Molecular determinants of monosodium urate crystal-induced murine peritonitis: a role for endogenous mast cells and a distinct requirement for endothelial-derived selectins.

Injection of monosodium urate (MSU) crystals, the etiological cause of gouty arthritis, into murine peritoneal cavities produced an intense recruitment of polymorphonuclear leukocytes (PMN). After 3 mg MSU crystal injection, cell influx was maximal (approximately 10 x 10[6] cells per mouse) at 6 hr postinjection and sustained up to the 24 hr time-point. In mice depleted of mast cells by administration of compound 48/80 72 hr before challenge with MSU crystals a lower PMN influx was measured (58% reduction). The occurrence of endogenous mast cell activation, in the MSU response, was validated by the observation that MSU challenge reduced by more than 90% the number of intact mast cells recovered in the peritoneal washes. Pretreatment of mice with a histamine H1 antagonist (tripolidine; 0.5 mg/kg) or a platelet-activating factor receptor antagonist (WEB2086; 10 mg/kg) significantly reduced by 50 to 60% the number of PMN recovered from the peritoneal cavities. The molecular determinants of this process of leukocyte recruitment were also investigated. Treatment of mice with an anti-CD62P or anti-CD62E monoclonal antibody (mAb; 100 microg i.v.) produced a distinct inhibition of PMN recruitment measured at 6 hr, whereas only a combined administration of both monoclonal antibodies was effective in reducing by 60% the influx of PMN caused by the MSU crystals within 24 hr. In conclusion, these data highlight a role for endogenous mast cells and for endothelial-derived selectins in MSU crystal-induced PMN recruitment into the peritoneal cavity, and may be useful to dissect molecular mechanism(s) which may be operating in gouty arthritis.

Viscosupplementation with hylan for the treatment of osteoarthritis: findings from clinical practice in Canada.

OBJECTIVE: To evaluate viscosupplementation with intraarticular hylan G-F 20 in current clinical practice. METHODS: A retrospective study of all patients with osteoarthritis of the knee treated with hylan by 5 Canadian clinicians over a period of 2.5 years. RESULTS: A total of 1537 injections were performed in 336 patients involving 458 knees. The overall response and the change of activity level were judged better or much better for 77 and 76% of the treated knees after the first course of treatment (3 weekly injections), and 87 and 84% after a 2nd course. The mean time elapsing between the first and 2nd course, 8.2 +/- 0.5 months, is an evaluation of the duration of benefits. Local adverse events were observed in 28 patients (32 knees), with an overall rate of 2.7% adverse events per injection, 7.0% per joint, and 8.3% per patient. No systemic adverse events were noted in any patient. The adverse events were characterized by pain and/or transient swelling of the injected joint, mostly mild or moderate in intensity, and 72% of the adverse events were considered to be possibly or probably related to the injection. The incidence of adverse events is significantly influenced by the injection technique: 5.2% adverse events per injection with a medial approach to a partially bent knee, and 2.4% (straight medial) and 1.5% (straight lateral). After an adverse event, clinical improvement still occurred in 69% of the affected knees. CONCLUSION: Hylan G-F 20 provided good clinical benefits and an acceptable safety profile in current clinical practice. The occurrence of adverse events after an intraarticular hylan injection is infrequent and unpredictable and is not necessarily hylan related, although injection related.

Paradoxical effects of colchicine on the activation of human neutrophilis by chemotactic factors and inflammatory microcrystal.

