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Section of Rheumatology, Department of Medicine, University of Illinois College of Medicine, Chicago, IL 60607
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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3-fold increase
of TIMP-2 protein levels. LXA4 inhibitory responses were
dose dependent and were abrogated by pretreatment with LXA4
receptor antiserum. LXA4-induced changes of IL-6 and TIMP
were accompanied by parallel changes in mRNA levels. These results
indicate that LXA4 in activated SF inhibits the synthesis
of inflammatory cytokines and MMP and stimulates TIMP production in
vitro. These findings suggest that LXA4 may be involved in
a negative feedback loop opposing inflammatory cytokine-induced
activation of SF. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1 are potent agonists of human
synovial fibroblast (SF) cell activation, a central mechanism of tissue
damage in the inflamed joint (3, 4). The active role of SF
in the formation of the invasive rheumatoid pannus has been recently
stressed by groups indicating the relevance of the synoviocyte
synthesis of matrix metalloproteinase (5, 6, 7) and release
of IL-6 (1, 8, 9, 10, 11), a cytokine that plays an essential
pathogenetic role in animal models of RA (12). Immunomodulatory cross-interactions existing between lipid and cytokine networks (13, 14) have led us to address the role of a novel class of antiinflammatory eicosanoids, the lipoxins, in regulating SF activation by IL-1ß. Lipoxins are trihydroxytetraene-containing eicosanoids that modulate leukocyte function (reviewed in Ref. (15)) and play a role in inflammation and wound healing (16). Lipoxin A4 (LXA4), the most potent isomer generated in mammalian cells (16), causes inhibition of neutrophil activation (17, 18), antagonism toward peptido-leukotrienes (19), and decrease of inflammatory infiltrates and edema in vivo (20). These antiinflammatory effects are exerted via specific high affinity receptors and a G protein, pertussis toxin-sensitive mechanism of signal transduction (21, 22, 23).
We report for the first time the identification of functional LXA4 receptors on human SF. These receptors, detected by RT-PCR and nested PCR using oligonucleotide primers derived from the sequence of the LXA4R expressed in cells of myeloid lineage (24), mediate LXA4 suppression of IL-1ß-induced activation of human SF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synthetic LXA4 was obtained from Cayman
Chemical (Ann Arbor, MI). DMEM and cell culture reagents were from
BioWhittaker (Walkersville, MD), with the exception of vitamin
supplement obtained from Life Technologies (Grand Island, NY).
Plasticware, agarose, and other biochemicals were from Fisher
Scientific (Houston, TX). Reverse transcription and PCR enzymes were
purchased from Promega (Madison, WI). Human rIL-1ß was purchased from
Boehringer Mannheim (Indianapolis, IN). Rabbit
LXA4 receptor antiserum
(LXA4R) was prepared as previously reported
(17).
Cell culture
Human primary SF were obtained from arthroscopic knee biopsies and grown under standard 5% CO2 humidified atmosphere at 37°C. Eagle’s MEM (EMEM) or DMEM containing 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and vitamin supplement were used for cell culture. After trypsinization, cells were propagated in 162-cm2 flasks until reaching passage 6. For experiments, SF were seeded in either 25-cm2 flasks or six-well plates and allowed to reach confluence. Twenty-four hours before each experiment, fresh medium containing 5% FBS was added to cultures. Only SF passages 7–11 were used in experiments to assure that cultures would be free of contaminating macrophages. Results were confirmed with monocyte-macrophage-depleted, fibroblast-enriched cultures, obtained by sequential negative plus positive selection steps involving a ferrit-bound microbead magnetic system using specific Abs directed against monocytes-macrophages and fibroblast cell surface markers, respectively (Miltenyi Biotec, Auburn, CA).
Measurement of IL-6, IL-8, MMP, and TIMP proteins
SF supernatants were collected at 24 h after the beginning of treatments, centrifuged (1500 rpm, 5 min), aliquoted and stored at -20°C. Samples were thawed at room temperature and, when necessary, diluted in 5% FBS-complete EMEM (Biowhittaker). Triplicate sample determinations using ELISA kits for human IL-6, IL-8 purchased from Endogen (Woburn, MA) were performed following manufacturer’s instructions. Human collagenase (matrix metalloproteinase-1 (MMP-1)), stromelysin (matrix metalloproteinase-3 (MMP-3)), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) ELISA kits were from Amersham Pharmacia Biotech (Arlington Heights, IL). In all experiments, a final vehicle ethanol concentration of <0.01% was used.
