(7)(8)).An experimental model of crystal synovitis in the dog and several methods for its quantification are described herein. Applications of these methods to the study of the host response mechanism are described in the accompanying paper (9) and in reference 10. MethodsGeneral Considerations.--The knee (stifle) joints of mongrel dogs weighing 12 to 25 kg were used. Light anesthesia was induced with intravenous sodium pentobarbital (25 to 35 mg/kg); additional small doses were given as needed for maintenance. One experiment was performed using the "physiologic" anesthetic chlorolose (50 mg/kg intravenously). Body temperature, monitored by rectal thermometer, was controlled with an electric blanket within a range of 2°C. In some experiments, the trachea was intubated and breathing was maintained with a me-*

Monosodium urate crystals added to human polymorphonuclear leukocytes are rapidly phagocytized. Crystals initially lie within phagosomes with a distinct, closely approximated phagosomal membrane. By 8 minutes phagolysosome distension is seen in some cells, degranulation begins, and phagolysosome membrane dissolution is seen. By 30 minutes there are many cell deaths with release of ingested crystals and cellular contents. Urate crystals have a profound and rapidly toxic effect on isolated leukocytes. The loss of phagolysosome membranes may be important in producing cell death.Polymorphonuclear leukocytes (PMNs) and crystals of monosodium urate (urate) are thought to be invariably present in the acutely inflamed gouty joint (1). The PMN is essential to the inflammatory response induced by urate crystals in the canine joint (2,3). In the present study, the effect of urate crystals on PMNs was studied by examining the sequential ultrastructural changes within human PMNs af- ter urate crystals were phagocytized, starting with changes that occurred immediately after the crystals were added to the cells. Others have described the electron microscopic appearance of urate crystals within synovial fluid leukocytes (-), but the duration of crystal-cell interaction was not known. Some urate crystals lie within lysophagosomes, but Schwarz et al (6) and Riddle et al (7,8) also noted that many urate crystals."*seemed to lie in the cytoplasm. Some were felt to be devoid of limiting membranes. We have shown that phagolysosome membranes are present early after in vitro crystal phagocytosis, with later disappearance of membranes being progressive. Increasing degranulation and cell necrosis were also seen. MATERIALS AND METHODSSynthetic monosodium urate crystals, ranging in length from one to 15 C, were prepared by the method of McCarty and Faires (9). The crystals were heated for 3 hours at l8O0C to remove any possible pyrogen contamination. Human leukocytes were separated from heparinized venous blood by dextran sedimentation (lo), and suspended in the

No abstract

Phagocytosis of monosodium urate (MSU) crystals by polymorphonuclear leukocytes (PMN) in vitro results in PMN degranulation, phagolysosome membrane dissolution, and cell death (1). Release of cytoplasmic as well as lysosomal enzymes has been reported to occur after in vitro phagocytosis of urate but not calcium pyrophosphate dihydrate (CPPD) crystals (2,3). Wallingford no distinct phagosome membranes around the urate crystals, although in pseudogout (calcium pyrophosphate crystal synovitis) easily demonstrable phagosomes have been described (5,6). These observations have suggested that the phagolysosome rupture seen with urates may not follow calcium pyrophosphate crystal phagocytosis.This report compares the ultrastructural findings after in vitro CPPD and MSU crystal phagocytosis. As judged by electron microscopy, synthetic CPPD crystals do not produce the rapid sequence of membrane lysis and cell death seen with MSU crystals. CPPD crystals, however, were also phagocytized less avidly than MSU. One possible factor in the decreased phagocytosis is the greater size of the CPPD. Grinding of CPPD or MSU to produce smaller crystals improved phagocytosis and may increase the injurious effect of CPPD on the PMN. MATERIALS AND METHODSSynthetic calcium pyrophosphate dihydrate crystals were prepared as described by Tse and Phelps (7). X-ray diffraction showed predominantly calcium pyrophosphate but also suggested slight contamination by material resembling hydroxyapatite. T h e preparation viewed by polarized light included both monodinic and triclinic crystals that appeared similar to those seen in human pseudogout. Crystals averaged 21 p i n length (95% of crystals were 10 to 26 p) and were wider than MSU crystals. A small amount of nonbirefringent globular material was also seen (Figure 1). The same preparation was used for all experiments.