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*
Department of Medicine, University of New South Wales, and
Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-stimulated PGE2 production by cultured synovial
fibroblasts. Enhancement of TNF--stimulated PGE2
production in synovial cells was accompanied by increased expression of
cyclooxygenase (COX)-2 and cytosolic phospholipase A2
(cPLA2)-. Blockade of COX-2 enzyme activity with the
selective inhibitor NS-398 prevented both TNF--stimulated and
sPLA2-IIA-amplified PGE2 production without
affecting COX-2 protein induction. However, both
sPLA2-IIA-amplified PGE2 production and
enhanced COX-2 expression were blocked by the sPLA2
inhibitor LY311727. Colocalization studies using triple-labeling
immunofluorescence microscopy showed that sPLA2-IIA and
cPLA2- are coexpressed with COX-2 in discrete
populations of CD14-positive synovial macrophages and synovial tissue
fibroblasts from RA patients. Based on these findings, we propose a
model whereby the enhanced expression of sPLA2-IIA by RA
synovial cells up-regulates TNF--mediated PG production via
superinduction of COX-2. Therefore, sPLA2-IIA may be a
critical modulator of cytokine-mediated synovial inflammation in
RA. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß,
play an important role in the pathogenesis of RA. Neutralization of
these cytokines alleviates synovial inflammation in both animal models
and human RA (1). These animal studies, together with
IL-1ß and TNF- in vivo gene deletion experiments, have shown that
IL-1ß is consistently important in mediating cartilage breakdown,
whereas TNF- is a key inducer of synovial inflammation
(2). Moreover, transgenic mice overexpressing TNF-
develop spontaneous arthritis (3). TNF- and IL-1ß
activate the transcription factor NF-
B (4) and the p38
and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK)
pathways (5) to induce a host of proinflammatory proteins.
Direct inhibition of NF-B (6), Jun/Fos (AP-1)
(7) or p38 MAPK (8) reduces disease severity
in animal models of arthritis, confirming the importance of these
signaling pathways in the inflammatory and/or the erosive component of
arthritis.
Prostaglandin E2 (PGE2)
contributes to pain and swelling during inflammation through induction
of hyperalgesia and increased vascular permeability (9)
and modulates bone resorption through stimulation of osteoclast
formation from precursor stem cells (10).
PGE2 production by cultured rheumatoid synovial
fibroblasts (RSFs) is induced within hours by IL-1ß mediated by
NF-B- and MAPK-dependent coordinate induction of cytosolic
phospholipase A2
(cPLA2)- and cyclooxygenase (COX)-2 (11, 12). Although COX-1 is constitutively expressed by RSFs,
IL-1ß-stimulated PGE2 production occurs
exclusively via COX-2 (13). In RA synovium both COX-1 and
-2 are expressed, with COX-2 expression elevated in relation to the
degree of inflammation in synovial tissue (14). Recently,
COX-2-selective inhibitors that maintain the anti-inflammatory
properties of nonsteroidal anti-inflammatory drugs (NSAIDs)
(15, 16) have been developed; but, unlike the latter
compounds, they have a favorable gastrointestinal side effect profile.
A low molecular mass (14 kDa) human type IIA secretory phospholipase A2 (sPLA2-IIA) has been identified in rheumatoid synovium (17); however, the importance of this enzyme in synovial pathology is poorly defined. sPLA2-IIA, first purified from the synovial fluid of patients with RA (18), is found at high levels in the colonic mucosa of patients with ulcerative colitis and Crohn’s disease (19), in the bronchoalveolar lavage fluid of patients with asthma following Ag challenge (20) and in the serum of septic shock patients (21). Serum sPLA2-IIA concentrations are elevated in patients with RA (22) and correlate with severity of disease (23). Enzyme expression is increased in RA synovial macrophages and fibroblasts relative to synovium from nonarthritic patients and correlates with histological markers of synovial inflammation (17). Human sPLA2-IIA is acutely inflammatory when injected into rabbit joints (24); however, transgenic mice overexpressing sPLA2-IIA do not develop arthritis (25).