Actinomycin D pulse experiments
Transcription inhibition was achieved with actinomycin D or with 5,6-dichlorobenzimidazole riboside (DRB), both from Sigma (St. Louis, MO). SF were treated with IL-1ß, LXA4, or vehicle for 24 h before the addition of actinomycin D (0.5 µg/ml) or DRB (10 µg/ml). At 0, 3, 6, 12, and 24 h after actinomycin D addition, supernatants were used for ELISA, and RNA was extracted from cell pellets for mRNA.
RNA isolation, reverse transcription, and RT-PCR
Total RNA extraction was performed using TriZol reagent (Life Technologies) followed by DNase treatment (RQ1 RNase-free DNase, Promega), phenol-chloroform extraction, and ethanol precipitation (Fisher Scientific, Houston, TX). RNA purity was checked by spectrophotometry, and RNA integrity was confirmed by visualization of 28S and 18S bands on agarose gel. Next, 1 µg of RNA was transcribed using the reverse transcription system (avian myeloblastosis virus RT, Promega).
RT-PCR analyses were performed with the following sets of primers and conditions: for LXA4R, set 1: forward, 5'-CAC CAG GTG CTG CTG GCA AG-3' (bp -20 to -1), reverse, 5'-AAT ATC CCT GAC CCC ATC CTC A-3' (bp 1055–1076) amplifying a 1095-bp fragment including the full open reading frame (ORF) (annealing temperature, 60°C); set 2: forward, 5'-TGC TTG GGG TCA CCT TTG TC-3' (bp 95–114), reverse, 5'-TGA AGC AGA ATT GGC AGC CG-3' (bp 1004–1023) generating a 928-bp fragment (annealing temperature, 58°C). The ß-actin signal was determined to verify equal loading and consistency of the RT-PCR amplification. The following specific primers were used: forward, 5'-CAT GTG CAA GGC CGG CTT CG-3' (bp 86–105), reverse, 5'-GAA GGT GTG GTG CCA GAT TT-3' (bp 292–311), amplifying a 226-bp fragment (annealing temperature, 56°C). Primer set 1 for the LXA4 receptor gene has been used previously for the amplification of LXA4R mRNA from myeloid cell types (25). The second set of LXA4 receptor primers has been selected by software-aided analysis (Vector NTI version 5.0, Informax, Bethesda, MD) of the formyl peptide receptor (FPR)-related gene family of receptors. This has allowed us to identify optimal PCR primers within the ORF region of the intronless LXA4R gene carrying minimal homology to other members of the formyl peptide receptor (FPR) family. BLAST homology searches with the GenBank databases were also performed with all primers to exclude random matching with known genomic sequences. The IL-6 RT-PCR was done with the following set of primers: forward, 5'-GCC TTC GGT CCA GTT GCC TT-3' (bp 1398–1418 within exon 1) and reverse, 5'-AGT GCC TCT TTG CTG CTT TCA C-3' (bp 2677–2655 within exon 2) at an annealing temperature of 51°C. The IL-6 fragment amplified was 232 bp long. TIMP-1 and TIMP-2 RT-PCR primers were all 25-mers. The sequence of the TIMP-1 forward was 5'-ATC CTG TTG TTG CTG TGG CTG ATA G-3' and reverse, 5'-TGC TGG GTG GTA ACT CTT TAT TTC A-3' and generated a 629-bp fragment. The TIMP-2 forward primer, 5'-AAA CGA CAT TTA TGG CAA CCC TAT C-3' and reverse, 5'-ACA GGA GCC GTC ACT TCT CTT GAT G-3' generated a 430-bp fragment (annealing temperature, 64°C).
