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Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1769-1776
Monosodium Urate Microcrystals Induce Cyclooxygenase-2 in Human
Monocytes
By
Marc Pouliot,
Michael J. James,
Shaun R. McColl,
Paul H. Naccache, and
Leslie G. Cleland
From the Rheumatology Unit, Royal Adelaide Hospital, Adelaide;
Department of Microbiology and Immunology, The University of Adelaide,
Adelaide, South Australia; Centre de Recherche en
Rhumatologie et Immunologie, Centre de Recherche du CHUL, Laval
University, Ste-Foy, Québec, Canada.
 |
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.
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INTRODUCTION |
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
(TNF ).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.16
COX-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.
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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. TXB2
antiserum was prepared from a rabbit immunized with thromboxane
conjugated to thyroglobulin and has been used in previous
studies.23 Peroxidase-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
methods1,24,25 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.26
Viability 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 × 106
cells/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.23 Cross-reactivities in the
TXB2 RIA were 0.06% for PGE2, 0.05% for
6-keto PGF1 , and less than 0.05% for
PGF2. Cross-reactivities in the PGE2 RIA
were less than 0.001% for TXB2, 4.6% for 6-keto
PGF1, and 3.8% for PGF2. 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%  -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 × 106
cells 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 when
P was less than .05.
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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 (PGE2
and TXA2) synthesis. Prostanoid synthesis increased with
increasing concentrations of MSU crystals, up to 1 mg/mL (Fig
1A). 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.
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| Fig 1.
Effect of inflammatory microcrystals on prostanoid
synthesis by human monocytes. (A) Monocytes were incubated for 24 hours with either MSU or CPPD crystals at the indicated concentrations and
cell-free supernatants were analyzed for prostanoid (PGE2 and TXB2) synthesis. Data obtained from 3 donors are
presented, and values are the mean ± SD of duplicate determinations.
(B) Monocytes were incubated with MSU crystals (0.6 mg/mL) for the indicated times and prostanoid synthesis was measured. Data obtained from 3 donors are presented, and values are the mean ± SD of
duplicate determinations. (C) Monocytes were stimulated in the absence
(C), or presence of either MSU or CPPD crystals (0.6 mg/mL) for 24 hours and prostanoid synthesis was measured. When compared with control
cells, MSU microcrystals induced significant prostanoid synthesis,
*P < .01 (by t test). Data presented are the mean ± SD from at least seven experiments.
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In time-course experiments, cells were incubated with 0.6 mg/mL MSU
crystals for up to 24 hours. After an observed lag period of 3 to 6 hours, prostanoid synthesis was detected in MSU crystal-stimulated monocytes, and this synthesis increased with incubation time, up to 24 hours (Fig 1B). Monocytes incubated with CPPD crystals under similar
conditions failed to consistently produce PGE2 or TXA2 (data not shown).
Integrated data from seven experiments in which cells were incubated
with crystals at a concentration of 0.6 mg/mL for 24 hours are
summarized in Fig 1C. Whereas levels of prostanoids synthesized by
nonstimulated monocytes were often under the limit of detection, MSU
crystal-stimulated monocytes produced more than 15 ng/mL of
PGE2 and approximately 100 ng/mL TXA2. In some
experiments, CPPD crystal-stimulated monocytes produced relatively low
levels of prostanoids. However, results did not reach statistical
significance (P > .05) when compared with nonstimulated
monocytes.
Effect of microcrystals on COX-2 protein expression in human
monocytes.
Cell samples from the aforementioned dose-response experiments were
processed for immunoblots to evaluate COX-2 protein expression. When
using an antibody that specifically recognizes the COX-2 isoform22,26 a characteristic doublet that likely
represents differentially glycosylated forms of the
enzyme,54 with a relative molecular mass of 72 to 74 kD,
was detected in samples from MSU crystal-stimulated monocytes (Fig
2), but not from CPPD crystal-stimulated monocytes. The intensity of the COX-2 doublet increased with increasing concentrations of MSU crystals (Fig 2A) within the range 0.1 to 1 mg/mL, and consistently decreased at the highest concentration used (3 mg/mL) in approximate correlation with the level of prostanoid production. In time-course experiments in which monocytes were incubated with MSU crystals, COX-2 protein could be observed after 3 to
7 hours of incubation with MSU crystals, and the intensity of the two
bands was maximal after 17 hours (Fig 2B). Barely detectable levels of
COX-1 protein were present in elutriated monocytes, and no significant
change in immunoreactivity could be detected following crystal
stimulation (data not shown).
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| Fig 2.
Effect of inflammatory microcrystals on the protein
expression of COX-2 in human monocytes. Monocytes were incubated for
(A) 24 hours with either MSU or CPPD crystals at the indicated
concentrations, or (B) with crystals (0.6 mg/mL) for the indicated
times and centrifuged. The cell pellets were then processed for
evaluation of COX-2 protein expression by immunoblotting as described
in Materials and Methods. A representative immunoblot is shown.