Given the presence of sPLA2-IIA at concentrations
up to several micrograms per milliliter in RA synovial fluid
(26), and the established inflammatory activities of PGs
(9), we have examined here both the relationship between
sPLA2-IIA and PGE2
production in cultured RSFs and the cellular localization of
sPLA2-IIA and COX-2 in rheumatoid synovial
tissue. The results of these studies demonstrate that concentrations of
sPLA2-IIA found in RA synovial fluid enhance
TNF--stimulated PGE2 production in RSFs by
superinducing COX-2 protein levels. Further,
sPLA2-IIA and COX-2 colocalize in discrete
subpopulations of rheumatoid synovial macrophages and fibroblasts.
These findings indicate that sPLA2-IIA may be an
important amplifier of cytokine-mediated PG production and may thereby
contribute to the severity of the synovial inflammatory response
in RA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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sPLA2-IIA was purified from the conditioned medium and cell pellets of a Chinese hamster ovary cell line (5A2) stably expressing human sPLA2-IIA cDNA (27) by immunoaffinity chromatography on an AKTA explorer system (Pharmacia Biotech, Uppsala, Sweden) and quantified by ELISA (22). sPLA2-IIA was a single 14-kDa band on silver-stained PAGE gels, and N-terminal amino acid sequence analysis (27) confirmed its identity. sPLA2-IIA contained <0.1 ng endotoxin/mg protein (Limulus amebocyte lysate pyrochrome, Associates of Cape Cod, Falmouth, MA) and was enzymatically active in a [3H]arachidonate-labeled Escherichia coli membrane assay (27).
Fibroblast culture
Synovial tissues were obtained as described (17) using procedures approved by the St. Vincent’s Hospital Ethics Committee. RSFs were isolated by trypsin (0.5%)/EDTA (5.3 mM) digestion (15 min, 37°C in DMEM/Ham’s F12 medium), followed by collagenase (200 U/ml; Life Technologies, Gaithersburg, MD), and cells were grown to confluence twice in medium containing 10% FBS (Commonwealth Serum Laboratory, Melbourne, Australia), penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (0.125 µg/ml) (Life Technologies), before storage in liquid nitrogen. Cells were phenotypically stable (CD14-negative, prolyl-5-hydroxylase-positive by immunofluorescence microscopy) to passage and were used from passage 4 to 10. The human neonatal lung fibroblast (NLF) cell line CCD34Lu (American Type Culture Collection, Manassas, VA) was grown in 2% FBS to synchronize the growth rate with that of the RSFs. For all experiments, confluent cell monolayers were grown antibiotic-free and received fresh medium containing 0.1% BSA (endotoxin-free, fatty acid-free; Boehringer Mannheim, Sydney, Australia) before stimulation.
PGE2 studies
Inhibitors LY311727 (28) and NS-398
(29) (Cayman Chemical, Ann Arbor, MI) were prepared as
10-mM stocks in DMSO. Both control and inhibitor-treated cultures
contained a final solvent concentration of 0.1% (v/v) DMSO. LY311727
was inhibitory toward sPLA2-IIA
(IC50 2.5 µM at an
sPLA2-IIA concentration of 10 ng/ml,
[3H]E. coli membrane assay
(27)), whereas NS-398 was noninhibitory up to 100 µM
(data not shown). Confluent fibroblast monolayers were stimulated with
sPLA2-IIA, human rTNF- (PeproTech, Rocky Hill,
NJ), or IL-1ß (R&D Systems, Minneapolis, MN) alone or in combination,
in the presence or absence of inhibitors for 24 h, and media were
stored at -80°C. PGE2 was quantified in
triplicate at three dilutions by enzyme immunoassay (Cayman
Chemical).
Western blot analysis
Cell lysates were prepared by resuspension in PBS containing 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EGTA,
aprotinin (50 µg/ml), leupeptin (200 µM), and PMSF (1 mM) followed
by repeated passage through a 21-gauge needle, incubation on ice (30
min), centrifugation (20 min, 13,800 x g, 4°C), and
storage of supernatants at -80°C. Lysates were analyzed by Western
blotting following SDS-PAGE (4–20% gradient gels; Novex, San Diego,
CA). Primary Abs were anti-ovine COX-1 mAb (cat. no. 160110, 1.7
µg/ml; Cayman Chemical), anti-human COX-2 mAb (cat. no. 160112,
0.5 µg/ml; Cayman Chemical), anti-human
cPLA2- mAb (cat. no. sc-454, 0.1 µg/ml;
Santa Cruz Biotechnology, Santa Cruz, CA), anti-ß-actin mAb
(clone AC-15, 0.3 µg/ml; Sigma, St. Louis, MO), and anti-human
ICAM-1 goat polyclonal Ab (cat. no. sc-1510, 0.2 µg/ml; Santa Cruz
Biotechnology). Proteins were visualized using enhanced
chemiluminescence (NEN, Boston, MA) and quantified by densitometry
(Molecular Dynamics, Sunnyvale, CA).