Quantification of mRNA transcripts
Quantitation of IL-6 and ß-actin mRNA transcripts was also
performed using the Xplore TM system (Endogen). Briefly, two
oligonucleotide probes (invader probe and signal probe) hybridize to a
specific mRNA target splice junction. They overlap with one base at the
hybridization site, and the enzyme cleavase VII, which clips off the
5'-fragment of the signal probe, recognizes this overlapping. The
released fragment accumulation is directly proportional to the number
of target mRNA transcripts present in the total RNA sample. A
biotinylated oligonucleotide captures (capture oligo) the signal probe
fragments on a streptavidin-coated plate and forms a primer-template
substrate for DNA polymerase, which extends the signal probe fragment
with fluorescein-dUTP. Final detection is achieved with an
anti-fluorescein alkaline phosphatase-conjugated Ab and a
chemifluorescent substrate. This novel method of mRNA detection was
preferred to quantitative PCR techniques on the basis of its
independence from RT amplification of template RNA and its direct
quantitation of target RNA molecules. In addition, two other advantages
for this new Xplore technology are the very low amounts of total RNA
required for the measurements (as little as 100 ng) and the very
high sensitivity of the assay (with lower detection limits in the
5-amol range). Known amounts of nucleic acid standards are used to
define a standard curve for the signal of interest, whereas
availability of ß-actin and GAPDH assays allow for normalization of
results. Densitometric analysis of PCR data was performed using a Gel
Doc 1000 system integrated with the Molecular Analyst software
(Bio-Rad, Hercules, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of specific high affinity LXA4R
is required for LXA4-induced modulation of
leukocyte functions (17). We examined whether
LXA4R is expressed in human SF using primers
specific for LXA4R. LXA4R
mRNA expression was demonstrated by RT-PCR amplification of SF-derived
total RNA (Fig. 1
, lane 1).
mRNA expression was markedly stimulated by IL-1ß (lane
5), LXA4 (lane 2), or
combinations of IL-1ß and LXA4
(lanes 3 and 4). Retinoic
acid-differentiated HL-60 cells (lane 8), from which
LXA4R has been cloned, were used as positive
control (24). Semiquantitative PCR amplification of
LXA4R mRNA, with primers encompassing the full
length ORF, yielded a mix of PCR products resolved in multiple bands of
molecular size ranging from 1350 to 350 bp (Fig. 1A,
top). The HL-60 cell-derived signal was consistent with
previous RT-PCR results obtained with use of these primers in myeloid
cells (25).
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A, middle PCR). Also, a third set of
LXA4R primers, yielding a smaller 359-bp
fragment, was designed to achieve the lowest homology with other
sequences in the FPR gene cluster and used in a nested PCR step to
confirm the expression of a true LXA4R on human
SF (data not shown). Densitometric analysis of the semiquantitative PCR
results indicates that IL-1ß can up-regulate
LXA4R mRNA expression by 3-fold (Fig. 1B). LXA4 inhibits IL-1ß-induced synthesis of IL-6, IL-8, and MMP-3
We next investigated SF responses to LXA4.
Because IL-6 has been implicated in the development of inflammatory
synovitis, we determined the effect of LXA4 on
IL-6 production. As shown in Fig. 2A, no effect was observed
with LXA4 alone, whereas IL-1ß-induced IL-6
release was inhibited by LXA4 at nanomolar
concentrations (Fig. 2A). Inhibition by
LXA4 was dose dependent and was abrogated by
specific LXA4R sera (Fig. 2A,
inset). These results indicate that LXA4
inhibits IL-1ß-induced IL-6 release in human SF via functional
LXA4R. After IL-1ß addition, release of IL-8, a
potent chemokine that stimulates angiogenesis and induces leukocyte
recruitment at inflammatory sites of events, was also inhibited by
LXA4 in a dose-dependent manner (Fig. 2B). The inhibition was reversed by
LXA4R (Fig. 2B, inset)
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C indicate that at subnanomolar
concentrations, LXA4 potently inhibits MMP-3
synthesis. LXA4 modulation of MMP-3 synthesis was
effectively prevented by LXA4R serum (Fig. 2C, inset). Results also indicated that
LXA4 did not significantly modify the expression
of MMP-1 (data not shown). LXA4 is a potent agonist of TIMP synthesis
During physiological tissue remodeling, activation of enzymes such
as MMP is controlled by specific inhibitors. TIMP-1 and TIMP-2
regulation by LXA4 was next determined. As shown
in Fig. 3, LXA4
stimulated the synthesis of TIMP-1 and TIMP-2, reversed the slight
inhibition of TIMP-2 by IL-1ß stimulation and induced up to 5-fold
increase in TIMP-2 protein levels (Fig. 3B). As shown in
Figs. 3 and 4,
LXA4R serum only partially affected
LXA4 stimulation of TIMP synthesis, resulting in
a modest reduction of both mRNA and protein levels (Figs. 3 and 4).