Neutrophil activation by chemotactic factors and by inflammatory microcrystals is accompanied by increases in protein tyrosine phosphorylation and by the activation of the NADPH oxidase. The addition of colchicine inhibited both responses induced by triclinic monosodium urate or calcium pyrophosphate crystals. On the other hand, colchicine enhanced the tyrosine phosphorylation of specific protein in neutrophils stimulated by chemotactic factor and augmented the production of superoxide anions induced by these same agonists. The effects of colchicine were shared by other anti-microtubule agents (nocodazole and vinblastine) but not by its inactive analogue beta-lumicolchicine, trimethylcolchicinic acid, indomethacin, or phenylbutazone. Furthermore, the (enhancing as well as inhibitory) effects of colchicine on tyrosine phosphorylation and superoxide anion production were reversed by taxol. Finally, in human cytoplasts colchicine again inhibited microcrystal-stimulated tyrosine phosphorylation but did not change chemotactic factor-stimulated phosphorylation. These data strongly support the hypothesis that microtubule-related mechanisms are involved in the modulation of the tyrosine phosphorylation response in human neutrophils, and suggest that a relationship may exist between the augmentation of tyrosine phosphorylation and of the stimulation of the NADPH oxidase induced by chemotactic factors.

Monosodium urate crystals promote neutrophil adhesion via a CD18-independent and selectin-independent mechanism.

The primary objective of this study was to determine whether monosodium urate (MSU) crystals induced neutrophil adhesion to cellular substrata and, if so, then to elucidate the molecular mechanisms involved. Human umbilical vein endothelial cells (HUVEC), as well as various other cellular substrata, were treated with various sized MSU crystals, washed, and then coincubated in the presence of neutrophils for 60 min. HUVEC exposed to MSU crystals but not to silica crystals or uric acid promoted neutrophil adhesion in a dose- and size-dependent manner, an event also observed with monolayers of rabbit synovial cells and rat intestinal epithelial cells. The increased neutrophil adhesion could not be attenuated by anti-CD18, anti-intracellular adhesion molecule-1, or various anti-selectin antibodies, despite the fact that scanning electron microscopy revealed that neutrophils were adhering primarily to the endothelial cells rather than to exposed crystals. CD18-deficient neutrophils adhered to MSU crystal-treated HUVEC as effectively as their CD18-positive counterparts. The neutrophil adhesion was temperature dependent but did not require protein synthesis. Additionally, HUVEC phagocytosis of crystals was necessary for subsequent neutrophil-endothelial cell interactions to transpire. Pretreatment of endothelial cells and neutrophils with colchicine significantly reduced the adhesive interaction. Our data demonstrate that exposure of endothelial and other cells to MSU crystals promotes neutrophil adhesion that occurs by a firm CD18-independent and selectin-independent adhesive mechanism.

Crystal-induced neutrophil activation. V. Differential production of biologically active IL-1 and IL-1 receptor antagonist.

Neutrophils produce IL-1 when stimulated by monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) crystals. Neutrophils also generate the IL-1R antagonist (IL-1Ra), especially when incubated with granulocyte-macrophage CSF (GM-CSF) or TNF-alpha. We studied the simultaneous expression of IL-1 and IL-1Ra by GM-CSF- or TNF-alpha-treated neutrophils activated by MSU or CPPD. Neutrophils incubated with GM-CSF or TNF-alpha produced approximately 300 or 200 times more IL-1Ra than IL-1, respectively. Suboptimal concentrations of MSU or CPPD induced low amounts of IL-1 without affecting IL-1Ra. Interaction of GM-CSF- and TNF-alpha-treated neutrophils with MSU or CPPD up-regulated IL-1 while simultaneously down-regulating IL-1Ra. As a result, the bioactivity of IL-1 secreted was enhanced. Synergistic increases of IL-1 (but not IL-1Ra) mRNA levels were noted in GM-CSF- or TNF-alpha-treated neutrophils exposed to CPPD. Treatment of neutrophils with colchicine before incubation with GM-CSF or TNF alpha, inhibited crystal-induced IL-1 by 50 to 55%, but failed to significantly affect IL-1Ra. The IL-1Ra to IL-1 ratio was significantly increased by 185 to 220%. These results demonstrate that IL-1 and IL-1Ra production by human neutrophils are differentially regulated, that the combined presence of GM-CSF or TNF-alpha and microcrystals favor the production of biologically active IL-1 over that of IL-1Ra, and that colchicine selectively inhibits IL-1 without affecting IL-1Ra production.

Acute apatite podagra with negative birefringent spherulites in the synovial fluid.

