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ROMOS
Abstract
The formation and deposition of monosodium urate (MSU) microcrystals in articular and periarticular tissues is the causative agent of acute or chronic inflammatory responses known as gouty arthritis. Mononuclear phagocyte activation is involved in early triggering events of gout attacks. Because stimulated mononuclear phagocytes can constitute an important source of the inducible isoform of cyclooxygenase (COX-2), we evaluated the effects that proinflammatory microcrystals might have on COX-2 protein expression in crystal-stimulated monocytes. We found that MSU crystals, but not calcium pyrophosphate dihydrate (CPPD) crystals, induced COX-2, which correlated with the synthesis of prostaglandin E2 (PGE2) and thromboxane A2(TXA2). Crystal-induced de novo synthesis of COX-2 was dependent on transcriptional and translational events. Inhibition of tyrosine phosphorylation, by herbimycin A, blocked crystal-induced COX-2. Similarly, an inhibitor of the p38 mitogen-activated protein kinase, SB 203580, inhibited the stimulation of COX-2. Colchicine inhibited crystal-induced COX-2. In all cases, prostanoid synthesis was concomitantly inhibited. Taken together, these results implicate COX-2 in the development of MSU-induced inflammation.
DEPOSITION OF monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) microcrystals in articular and periarticular tissues is the cause of acute or chronic inflammatory responses known as gouty arthritis and chondrocalcinosis, respectively.1-3 Clinical symptoms of these inflammatory responses are characterized by severe pain, edema, and erythema in the joint. Cell activation by MSU microcrystals is a central feature of acute gouty arthritis4 and proinflammatory microcrystals can interact with all of the major synovial cell types, including neutrophils, monocytes/macrophages, and fibroblast-like (type B) synoviocytes.3 In monocytes, for example, microcrystals stimulate the synthesis of a number of proinflammatory cytokines, such as interleukin-1 (IL-1), IL-6, IL-8, and tumor necrosis factor alpha (TNFα).4-8 Monocytes-macrophages exposed to MSU crystals in vitro also release prostaglandins. Prostanoids are found in the synovial fluid of gouty arthritis patients and prostaglandin E2 (PGE2) has been shown to be involved in crystal-induced inflammation.9 Early vasodilation, enhanced vascular permeability, and pain in gouty arthritis are likely to be mediated, at least in part, by vasoactive prostaglandins, including PGE2.10 Similarly, rats deficient in essential fatty acids, biologic precursors of prostaglandins, develop less footpad swelling than do normal rats following injection of MSU microcrystals,11 which suggests an involvement of prostanoids in crystal-induced inflammation.
Cyclooxygenase (COX) catalyzes the conversion of arachidonic acid to prostaglandin H2, the first committed step in the biosynthesis of prostanoids.12 13 To date, two COX isoforms, each encoded by distinct genes,14 have been described in mammalian cells.15 The COX-1 isozyme is expressed constitutively in most tissue types, often at low levels, and appears to be a “housekeeping” enzyme that supports the levels of prostanoid biosynthesis required for maintaining organ and tissue homeostasis.13 15 The second isoform, COX-2, is only expressed in a limited number of cells, including monocytes/macrophages, synovial cells, and fibroblasts.16COX-2 can be induced by various proinflammatory agents, including endotoxin, cytokines, mitogens, and lipid mediators,17 and is thought to be the predominant COX isoform involved in the inflammatory response.18 19
Stimulated human monocytes constitute an important source of COX-2–derived prostanoids, including PGE2 and thromboxane A2 (TXA2).20 Since a phlogistic role has been established for prostaglandins in gouty arthritis, and since monocyte activation appears to be involved in triggering events in gout attacks,21 we evaluated the effects that proinflammatory microcrystals might have on COX-2 protein expression in human monocytes. We now report that MSU microcrystals induce COX-2, as well as the production of prostanoids in these cells. The results of this study indicate that MSU crystals upregulate COX-2 levels in monocytes and potentially identifies a novel site of action of MSU crystals in the pathogenesis of gouty arthritis.
MATERIALS AND METHODS
REAGENTS.