These results are in contrast to complete abrogation of
LXA4 inhibition of IL-6, IL-8, and MMP-3 release
by LXA4R (Fig. 2), suggesting that in SF
LXA4 modulatory activities might involve novel
signaling pathways in addition to those triggered by activation of
functional LXA4R.
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Next, TIMP-1, TIMP-2, and IL-6 mRNA expression was examined to
determine whether LXA4 effect involved pre- or
posttranscriptional events. Results shown in Figs. 4 and 5 confirmed those obtained for protein
synthesis (Fig. 3), with IL-1ß inducing a slight increase of TIMP-1
and a modest reduction of TIMP-2. LXA4 alone, or
in combination with IL-1ß, increased mRNA expression of both TIMPs.
Notably, LXA4 up-regulation of TIMP-2 mRNA was in
the order of 3- to 6-fold compared with control cells, whereas a 2-fold
increase was measured for TIMP-1 (Figs. 4 and 5). As indicated in Fig. 5, basal TIMP-1 mRNA expression in unstimulated cells was higher than
TIMP-2 with a significant increase for TIMP-2 mRNA expression occurring
24 h after LXA4 addition. Conversely, lower
basal TIMP-2 mRNA levels in unstimulated cells were accompanied by
significant increase after 4 h of exposure to
LXA4, and a substantial 4- to 6-fold increase was
reached after 24 h. As indicated for IL-6 (Table I), increased levels of TIMP protein and
mRNA were observed until reaching plateau at 120 h (TIMP-1 1
µg/ml and TIMP-2 50 ng/ml, after LXA4
stimulation). whereas relative ratios among different treatments were
comparable with those observed at 24 h (data not shown).
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25–40% reduction of the IL-6 protein and mRNA levels induced by
IL-1ß (Table I). Actinomycin D or DRB, two inhibitors of nucleic acid
synthesis, were used to investigate the transcriptional step
potentially modulated by LXA4. Actinomycin D was
added 24 h after SF incubation with the indicated combinations of
IL-1ß and LXA4 to block RNA synthesis. Results
indicated a mRNA half-life of 3.7 h for both IL-1ß and IL-1ß
plus LXA4-treated cells (Fig. 6). The decrease of mRNA levels observed
for the 24 h after actinomycin D additions was not reflected on
IL-6 protein levels that continued to increase slowly (Fig. 6A). As shown in Fig. 6, B and C, use
of semiquantitative RT-PCR analysis confirmed the results obtained with
the Xplore assay. RT-PCR data also indicate similar
LXA4 activities toward IL-8 and MMP-3 (data not
shown). These data indicate that in human SF LXA4
dose-dependent inhibition of IL-1ß-induced IL-6, IL-8, and MMP-3 mRNA
levels does not involve posttranscriptional regulation steps such as
modulation of mRNA stability, suggesting that earlier transcriptional
events are affected by LXA4.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-fold upon stimulation
with IL-1ß when compared with control cells (Fig. 1). Because several
LXA4 antiinflammatory activities are mediated via
signaling pathways involving LXA4R cell surface
expression (17, 22), the noted IL-1ß up-regulation
suggests that this cytokine is activating potential negative feedback
pathways along with the proinflammatory modification that it exerts on
SF. Among IL-1ß effects on LXA4R expression the
appearance during LXA4R RT-PCR amplification of
multiple bands, with sizes above and below the expected amplimer, was
also addressed. These signals cannot represent immature nuclear forms
of LXA4R mRNA transcripts because the human
LXA4R ORF is encoded by an intronless gene;
rather, they suggest the presence of LXA4R
related sequences. In view of these data, we next performed a nested
PCR confirming the identity of the target RNA strands being
investigated (Fig. 1).
Detection of LXA4R mRNA by RT-PCR does not
exclude that other pathways might be involved in mediating
LXA4 antiinflammatory activities. In myeloid
cells, we have previously demonstrated that LXA4
inhibition of homotypic aggregation and ß2
integrin up-regulation in activated polymorphonuclear cells (PMN) is
abrogated by use of LXA4R antisense
oligonucleotides and specific blocking LXA4R
sera (17). In the present study (Fig. 2, insets), LXA4R serum was used to
test the consequence of blocking LXA4R-mediated
signaling in SF. Results confirm that LXA4R
molecules are functionally expressed in human SF and that their
activation is responsible for the inhibitory actions of
LXA4 on IL-1ß-induced cell activation.