We describe a young male patient who presented with acute podagra after jogging activity. He had no underlying pathology. Polarized light microscopy of the synovial fluid from his first metatarsophalangeal joint revealed numerous negative birefringent spherulites about 6 microns in diameter, presenting the typical appearance of Maltese crosses. The molar calcium/phosphorus ratio of these spherulites as determined by X-ray energy dispersive analysis was virtually identical to that of synthetic or pathologic apatite.

Crystal-induced neutrophil activation. IV. Specific inhibition of tyrosine phosphorylation by colchicine.

We recently demonstrated that pathologically relevant inflammatory microcrystals, namely triclinic monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals, potently stimulate a characteristic protein tyrosine phosphorylation pattern in human neutrophils that differed from that observed in response to other soluble or particulate agonists. In this study, the effects of colchicine on protein tyrosine phosphorylation induced by MSU and CPPD crystals in human blood neutrophils were investigated. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that colchicine dose-dependently inhibited the tyrosine phosphorylation of all the proteins phosphorylated in response to MSU and CPPD crystals. Other microtubule-disruptive agents such as vinblastine, nocodazole, and colcemid also inhibited crystal-induced protein tyrosine phosphorylation while lumicolchicine and trimethylcolchicinic acid were without effect. Indomethacin and phenylbutazone were similarly without effect on microcrystal-induced tyrosine phosphorylation. Colchicine, as well as the other active alkaloids, failed to inhibit the protein tyrosine phosphorylation elicited by FMLP, C5a, leukotriene B4, and unopsonized zymosan. Overall, these results demonstrate that colchicine specifically and significantly inhibits the protein tyrosine phosphorylation induced by MSU and CPPD crystals and suggest that its effects are associated, at least in part, with its interaction with microtubules. Furthermore, the use of microtubule-disrupting drugs demonstrate that the mechanisms implicated in the induction of protein tyrosine phosphorylation by microcrystals differed from those involved in response to other soluble or particulate agonists.

Crystal-induced neutrophil activation. III. Inflammatory microcrystals induce a distinct pattern of tyrosine phosphorylation in human neutrophils.

The activation of human neutrophils by monosodium urate and calcium pyrophosphate dihydrate crystals is believed to play a critical role in the pathogenesis of arthritides such as acute gout and pseudogout, respectively. In this study, we investigated the potential involvement of tyrosine phosphorylation in microcrystal-mediated activation of human neutrophils. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that triclinic monosodium urate and calcium pyrophosphate dihydrate crystals stimulated a time- and concentration-dependent tyrosine phosphorylation of at least five proteins (pp130, 118, 80, 70, and 60). While phosphoprotein (pp) 118 and pp70 were the major phosphorylated substrates, pp70 was the dominant one in reactivity with antiphosphotyrosine antibodies. When the temporal patterns, as well as the levels of tyrosine phosphorylation for both types of crystals were compared, monosodium urate crystals were found to be more potent activators than calcium pyrophosphate dihydrate crystals. The tyrosine phosphorylation patterns induced by microcrystals differed from those stimulated by other soluble (FMLP, C5a, or leukotriene B4) or particulate (unopsonized latex beads or zymosan) agonists which stimulated preferentially the tyrosine phosphorylation of pp118. The ratio of the intensities of pp118 and pp70 were specific of the stimulation with microcrystals when compared to those observed with the other soluble or particulate agonists. Colchicine, a drug used specifically in the treatment of gout and pseudogout, inhibited microcrystal-induced tyrosine phosphorylation, while beta- and gamma-lumicolchicine were without effect. On the other hand, colchicine failed to inhibit FMLP-induced tyrosine phosphorylation. Furthermore, while colchicine inhibited the activation of the NADPH oxidase by microcrystals, it, on the other hand, enhanced the production of superoxide anions by FMLP. Taken together, these results (a) demonstrate that tyrosine phosphorylation is involved in the mechanism of activation of human neutrophils induced by microcrystals; and (b) suggest, on the basis of the characteristics of the observed patterns of tyrosine phosphorylation, that this response may be specific to the microcrystals and relevant to their phlogistic properties.