Immunofluorescence and confocal microscopy
Synovial tissue from five RA patients receiving, alone or in combination, NSAIDs, auranofin, azathioprine, cyclosporin, sulfasalazine, or methotrexate, but not prednisolone, was examined by immunofluorescence microscopy as described (17). Sections were sequentially incubated with anti-human COX-2 goat polyclonal Ab (cat. no. sc-1745, 5 µg/ml, 45 min; Santa Cruz Biotechnology) or negative control goat IgG (5 µg/ml; Sigma), donkey anti-goat rhodamine red-X conjugate (1/100, 30 min; Jackson ImmunoResearch, West Grove, PA), a second primary Ab (45 min), and donkey anti-mouse-indodicarbocyanine (Cy5) conjugate (1/200, 30 min; Jackson ImmunoResearch). For double-labeling studies, second primary Abs were anti-human CD68 (clone EBM11, 4.3 µg/ml; Dako, Glostrup, Denmark), anti-human prolyl 4-hydroxylase (clone 5B5, 1.5 µg/ml; Dako), anti-human Von Willebrand factor (clone F8/86, 4.4 µg/ml; Dako), or isotype-matched (IgG1k) negative control mAb 81193 (Bioquest, Sydney, Australia).
For triple-labeling studies, second primary Abs were anti-human
sPLA2 (clone 9C1 (22), 2 µg/ml;
Bioquest), isotype-matched (IgG1k) negative control 81193 (2
µg/ml), anti-human cPLA2- (4 µg/ml),
or isotype-matched (IgG2b) negative control (clone MOPC-195, 4 µg/ml;
Immunotech, Marseille, France). The specificity of Ab 9C1 for
sPLA2-IIA in immunohistochemistry has been
partially but not exhaustively determined (13, 17). The Ab
recognizes a conformational epitope on sPLA2-IIA,
identifies sPLA2-expressing cells in rheumatoid
synovium with the same specificity as two other independent mAbs (4A1
and 10B2) raised to sPLA2-IIA, and a capture
ELISA using 9C1 together with Ab 4A1 does not recognize human
sPLA2-V or sPLA2-1B.
Sections were blocked (4% mouse serum, 30 min; Sigma) and incubated
with CD14 mAb-FITC conjugate (clone RMO52, monocyte marker, 1/5;
Immunotech) or isotype-matched (IgG2a) negative control mAb-FITC
conjugate (clone U7.27, 1/5; Immunotech). Sections were analyzed by
confocal microscopy as described (17). Images were
exported into Adobe Photoshop 5.0 (Adobe Systems, San Jose, CA) and
Canvas 5.02 (Deneba Software, Miami, FL) for presentation.
Statistical analysis
Statistical evaluations were performed on primary data using the Wilcoxon signed rank test or the Student’s unpaired t test with StatView software (Abacus Concepts, Berkeley, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-stimulated PGE2 production
To determine the responsiveness of the
cPLA2-/COX pathway to TNF-, RSFs were
stimulated with increasing concentrations of TNF-, then
PGE2 production and COX and
cPLA2- protein levels were determined. Basal
PGE2 production varied between cultures (range
46 ± 2 to 245 ± 10 pg/ml); however, in each of the five
cultures, TNF- dose-dependently stimulated
PGE2 production (range 0.32 ± 0.07 to
13.4 ± 3.1 ng/ml at 10 ng/ml TNF-) (Fig. 1
A). Increased
PGE2 production correlated with accumulation of
COX-2 protein (Fig. 1B). cPLA2- was
weakly but consistently up-regulated with COX-2, whereas COX-1 was
unaffected. The adhesion molecule ICAM-1 was near-maximally induced at
1 ng/ml TNF- (Fig. 1B).
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(10 ng/ml) in the presence of the
COX-2-selective inhibitor NS-398 (1 µM) (29) abrogated
TNF--stimulated PGE2 production whereas the
sPLA2-selective inhibitor LY311727
(28) reproducibly suppressed PGE2 by
30% (Fig. 2).