Specifically, LXA4 potently inhibits
IL-1ß-induced synthesis of IL-8 and MMP-3 and reduces by 35% IL-6
production, activities that are almost completely abrogated if cells
are exposed to the blocking LXA4R serum (Fig. 2, insets). Noticeably, LXA4
selectively impacted MMP-3 production without statistically significant
alteration of MMP-1 levels (data not shown).
Although LXA4 inhibition of SF responses resemble
events previously described in human PMN (17, 18, 27),
stimulation of TIMP-1 and TIMP-2 is a novel observation (Fig. 3). For
the first time, our results show that LXA4 has
agonist actions on human synovial fibroblasts expressing the specific
LXA4R receptor. The significance of these
bioactivities is corroborated by recent studies in human epithelial
cells, neutrophils, and murine inflammatory models where
LXA4 agonist functions lead to regulation of the
chemokine-cytokine axis (28). As previously noted,
exposing SF to LXA4R caused 90% reduction
of LXA4 effects on IL-6, IL-8, and MMP-3 but only
a 30% inhibition of LXA4-induced TIMP
stimulation. These data suggest that multiple signaling pathways are
activated and required for the expression of the full array of
LXA4 antiinflammatory activities in SF. Several
models, such as internalization of the receptor-ligand complex or
presence of specific LXA4 transporter proteins,
as we have previously documented in PMN (29), can be
hypothesized that would allow exogenous LXA4 to
reach the critical intracellular compartment where it may interact
directly with transcriptional apparatuses. To support a role for the
direct interaction of LXA4 with transcription
factors, recent studies have reported that LXA4
interacts directly with the aryl hydrocarbon receptor (AhR) determining
its activation (30). Our results would, however, indicate
that LXA4-positive regulation of TIMP (
Figs. 3–5) occurs at nanomolar concentrations of the agonist in contrast
with the micromolar amounts required for the activation of
AhR-dependent transcription. Thus, unless potential mechanisms for
intracellular accumulation of LXA4 in SF will be
experimentally verified, the present data would suggest that in human
SF, transcriptional mechanisms other than AhR may be involved in
LXA4 stimulation of TIMP synthesis.
Inhibition of IL-6 release by LXA4 is of interest
because significant elevation of IL-6 has been detected both
systemically and in the joint tissues in RA patients (8, 11). Experimental and clinical observations support a
pathogenetic role for IL-6 in RA (8, 11, 12, 31, 32). For
example, a functional IL-6 gene is necessary for development of
collagen type II-induced arthritis in mice (12), and a
link with IL-6 levels has been established for an Ag-induced arthritis
model (31). In the above-cited models, abrogated or
reduced IL-6 levels block the onset of RA. Results shown in Fig. 6 and
Table I indicate that nanomolar concentrations of
LXA4 can effectively antagonize IL-1ß-induced
synthesis of IL-6 mRNA. Time course studies shown in Table I indicate
that LXA4 caused a reduction of IL-6 mRNA
apparent half-lives (from 2.3 and 1.8 days, IL-1ß vs. IL-1ß plus
LXA4, respectively). However, actinomycin D pulse
experiments show that after IL-1ß stimulation the IL-6 mRNA half-life
of 3.7 h is not significantly affected by LXA4
(Fig. 6), suggesting that LXA4 triggered events
can impact the transcriptional regulation of the IL-6 gene without
altering its mRNA stability.
