3 Answers

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 PGF_1α, and less than 0.05% for PGF_2α. Cross-reactivities in the PGE₂ RIA were less than 0.001% for TXB₂, 4.6% for 6-keto PGF_1α, and 3.8% for PGF_2α. 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.

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