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-stimulated
PGE2 production
RSFs were treated with increasing concentrations of
sPLA2-IIA in the presence or absence of TNF-
(10 ng/ml). sPLA2-IIA alone did not stimulate
PGE2 production at any concentration examined
(Fig. 3A). Coaddition of
sPLA2-IIA with TNF- resulted in a
dose-dependent enhancement of TNF--stimulated
PGE2 production with a mean 3-fold augmentation
of the TNF- response over the five RSF cultures at 10 µg/ml
sPLA2-IIA (Fig. 3A). The response was
significant at both 1 and 10 µg/ml sPLA2-IIA,
even though only four of the five RSF cultures were responsive.
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A),
sPLA2-IIA (5 µg/ml) consistently increased
COX-2 protein to a level similar to that induced by TNF- (10 ng/ml)
(Fig. 3, B–D). Coaddition of
sPLA2-IIA with TNF- resulted in a synergistic
increase in COX-2 protein levels (Fig. 3, B–D).
The increase over sPLA2-IIA alone was clearly
detectable at 0.1 ng/ml TNF- (Fig. 3D). COX-1 protein was
not affected by these treatments, whereas a small increase in
cPLA2- protein levels was consistently
observed with TNF- (>0.1 ng/ml)/sPLA2-IIA
treatment (Fig. 3, B and D).
sPLA2-IIA did not increase TNF--stimulated
ICAM-1 protein expression (data not shown). Although
sPLA2-IIA also showed augmentation of
IL-1ß-stimulated PGE2 production and COX-2
induction (data not shown), the response was small relative to
sPLA2-IIA augmentation of the TNF--stimulated
response and did not occur consistently in RSF cultures.
To determine whether the responsiveness of RSFs to
sPLA2-IIA was a general feature of human
fibroblasts, comparable experiments were performed using a human NLF
cell line. In NLF cells, PGE2 production was
significantly but weakly increased by TNF- (10 ng/ml) from 71
± 3 pg/ml (n = 4) to 90 ± 10 pg/ml
(n = 2) (p < 0.05). However,
the weak induction was not due to a lack of responsiveness of the
pathway to stimulation because IL-1ß (0.1 ng/ml) stimulated basal
PGE2 production by 12-fold
(p < 0.001) (Student’s unpaired t
test). Also, COX-1, COX-2, cPLA2-, and ICAM-1
expression following TNF- or IL-1ß stimulation was similar to that
observed in RSFs (data not shown). NS-398 (1 µM) abrogated
TNF--stimulated PGE2 production, whereas
LY311727 (10 µM) had no effect. sPLA2-IIA (10
µg/ml) did not augment TNF--stimulated PGE2
production by the NLF cell line. In NLF cultures,
sPLA2-IIA, like TNF-, also increased COX-2
protein expression, whereas coaddition of
sPLA2-IIA and TNF- had only an additive effect
on COX-2 protein levels that was not associated with increased
PGE2 production over TNF- alone (data not
shown).
sPLA2-IIA-augmented PGE2 production is coupled to COX-2
RSFs were stimulated with TNF- (10 ng/ml) and increasing
concentrations of sPLA2-IIA in the presence or
absence of the sPLA2 inhibitor LY311727 (10
µM). LY311727 reduced both PGE2 production
(Fig. 4A) and
COX-2/cPLA2- protein (Fig. 4, B and
C) to levels observed in TNF--stimulated RSFs. The
COX-2-selective inhibitor NS-398 (1 µM) reduced
PGE2 production to basal levels (Fig. 4A) without affecting
TNF-/sPLA2-IIA-stimulated COX-2 protein (Fig. 4, B and C). Levels of COX-1 were not affected by
either treatment (Fig. 4B).
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To determine whether the expression of PLA2
enzymes was detectable in synovial tissue cells expressing COX-2, the
localization of COX-2 relative to sPLA2-IIA and
cPLA2- in RA synovial membrane sections was
examined using immunofluorescence confocal microscopy. The population
of cells expressing COX-2 was first defined by double
immunofluorescence with Abs to macrophage-like cells (CD68),
fibroblast-like cells (5B5), and endothelial cells (factor VIII) (data
not shown). COX-2 staining intensity in synovial sections derived from
five independent RA patients was consistently strongest in the
macrophage-like cells. Positive staining was also observed in both
synovial lining and subsynovial lining fibroblast-like cells, whereas
endothelial cells were usually negative or weakly COX-2 positive.