Materials were obtained from the following sources: Pyrogen-free Lymphoprep, Nycomed, Oslo, Norway; RPMI culture medium and fetal calf serum (FCS), Biosciences, Sydney, Australia; Minisorp polyethylene tubes, Nunc, Roskilde, Denmark; Rabbit PGE2 antiserum, actinomycin D (AD), cycloheximide (CHX), E-Toxa-Clean, and herbimycin A, Sigma Chemical, St Louis, MO; Trans-Blot transfer membranes, Bio-Rad, North Ryde, Australia; rabbit polyclonal PGHS-2 (human) antiserum raised to a synthetic human PGHS-2 peptide,22 and PGE2, Cayman Chemical, Ann Arbor, MI. TXB2antiserum was prepared from a rabbit immunized with thromboxane conjugated to thyroglobulin and has been used in previous studies.23Peroxidase-labeled donkey antirabbit and goat antimouse antibodies, and the enhanced chemoluminescence immunoblotting analysis system were obtained from Amersham International, Little Chalfont, England. [4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole] (SB 203580), and [2-(4-Methylsulfinyl)-3-[4-(2-methylpyridyl)]-6,7-dihydro [5H] pyrrolo [1, 2-a] imidazole] (SK&F 106978) were generously supplied by Dr John C. Lee (SmithKline Beecham, King of Prussia, PA).
PREPARATION OF MICROCRYSTALS.
MSU and CPPD microcrystals were prepared using previously described methods1 2425 with modifications and were generously provided by Dr R. De Médicis (Unité des maladies rhumatismales, CUSE, Sherbrooke, Québec, Canada). Briefly, a boiling MSU solution (0.03 mol/L pH 7.5) was prepared by dissolution of equimolar quantities of uric acid and sodium hydroxide (3 μmol/L) and filtered on an Acropor membrane filter (AN-3000; Gelman Sciences, Ann Arbor, MI). Sodium chloride (0.1 mol/L final concentration) was added to speed up and improve the uniformity of the crystallization. CPPD was obtained by mixing a calcium nitrate solution (0.1 mol/L final concentration) with an acidic solution of sodium pyrophosphate (final concentration, 0.025 mol/L of Na2P2O7 and 0.03 mol/L HNO3). The milky-white precipitate formed CPPD crystals after a 1-day incubation at 50 to 60°C. The crystals were characterized by x-ray diffraction (Rigaku Geigerflex D/max), by examination under phase and polarizing microscopy and by scanning electron microscopy. The MSU and CPPD crystals showed triclinic morphologic characteristics. Their dimensions as determined by scanning microscopy were 10 × 1 × 1 μm to 25 × 1.5 × 1.5 μm and 12 × 1.4 × 1.4 μm to 25 × 1.7 × 1.7 μm for MSU and CPPD, respectively. Crystal preparations were free of endotoxins (as assessed by by the Limulus Assay; Whittaker, Walkerville, MD) and used without opsonization. Before use, crystals were resuspended in endotoxin-free Hanks' balanced salt solution (HBSS).
MONOCYTE ISOLATION.
Packed-cell preparations were obtained fresh from the Red Cross Blood Center, Adelaide, South Australia. Mononuclear cells were collected following centrifugation (800g, 30 minutes) of the packed-cell fractions on pyrogen-free Lymphoprep. After multiple washings, the mononuclear cells were suspended in 10 mL running buffer (HBSS 1×, 0.01% EDTA, 0.1% glucose, 0.1% low-lipopolysaccharide FCS). In this solution, the cells underwent counter-current centrifugal elutriation (J-6M/E Elutriation System; Beckman, Palo Alto, CA) with constant rotor speed (2,050 rpm) and with a constant flow rate of 11 mL/min during 30 minutes. Purity of the obtained monocyte fraction (>85%) was assessed either by Giemsa staining of cytocentrifuged smears, or by FACS analysis using an anti-CD14 monoclonal antibody (clone FMC 32; Serotec, Adelaide, South Australia). Contaminating cells were essentially all lymphocytes; the relative absence of contaminating platelets in such preparations, as assessed by immunoblots for the detection of COX-1, was demonstrated previously.26Viability of the cells was consistently greater than 95% as determined by Trypan blue exclusion. For the maintenance of minimal lipopolysaccharide contamination, the mononuclear-cell isolation procedure was performed under sterile conditions; all glassware, plasticware, and elutriator tubing were treated with E-Toxa-Clean before each elutriation.