Crystal-induced neutrophil activation. II. Evidence for the activation of a phosphatidylcholine-specific phospholipase D.

OBJECTIVE: To investigate the involvement of phospholipase D in the signaling pathways activated by 2 pathologically relevant inflammatory microcrystals, monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD). METHODS: Human peripheral blood neutrophils were used throughout. Phospholipase D activity was monitored by measuring 3 separate indices: 1) the mass of phosphatidic acid, 2) the levels of alkyl-phosphatidic acid, and 3) the levels of formation, in the presence of ethanol, of phosphatidylethanol. The latter 2 parameters were measured in cells labeled with 1-0-3H-alkyl-2-acetyl-sn-glycero-3-phosphocholine. The cells were stimulated with microcrystals of triclinic morphology. RESULTS: Both MSU and CPPD crystals induced a time- and concentration-dependent accumulation of phosphatidic acid mass and elevation in levels of alkyl-phosphatidic acid and phosphatidylethanol in prelabeled cells. The activation of phospholipase D by the microcrystals was partially sensitive to colchicine and largely resistant to pertussis toxin. Inhibition of phosphatidic acid formation by wortmannin or ethanol reduced the microcrystal-stimulated production of superoxide anions. CONCLUSION: These results indicate that microcrystals stimulate phospholipase D in human neutrophils and that at least some of the functional consequences of neutrophil-microcrystal interactions may be dependent on this biochemical pathway.

Acute polyarthritis associated with birefringent lipid microspherules occurring in a patient with longstanding rheumatoid arthritis.

We describe a 52-year-old patient with longstanding rheumatoid arthritis (RA) who developed an acute polyarthritis of her hands and wrists. Synovial fluid analysis revealed the presence of intra and extracellular lipid microspherules with the typical appearance of Maltese crosses under polarized light microscopy. No other specific cause could be identified. This is the first description of an acute polyarthritis associated with lipid microspherules in RA.

Crystal-neutrophil interactions lead to interleukin-1 synthesis.

Normal human blood neutrophils were studied for their capacity to synthesize and release interleukin-1 (IL-1) species after phagocytosis of triclinic monosodium urate (MSU) and calcium pyrophosphate dihydrate crystals (CPPD). MSU crystals were more potent inducers of IL-1 generation than CPPD or unopsonized zymosan. Microcrystal-stimulated neutrophils characteristically secreted most of the newly synthesized IL-1. Colchicine partly inhibited the secretion of IL-1 by neutrophils during phagocytosis of solid particles. However, colchicine selectively inhibited IL-1 synthesis induced by microcrystals. These results suggest that neutrophil-derived IL-1 may contribute to the pathogenesis of crystal-induced arthritis.

Crystal-induced neutrophil activation. I. Initiation and modulation of calcium mobilization and superoxide production by microcrystals.

The effects of monosodium urate and calcium pyrophosphate dihydrate crystals on the levels of cytoplasmic free calcium and on the oxidative burst in normal human blood neutrophils were examined. The pattern of sensitivity to granulocyte-macrophage colony-stimulating factor, colchicine, cytochalasin B, pertussis toxin, diglyceride kinase, and protein kinase C inhibitors differentiated the mechanism(s) of neutrophil activation by the crystals from that involved in the responses to soluble chemotactic factors and indicated that individual crystals can use several activation pathways.

The use of a 51Cr technique to detect gastrointestinal microbleeding associated with nonsteroidal antiinflammatory drugs.