Overall, the COX-2 staining pattern observed was consistent with that
described previously for RA (14) with the exception that
strong COX-2-positive endothelial cell staining was not
consistently seen.
RA synovium from three patients was then triple-labeled with Abs to
COX-2, CD14, and sPLA2 (Fig. 5) or COX-2, CD14, and
cPLA2- (Fig. 6).
All cells positive for COX-2 were also
sPLA2-positive (Fig. 5D), although
sPLA2-positive/COX-2-negative cells were
observed. The majority of CD14-positive cells were COX-2-negative, and
a subpopulation of those cells were
sPLA2-positive (data not shown). A small number
of COX-2/CD14/sPLA2-positive cells were observed.
The pattern of staining described (Fig. 5) is consistent with that
observed in synovial sections derived from the two other RA patients
examined (data not shown). All COX-2-positive cells were also
cPLA2--positive, although
cPLA2--positive/COX-2-negative cells were
observed, notably the endothelial cells lining vessels (Fig. 6). As in
the sPLA2 studies, the majority of CD14-positive
cells were COX-2-negative, and a subpopulation of these were
cPLA2--positive. Several
COX-2/CD14/cPLA2--positive cells were
observed, as were CD14-negative, cPLA2--
positive, and COX-2-positive cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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alone,
sPLA2-IIA alone has no effect on
PGE2 production in RSFs, although it consistently
up-regulated COX-2 protein to levels comparable to those seen with
TNF- alone. These data suggest that COX-2 up-regulation is necessary
but insufficient for PGE2 production. Additional
signals are provided by TNF- stimulation, which
sPLA2-IIA alone cannot provide. It is possible
that sPLA2-IIA, although enzymatically active,
can neither supply substrate directly to COX-2 nor indirectly activate
cPLA2-. TNF- signaling likely results in
post-translational activation of cPLA2- at the
level of phosphorylation and/or mobilization to membranes.
Alternatively, TNF- may also regulate the recently cloned PGE
synthase (37) immediately distal to COX-2 in the
PGE2 pathway. Importantly, COX-2 has recently
been shown to mediate effects on cellular proliferation independently
of its enzyme activity (38). Thus,
sPLA2-IIA, via induction of COX-2 protein, may
have broader effects on cell function than modulation of PG synthesis
alone.
sPLA2-IIA amplifies TNF--induced
PGE2 production by RSFs at concentrations that
are found in the synovial fluid of patients with RA (26).
Importantly, this amplification occurs over a range of TNF-
concentrations, suggesting that expression of
sPLA2-IIA in synovium may sensitize synovial
cells to produce PGs at low concentrations of TNF-, thereby
contributing to the severity of the PG-mediated synovial inflammatory
response. This suggestion is supported by the observation that the
spontaneous arthritis resulting from transgenic overexpression of
TNF- in mice (3) is exacerbated and is earlier in onset
when human sPLA2-IIA is transgenically
overexpressed in combination with TNF- (39).
Our experiments with NLFs show that amplification of TNF--induced
PGE2 production by
sPLA2-IIA is not a general feature of human
fibroblasts, even though a functional cytokine-inducible
COX-2-dependent PGE2 production pathway is
present in these cells. Further, sPLA2-IIA did
not consistently amplify IL-1ß-stimulated PGE2
production in RSFs, suggesting that IL-1ß alone may be sufficient to
maximally stimulate the cPLA2-/COX-2 pathway
in these cells. The relative roles of TNF- and IL-1ß in
stimulating synovial inflammation is a matter of controversy. Notably,
both cytokines are capable of inducing synovial inflammation, and there
is data suggesting that each is dependent on the other. Also, TNF-
is important in the induction of synovial inflammation in RA, whereas
IL-1ß, although able to cause inflammation, is more consistently
potent at mediating cartilage degradation (2). It is
likely that there is significant variability in the cytokine profiles
of patients with RA depending on both genetic and environmental
factors. Consequently, sPLA2-IIA effects may also
vary depending on the local synovial cytokine environment.