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RNA extraction from crystal-stimulated monocytes presented serious
technical problems and did not enable evaluation of the potential
effects of these crystals on the expression of COX-2 mRNA in monocytes
(data not shown).
Effect of metabolic inhibitors on prostanoid synthesis and COX-2
protein expression in MSU crystal-stimulated human monocytes.
Monocytes were incubated with MSU crystals, alone or in the presence of
an inhibitor of protein synthesis, CHX, an inhibitor of transcription,
AD, or an inhibitor of tyrosine kinases, herbimycin A. Both CHX and AD
inhibited prostanoid synthesis by at least 80% when compared with MSU
crystal-stimulated monocytes (Fig 3A).These inhibitors also decreased the COX-2 protein expression under the limit of detection, which suggests that both translational and transcriptional events are required for the stimulation of COX-2 protein expression (Fig 3B). Herbimycin A decreased MSU
crystal-stimulated prostanoid synthesis by more than 90%, an event
that was also paralleled by an inhibition of COX-2 protein expression.
SB 203580, a specific inhibitor of p38 mitogen-activated protein
kinase, was also assessed. The presence of SB 203580 decreased the
production of PGE2 by approximately 80% and production of
TXB2 by 100%. SB 203580 decreased COX-2 protein expression
below the limits of detection. In contrast, an inactive structural
analog of SB 203580, SK&F 106978, had little effect on MSU
crystal-stimulated prostanoid synthesis or COX-2 protein expression.
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| Fig 3.
Effect of metabolic inhibitors on the MSU crystal-induced
prostanoid synthesis and protein expression of COX-2 in human
monocytes. Monocytes were incubated with diluent (C), or MSU crystals
(M; 0.3 mg/mL) for 24 hours alone or in combination with a protein synthesis inhibitor, cycloheximide (CHX; 10 µg/mL); an inhibitor of
transcription, actinomycin D (AD; 5 µg/mL), or a specific inhibitor of tyrosine kinases, herbimycin A (HA; 100 nmol/L). A p38
mitogen-activated protein kinase inhibitor, SB 203580 (SB; 10 µmol/L), as well as an inactive structural analog, SK&F 106978 (SKF;
10 µmol/L) were also used under similar conditions. (A) Cell-free
supernatants were collected and analyzed for prostanoid synthesis as
indicated in the Materials and Methods. Results are the mean ± SD
from 3 separate experiments and are expressed as percent of MSU
crystal-stimulated monocytes in the absence of metabolic inhibitor. (B)
Corresponding cell samples were processed for evaluation of
immunoreactive COX-2 by immunoblot as described in Materials and
Methods. A representative immunoblot is shown.
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Effect of colchicine on prostanoid synthesis and COX-2 protein
expression in human monocytes.
Monocytes were incubated with MSU crystals in the presence or absence
of different concentrations of colchicine. This antiphlogistic agent
effectively blocked prostanoid production (both PGE2 and TXA2) by approximately 90% and inhibited MSU
crystal-induced COX-2 protein expression at 100 nmol/L, the lowest
concentration used (Figs 4 and
5). In
contrast, two inactive structural analogs of colchicine, - and
 -lumicolchicine, had little effect either on the protein expression
of COX-2 or on the production of prostanoids, stimulated by MSU
crystals, up to a concentration of 10 µmol/L (Figs 4 and 5).
Colchicine did not inhibit monocyte COX-2 protein expression,
stimulated by either serum-treated zymosan or lipopolysaccharide (Fig
5B and C).
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| Fig 4.
Effect of colchicine on microcrystal-induced prostanoid
synthesis by human monocytes. Monocytes were incubated with diluent, MSU crystals (0.6 mg/mL), or CPPD (0.6 mg/mL) for 24 hours, alone or in
combination with various concentrations of colchicine (col), or with
its inactive analogs, - and -lumicolchicine (L-col), both at a
concentration of 10 µmol/L. Cell-free supernatants were collected and
analyzed for prostanoid synthesis. The results are the mean ± SD from
three separate experiments and are expressed as percent of MSU
crystal-stimulated monocytes in the absence of metabolic inhibitor.
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| Fig 5.
Effect of colchicine on microcrystal-induced COX-2
protein expression in human monocytes. Monocytes were incubated without or with MSU crystals (0.6 mg/mL) for 24 hours alone or in combination with various concentrations of colchicine (col) or with its inactive analogs, - and -lumicolchicine (L-col), both at a concentration of 10 µmol/L. Cell samples were then processed for evaluation of
immunoreactive COX-2 by immunoblot as described in Materials and
Methods. (A) MSU crystal-stimulated cells. (B) Serum-treated zymosan
(STZ; 100 µg/mL; 24 hours)-stimulated cells, in the presence of
various concentrations (µmol/L) of colchicine. (C) Lipopolysaccharide (LPS; 2 µg/mL; 24 hours)-stimulated cells, in the presence of various
concentrations (µmol/L) of colchicine. In each case, an immunoblot
representative of at least three separate experiments is shown.