Of techniques used to evaluate gastrointestinal (GI) bleeding, use of radiochromium (51Cr)-tagged erythrocytes is the most quantitative and scientifically acceptable method. The value of this technique as well as systematic errors possible with its use are discussed. The medical literature concerning 51Cr evaluation of GI microbleeding with naproxen therapy is critically reviewed. We suggest that future studies using this technique be parallel, randomized, double-blind, and include a 1-week placebo baseline phase for all subjects. Treatment with nonsteroidal antiinflammatory drugs (NSAIDs) should last 3 to 4 weeks. A parallel group of subjects should receive placebo throughout the study. For valid statistical analyses, randomization must achieve baseline comparability of weight, height, age, and sex in the treatment groups. Data transformations may be necessary to satisfy the assumptions of the statistical model. Following these guidelines will enable investigators to better evaluate GI microbleeding during treatment with naproxen or other NSAIDs, and, hopefully, to establish the safety profiles of these drugs.

Monosodium urate and calcium pyrophosphate crystals differentially activate the excitation-response coupling sequence of human neutrophils.

The activation patterns of human neutrophils elicited by unopsonized monosodium urate and calcium pyrophosphate dihydrate crystals were investigated. The parameters chosen, the mobilization of calcium and the synthesis of leukotrienes, are generally accepted to be relevant to the activation of the cells and their pathophysiological roles. Both particles were found to elicit increases in cytoplasmic free calcium and leukotriene synthesis. However, the rank order of potency of these two stimuli was found to be sharply dependent on the test chosen. Monosodium urate crystals were significantly more effective than calcium pyrophosphate dihydrate crystals in terms of calcium mobilization, while the latter are more potent at inducing leukotriene synthesis. These results demonstrate that these two phagocytic particles which are related to separate inflammatory joint diseases differentially activate the excitation-response coupling sequence of human neutrophils.

Reducing property of some slow acting antirheumatic drugs.

Many slow acting antirheumatic drugs and several other drugs without antirheumatic activity possess a potential thiol function, i.e., a free SH group or one that is generated by hydrolysis. Since drugs with effective SH activity may react with intracellular disulfides, we evaluated their reducing properties by measuring their redox potential and their ability to react with glutathione, the most prevalent intracellular thiol, and dithiobis (nitrobenzoic acid), a specific reactant for thiols. Drugs containing aromatic thiols are poor reducers and are generally devoid of antirheumatic activity. Antirheumatic drugs, such as D-penicillamine, levamisole, gold salts, thiopronine and captopril, are potential aliphatic thiols with strong reducing properties. These different antirheumatic drugs may therefore operate by a common mechanism through an altered cellular redox equilibrium and sulfide-disulfide exchanges.

Adjuvant arthritis: influence of the adjuvant volume and composition on the established arthritis.

Adjuvant arthritis remains an interesting model for the evaluation of therapeutic agents and for the study of inflammatory mechanisms. The severity and time course of the primary nonspecific inflammation and of the induced polyarthritis are influenced by the composition and volume of the injected adjuvant. The injection of complete adjuvant produces always an oedema which is much greater than the sum of the oedemas induced by mineral oil or mycobacteria alone. In Lewis rats, highly susceptible to adjuvant arthritis, the intensity of the induced lesions increases with the injected oil volume and for equal volumes is maximal with 500 micrograms of Mycobacterium butyricum. In the injected paw the specific immune character of the inflammation is more important when a small volume of adjuvant is used and is maximal for 500 micrograms of mycobacteria in 0.1 ml of mineral oil.

Correlation between ossification and inflammation using a rat experimental model.

In seronegative spondylarthropathies both inflammation and ossification can be demonstrated. Inflammation is a hallmark of diseases associated with antigen HLA-B27 in ankylosing spondylitis, Reiter's syndrome, and acute uveitis. Ossification is traditionally considered the end product of inflammation, but clinical examination does not show that this is always the case in man. The relationship between inflammation and ossification is not demonstrated in experiments on spinal involvement in adjuvant arthritis in the rat. Using that experimental model, we tested the efficacy of 3 nonsteroidal antiinflammatory drugs (indomethacin, naproxen, and phenylbutazone) given at dosages comparable to those employed in clinical practice, but at a lower level than those used by drug companies in animals. Results show that the drug exhibiting almost no antiinflammatory activity in the rat at the dosage used, phenylbutazone, was the most powerful inhibitor of ossification. Another mechanism of local osteogenesis must be sought to explain such a phenomenon.