Both TNF--stimulated and sPLA2-IIA
augmentation of TNF--stimulated PGE2
production by RSFs is COX-2-dependent. This finding is consistent with
the effects of exogenous sPLA2 in studies in
model rodent cell lines using other agonists such as nerve growth
factor stimulation of rat mast cells (40, 41). In
addition, in some, but not all cases where sPLA2
has been reported to augment agonist-stimulated PG production, COX-2
protein is also up-regulated (40, 41) as has been shown
here. Our immunofluorescence studies show that
sPLA2 is coexpressed with COX-2 in specific
subpopulations of fibroblast and CD14-positive macrophages in
rheumatoid synovium, supporting the relevance of our observations with
synovial cells in culture to synovium. From these studies, it is likely
that the selective COX-2 inhibitors now in clinical use
(16) would effectively block
sPLA2-IIA amplification of TNF--stimulated PG
production in synovium.
The sPLA2 inhibitor LY311727, which binds in the
active site channel of sPLA2 to block enzyme
activity (28), suppressed both
sPLA2-IIA-augmented PGE2
production and the concomitant up-regulation of COX-2, suggesting that
regulation of COX-2 protein levels requires sPLA2
enzyme activity. However, an activity-independent mechanism of
sPLA2-IIA-mediated up-regulation of COX-2 protein
levels in RSFs cannot be ruled out in light of a recent report in rat
serosal mast cells, where an "activity-dead"
sPLA2-IIA mutant enzyme still augmented COX-2
up-regulation in response to nerve growth factor even though induction
of COX-2 by nerve growth factor was suppressible by LY311727
(42). Importantly, in contrast to findings in a murine
osteoblast cell line (41), the insensitivity of the
TNF-/sPLA2-IIA-mediated induction of COX-2 to
NS-398 rules out an autocrine effect of PGE2 on
this pathway in RSFs.
Cytokine induction of the genes encoding COX-2 and
cPLA2- is NF-B-dependent in RSFs (12);
however, sPLA2-IIA does not affect
TNF--induced mobilization of NF-B into the nucleus as determined
by EMSAs, Western blots for nuclear p65, or IB degradation assays
(M. J. Bidgood, M. L. Taberner, and K. F. Scott, manuscript
in preparation). COX-2 is also post-transcriptionally regulated via a
p38-MAPK-dependent mechanism in human monocytes (43). It
is known that TNF- can signal through the p38 pathway in
chondrocytes (5), and it is possible that
sPLA2-IIA costimulates this pathway in RSFs.
However, in our hands, sPLA2-IIA does not induce
phosphorylation of p38 MAPK nor does it stimulate TNF--mediated
phosphorylation of p38 MAPK in RSFs (M. J. Bidgood, M. L.
Taberner, and K. F. Scott, manuscript in preparation).
Consequently, the mechanism by which sPLA2-IIA
amplifies COX-2 protein expression in RSFs remains to be
established.
Our studies support the view that sPLA2-IIA is
one factor that may amplify TNF--dependent pathways in rheumatoid
synovium and that the level of expression of
sPLA2-IIA in synovium, together with that of
TNF-, may contribute to the severity of the PG-mediated inflammatory
response. Levels of sPLA2-IIA are also
increased in several other immune-mediated conditions. It is
possible that sPLA2-IIA may be a severity factor
in these conditions also. Genome scanning studies have shown that
several non-HLA loci contribute to disease susceptibility and severity
in humans and in animal models of immune-mediated disease.
Susceptibility loci identified in several autoimmune disorders,
including RA (44), cluster to discrete chromosomal regions
(45), suggesting that immune-mediated diseases may have
genetic features that are shared, despite their diverse clinical
manifestations. One of these loci (human chromosome 1p36-ter),
identified as a susceptibility locus in multiple sclerosis and Crohn’s
disease (45) and later in RA (44), overlap
with the chromosomal location of the genes encoding
sPLA2-IIA, -IID, -IIE, and -V, 1p35–36 (33, 34, 46). Thus, as suggested by our biochemical studies and by
transgenic overexpression of sPLA2-IIA with
TNF- (39), sPLA2-IIA may be one
among several candidate genes in this region in which mutations that
lead to variation in expression may contribute to onset and/or severity
of immune-mediated inflammatory diseases in humans.