CELL STIMULATION.
Elutriated monocytes were resuspended (2 × 106cells/mL) in RPMI 1640 supplemented with 10% low-lipopolysaccharide FCS and penicillin/streptomycin. Cells were distributed in 1-mL aliquots, in Minisorp tubes (Nunc, Roskilde, Denmark) to minimize adhesion, and incubated at 37°C with 5% CO2 in a humidified atmosphere. Where mentioned, cells were preincubated for 10 minutes with the appropriate pharmacologic agent prepared in either ethanol or dimethylsulfoxide, or with an equal volume of diluent before stimulation. Concentration of organic solvent never exceeded 0.1%. For prostanoid (PGE2 and TXA2) measurements, cell suspensions were centrifuged and cell-free supernatants were stored at −20°C.
TXA2 AND PGE2 MEASUREMENT.
TXA2 has a half-life of approximately 30 seconds under physiologic conditions and is readily converted to the stable metabolite TXB2, which was measured. TXB2 and PGE2 levels were determined by radioimmunoassay (RIA), as previously described.23Cross-reactivities in the TXB2 RIA were 0.06% for PGE2, 0.05% for 6-keto PGF1α, and less than 0.05% for PGF2α. Cross-reactivities in the PGE2 RIA were less than 0.001% for TXB2, 4.6% for 6-keto PGF1α, and 3.8% for PGF2α. Internal controls were performed that confirmed that both PGE2 and TXB2 are metabolites that are readily released from the cells following their synthesis. Moreover, the presence of crystals, up to a concentration of 3 mg/mL, did not interfere with the detection of these prostanoids (data not shown).
IMMUNOBLOTS.
Following the desired treatment, cell pellets were processed for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots as described previously,27 with the following modifications. Cell pellets (5 × 106 cells) were resuspended in 75 μL of ice-cold lysis buffer (HEPES-buffered HBSS pH 7.4, 0.5% Triton X-100, 10 μg/mL phenylmethylsulfonyl fluoride [PMSF], 10 μg/mL leupeptin, 10 μg/mL aprotinin). Seventy-five microliters of 2× sample buffer (0.125 mol/L Trizma base, pH 6.8, 20% glycerol, 4% SDS, 10% β-mercaptoethanol) were then added and the samples were boiled for 5 minutes. Samples were loaded on a 9% acrylamide gel. Typically, the equivalent of 1.7 × 106cells were loaded in each well. Proteins were transferred at 4°C for 16 hours, at 300 mA current setting onto a Trans-Blot membrane. Equal protein loading and transfer efficiency were visualized by Ponceau red staining. The membranes were soaked for 30 minutes at 25°C in Tris-buffered saline (TBS; 25 mmol/L Tris-HCl pH 7.6, 0.2 mol/L NaCl, 0.15% Tween 20) containing 5% dried milk (wt/vol), and subsequently exposed to antibodies reacting with either human COX-1 or COX-2. The membranes were then washed twice with TBS, and incubated either with a horseradish peroxidase–linked donkey antirabbit, or goat antimouse antibody. Bound antibodies were revealed with the enhanced chemoluminescence reagent, following the manufacturer's protocol (Amersham).
STATISTICAL ANALYSIS.
Statistical analysis was performed by Student's paired t-test (two-tailed), and significance was considered to be attained whenP was less than .05.
RESULTS
EFFECT OF MICROCRYSTALS ON PROSTANOID PRODUCTION IN HUMAN MONOCYTES.
Elutriated human monocytes were incubated with increasing concentrations of inflammatory microcrystals for 24 hours, and cell-free supernatants were analyzed for prostanoid (PGE2and TXA2) synthesis. Prostanoid synthesis increased with increasing concentrations of MSU crystals, up to 1 mg/mL (Fig1A). Typically, prostanoid synthesis was first observed at a concentration of 0.3 mg/mL and was maximal at concentrations between 0.6 mg/mL and 1 mg/mL. Higher MSU crystal concentration (3 mg/mL) was associated with a diminution in prostanoid synthesis. Monocytes incubated with CPPD crystals up to a concentration of 3 mg/mL failed to consistently produce PGE2 or TXA2.
http://www.bloodjournal.org/content/91/5/1769?sso-checked=true
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