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DISCUSSION |
The formation and/or deposition of MSU microcrystals in
articular and periarticular tissues is the cause of gouty arthritis. These microcrystals interact with all of the major synovial cell types,
including neutrophils, fibroblasts, and monocytes/macrophages to
produce a variety of inflammatory mediators.2,3 Clinical hallmarks of this inflammatory condition include severe pain, edema,
and erythema in the joint, all of which are biologic actions of, among
other agents, prostanoids such as PGE2, with its well-known role in pain. The results of the present study show that inflammatory MSU crystals, but not CPPD crystals, stimulate COX-2 protein expression in human monocytes in a dose- and time-dependent manner and that this
is associated with the production of PGE2 and
TXA2. Studies using various metabolic inhibitors indicate
that both transcriptional and translational processes, as well as
activity of tyrosine kinases, are necessary for this stimulation to
occur. At supraoptimal concentrations of MSU crystals, induction of
COX-2 and prostanoid synthesis were reduced, an effect that is likely
to be related to the known cytolytic effects of these crystals.
Stimulated human blood monocytes constitute an important source of
COX-2-derived prostanoids, including PGE2 and
TXA2.20 The use of elutriation to isolate human
monocytes allows the purification of a cell population essentially free
of platelets, a major source of COX-1 derived prostanoids. In fact, in
resting or crystal-stimulated monocytes isolated by elutriation, COX-1
was only weakly if at all detected when assessed by immunoblotting
(Pouliot, unpublished observations, April 1996), supporting the
relative absence of platelets in these monocyte preparations. To
confirm the involvement of COX-2 in our
system, signal transduction pathways that are critically involved in
the expression of COX-2, but not of COX-1,26,28-35 were
blocked with the use of specific inhibitors. The tyrosine kinase
inhibitor, herbimycin A, as well as the p38 mitogen-activated protein
kinase inhibitor, SB 203580, both decreased this stimulated production
of PGE2 and essentially eliminated all detectable
TXB2 production, in parallel to preventing the induction of
COX-2. Moreover, MSU-induced prostanoid synthesis was blocked by
inhibitors of transcription and of translation, revealing the
requirement of de novo protein synthesis for increased prostanoid
production to occur in this system. Collectively, these data
demonstrate the preeminent role of a monocyte-derived and inducible COX
isoform over the constitutive isoform in MSU-stimulated monocytes. In allowing discrimination between COX-1- and COX-2-mediated prostanoid production in human monocytes, elutriation represents a significant improvement over the adhesion technique, which produces samples that
contain platelets as the primary contaminant.
The stimulation of cytokine production by monocytes is not universal to
all types of inflammatory crystals. For example, it has been
demonstrated that MSU-stimulated human monocytes and, to a lesser
extent, cells treated with hydroxyapatite crystals, released
significant amounts of TNF, whereas cells incubated with CPPD
crystals failed to produce TNF.8 Similarly, MSU, but not
CPPD crystals, induced the production of IL-1 by human monocytes.36 Another study showed that MSU crystals were
approximately 50 times more potent than CPPD crystals for optimal IL-6
production by human monocytes. However, in that same study, CPPD were
as potent as MSU crystals for the induction of IL-6 production by human
synoviocytes.7 This indicates a cell-specific type of response to inflammatory crystals. These observations also suggest that
MSU crystals may be more potent for the induction of early-response genes in human monocytes than CPPD crystals, an hypothesis that our
current results further support.
A class of pyridinyl imidazole compounds that inhibit cytokine
production has been developed37 and SB 203580 is a recent representative. These compounds have been found to inhibit IL-1 and
TNF production in human monocytes. They also have antiinflammatory effects in animal models such as arachidonic acid-induced edema and
collagen-induced arthritis in mice, and they inhibit inflammatory cell
infiltration induced in mice by MSU crystals or
carrageenan.38,39 Recently, we have shown that these
compounds specifically inhibit the expression of COX-2 in human
monocytes stimulated with different stimuli, including serum-treated
zymosan, TNF-, IL-1, phorbol myristate acetate, and
lipopolysaccharide.26 In the present study, SB 203580 also
inhibited COX-2 protein expression induced by MSU crystals, which
further indicates an important role for p38 mitogen-activated protein
kinase in the induction of the enzyme. This result also suggests that
SB 203580 affects events in the signal transduction pathways that lead
to COX-2 expression and are common for different classes of
monocyte-stimulating agents.