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Acknowledgments |
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Footnotes |
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2 Current address: Executive Dean, Faculty of Health Sciences, University of Queensland, Edith Cavell Building, Royal Brisbane Hospital, Herston, QLD 4029, Australia.
3 Address correspondence and reprint requests to Dr. Kieran F. Scott, Department of Medicine, University of New South Wales, St. Vincent’s Hospital, Level 10, Garvan Institute Building, 384 Victoria Street, Sydney, New South Wales 2010, Australia.
4 Abbreviations used in this paper: RA, rheumatoid arthritis; MAPK, mitogen-activated protein kinase; RSF, rheumatoid synovial fibroblast; cPLA2, cytosolic phospholipase A2; COX, cyclooxygenase; NSAID, nonsteroidal anti-inflammatory drug; sPLA2-IIA, human type IIA secretory phospholipase A2; NLF, neonatal lung fibroblast.
Received for publication January 28, 2000. Accepted for publication June 8, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation provides the potential link between inflammation and hyperplasia in the arthritic joint. Proc. Natl. Acad. Sci. USA 95:13859., and tumor necrosis factor- increase human osteoclast formation and bone resorption in vitro. Endocrinology 139:3157.B proteins alters interleukin-1ß-induced human rheumatoid synovial fibroblast prostaglandin E2 formation. J. Biol. Chem. 271:31496.This article has been cited by other articles:
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 S. Beck, G. Lambeau, K. Scholz-Pedretti, M. H. Gelb, M. J. W. Janssen, S. H. Edwards, D. C. Wilton, J. Pfeilschifter, and M. Kaszkin Potentiation of Tumor Necrosis Factor {alpha}-induced Secreted Phospholipase A2 (sPLA2)-IIA Expression in Mesangial Cells by an Autocrine Loop Involving sPLA2 and Peroxisome Proliferator-activated Receptor {alpha} Activation J. Biol. Chem., August 8, 2003; 278(32): 29799 - 29812. [Abstract] [Full Text] [PDF]
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 H. S. Smith and W. Baird Meloxicam and selective COX-2 inhibitors in the management of pain in the palliative care population American Journal of Hospice and Palliative Medicine, July 1, 2003; 20(4): 297 - 306. [Abstract] [PDF]
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D.-W. Park, J.-R. Kim, S.-Y. Kim, J.-K. Sonn, O.-S. Bang, S.-S. Kang, J.-H. Kim, and S.-H. Baek Akt as a Mediator of Secretory Phospholipase A2 Receptor-Involved Inducible Nitric Oxide Synthase Expression J. Immunol., February 15, 2003; 170(4): 2093 - 2099. [Abstract] [Full Text] [PDF]
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M. W. Anthonsen, A. Solhaug, and B. Johansen Functional Coupling between Secretory and Cytosolic Phospholipase A2 Modulates Tumor Necrosis Factor-alpha - and Interleukin-1beta -induced NF-kappa B Activation J. Biol. Chem., August 3, 2001; 276(32): 30527 - 30536. [Abstract] [Full Text] [PDF]
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W. B. Church, A. S. Inglis, A. Tseng, R. Duell, P.-W. Lei, K. J. Bryant, and K. F. Scott A Novel Approach to the Design of Inhibitors of Human Secreted Phospholipase A2 Based on Native Peptide Inhibition J. Biol. Chem., August 24, 2001; 276(35): 33156 - 33164. [Abstract] [Full Text] [PDF]
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 M. Hernandez, L. Fuentes, F. J. Fernandez Aviles, M. S. Crespo, and M. L. Nieto Secretory Phospholipase A2 Elicits Proinflammatory Changes and Upregulates the Surface Expression of Fas Ligand in Monocytic Cells: Potential Relevance for Atherogenesis Circ. Res., January 11, 2002; 90(1): 38 - 45. [Abstract] [Full Text] [PDF]
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) or
in combination with TNF-
). Unstimulated PGE2
levels ranged from 46 ± 2 to 245 ± 10 pg/ml. **,
p < 0.01; ***, p <
0.001 between LY311727-treated cells and
sPLA2-IIA/TNF-
, sPLA2/COX-2-positive,
CD14-negative cell; 
, sPLA2-positive,
COX-2/CD14-negative cell; and 
,
sPLA2/COX-2/CD14-positive cell. Adjacent sections were used
for control staining with isotype-matched irrelevant primary Ab
controls: goat IgG (E), IgG1