Although the effectiveness of colchicine in crystal-associated
rheumatic diseases40 has been reported as early as the
sixth century,41 the basis of its antiphlogistic action
remains unclear. The major accepted effect of colchicine on cells is
inhibition of microtubule assembly.42 However, it is
uncertain whether this site of action is responsible for the in vivo
antiinflammatory effects of colchicine, since concentrations greater
than those achieved during therapy (1 µmol/L v 100 nmol/L)
are required to inhibit microtubule-dependent functions in
vitro.43-46 On the other hand, concentrations of colchicine
required to inhibit outside-in-signaling through the microtubular
system can occur at lower concentrations than required to inhibit some
effector functions.47
In a number of different cell types, including human monocytes,
tyrosine phosphorylation is central to COX-2
induction.28-35 This was further confirmed in the present
study, where herbimycin A effectively blocked the protein expression of
COX-2 and synthesis of prostanoids. MSU crystals induce a specific
pattern of tyrosine phosphorylation in neutrophils which is inhibited
by colchicine.48,49 In our study, specific inhibition of
monocyte COX-2 protein expression and prostanoid synthesis by
colchicine has been observed at similar concentrations (submicromolar)
to those which inhibit tyrosine phosphorylation, concentrations which
may be achieved therapeutically. Also, the inhibitory effect of
colchicine on COX-2 was specific to MSU crystals, in that it did not
inhibit lipopolysaccharide- or zymosan-stimulated COX-2 protein
expression even at the highest concentration tested. This result is in
good accordance with recent findings in human neutrophils, in which
colchicine blocked tyrosine phosphorylation stimulated by MSU crystals,
but not by chemotactic agents or by zymosan.49 In addition,
recent data indicate that colchicine inhibits MSU- and CPPD-induced
IL-8 production by human neutrophils, but not that induced by
granulocyte-macrophage colony-stimulating factor (GM-CSF) or TNF
(McColl & Naccache, unpublished data, June 1996). Taken
together, these results provide further evidence for a proposed
antiprostaglandin action of colchicine in crystal-induced inflammation,50 and give further support to the concept
that beneficial therapeutic effects of colchicine are considered to be
relatively limited to inflammatory conditions involving inflammatory microcrystals.50,51
In addition to its pain-triggering role, PGE2 may
participate in several ways to the symptoms that are marks of gouty
arthritic flares, such as early vasodilation, edema, and leukocyte
migration. In an in vivo model of inflammation, PGE2
prevented leukotriene (LT)C4-induced vasoconstriction and
cause a marked potentiation of LTC4-induced plasma
extravasation in Syrian hamsters. PGE2 also potentiated
histamine-induced leakage, as well as LTB4-induced leukocyte-associated leakage, from both postcapillary and larger venules.52 It appears therefore that effects of
PGE2 can be potentiated by the presence of other factors
including lipoxygenase-derived metabolites such as the LTs.
Interestingly, MSU crystals can also trigger the formation of LTs and
of hydroxy acids by human neutrophils and platelets.53
However, it must be stressed that PGE2 can exert both
proinflammatory and antiinflammatory properties52; this may
help explain conflicting effects of nonsteroidal antiinflammatory drugs
in different models. The study of other factors, such as the site of
synthesis, and how the balance of synthesized lipid mediators can
influence the final response, may increase our understanding of the
underlying mechanisms that govern gouty arthritis episodes.
In the present study, we have demonstrated that MSU microcrystals can
specifically induce COX-2 in human monocytes. These results identify
COX-2 expression as a new factor involved in MSU-induced inflammation.
The observed inhibition by colchicine of COX-2 protein expression and
of prostanoid synthesis in MSU crystal-stimulated cells provides a
possible mechanism for the antiphlogistic action of this therapeutic
agent. Taken together, these results provide greater understanding of
the mechanisms involved in early events that trigger gout attacks.
Further studies are in progress to identify which signal transduction
pathways are specifically involved in the interaction of monocytes with MSU microcrystals.
| |
FOOTNOTES |
Submitted August 20, 1997;
accepted October 20, 1997.
Supported by project grants from the National Health & Medical Research
Council of Australia and by a grant from the Arthritis Society of
Canada. M.P. is the recipient of a fellowship from the Medical Research
Council of Canada.
Address reprint requests to Marc Pouliot, PhD, Centre de Recherche en
Rhumatologie et Immunologie, Centre de Recherche du CHUL, Room
T-1-49, 2705 Boulevard Laurier, Ste-Foy, Québec,
Canada, G1V 4G2.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr John C. Lee (SmithKline Beecham, King of Prussia, PA) for
generously supplying the p38 mitogen-activated protein kinase
inhibitor.
| |
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Abstract
Introduction
Abstract