Overall, LXA4 modulation of SF activation could greatly impact the progression of inflammatory processes where reduction of SF-derived IL-8 can lead to reduced migration of inflammatory leukocytes in the joint tissues whereas lower levels of MMP-3 could limit the extent of tissue destruction. Increased synthesis of TIMP-1 and TIMP-2 would further enhance LXA4-mediated mechanisms of tissue protection. The in vivo significance of the observed responses to LXA4 by SF remains to be determined. However, the concerted inhibition of inflammatory cytokine-induced MMP expression, IL-6 and IL-8 release, combined with stimulation of TIMP-1 and TIMP-2 expression strongly suggest that the immunomodulatory actions of LXA4 may play a major role in counteracting the development of inflammatory arthritides. In vivo studies have recently demonstrated that LXA4 significantly decreases inflammatory infiltrates and edema in a mouse model of inflammation (20). In this model, stable LXA4 analogues interact with a mouse homologue of the human LXA4R, leading to antiinflammatory activities that are more potent than those observed with equimolar concentrations of dexamethasone (20). Similarly, our results with human SF strongly suggest that LXA4 possesses antiinflammatory effects that may be involved in regulating critical pathophysiological processes in the development of inflammatory arthritides.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stefano Fiore, Section of Rheumatology, M/C 733, Molecular Biology Research Building, Room 1252, University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, IL 60607-7171. E-mail address:
3 Abbreviations used in this paper: RA, rheumatoid arthritis; EMEM, Eagle’s MEM; LXA4, lipoxin A4, 5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid; LXA4R, lipoxin A4 receptor; LXA4R, rabbit serum raised against the lipoxin A4 receptor; MMP, matrix metalloproteinases; MMP-1, collagenase, matrix metalloproteinase-1; MMP-3, stromelysin, matrix metalloproteinase-3; FPR, formyl peptide-related receptor; ORF, open reading frame; SF, synovial fibroblast; TIMP-1, tissue inhibitor of matrix metalloproteinase-1; TIMP-2, tissue inhibitor of matrix metalloproteinase-2; DRB, 5,6-dichlorobenzimidazole riboside; PMN, polymorphonuclear cells; AhR, aryl hydrocarbon receptor.
Received for publication August 19, 1999. Accepted for publication December 13, 1999.
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 S. Gangemi, G. Luciotti, E. D'Urbano, A. Mallamace, D. Santoro, G. Bellinghieri, G. Davi, and M. Romano Physical exercise increases urinary excretion of lipoxin A4 and related compounds J Appl Physiol, June 1, 2003; 94(6): 2237 - 2240. [Abstract] [Full Text] [PDF]
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 J Martel-Pelletier, D Lajeunesse, P Reboul, and J-P Pelletier Therapeutic role of dual inhibitors of 5-LOX and COX, selective and non-selective non-steroidal anti-inflammatory drugs Ann Rheum Dis, June 1, 2003; 62(6): 501 - 509. [Abstract] [Full Text] [PDF]
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 C. Bonnans, B. Mainprice, P. Chanez, J. Bousquet, and V. Urbach Lipoxin A4 Stimulates a Cytosolic Ca2+ Increase in Human Bronchial Epithelium J. Biol. Chem., March 21, 2003; 278(13): 10879 - 10884. [Abstract] [Full Text] [PDF]
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 R. He, H. Sang, and R. D. Ye Serum amyloid A induces IL-8 secretion through a G protein-coupled receptor, FPRL1/LXA4R Blood, February 15, 2003; 101(4): 1572 - 1581. [Abstract] [Full Text] [PDF]
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 T. E. Van Dyke and C.N. Serhan Resolution of Inflammation: A New Paradigm for the Pathogenesis of Periodontal Diseases Journal of Dental Research, February 1, 2003; 82(2): 82 - 90. [Abstract] [Full Text] [PDF]
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H. Perlman, K. Bradley, H. Liu, S. Cole, E. Shamiyeh, R. C. Smith, K. Walsh, S. Fiore, A. E. Koch, G. S. Firestein, et al. IL-6 and Matrix Metalloproteinase-1 Are Regulated by the Cyclin-Dependent Kinase Inhibitor p21 in Synovial Fibroblasts J. Immunol., January 15, 2003; 170(2): 838 - 845. [Abstract] [Full Text] [PDF]
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T C A Tolboom, E Pieterman, W H van der Laan, R E M Toes, A L Huidekoper, R G H H Nelissen, F C Breedveld, and T W J Huizinga Invasive properties of fibroblast-like synoviocytes: correlation with growth characteristics and expression of MMP-1, MMP-3, and MMP-10 Ann Rheum Dis, November 1, 2002; 61(11): 975 - 980. [Abstract] [Full Text] [PDF]
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 S. Mitchell, G. Thomas, K. Harvey, D. Cottell, K. Reville, G. Berlasconi, N. A. Petasis, L. Erwig, A. J. Rees, J. Savill, et al. Lipoxins, Aspirin-Triggered Epi-Lipoxins, Lipoxin Stable Analogues, and the Resolution of Inflammation: Stimulation of Macrophage Phagocytosis of Apoptotic Neutrophils In Vivo J. Am. Soc. Nephrol., October 1, 2002; 13(10): 2497 - 2507. [Abstract] [Full Text] [PDF]
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 L. Jozsef, C. Zouki, N. A. Petasis, C. N. Serhan, and J. G. Filep Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 inhibit peroxynitrite formation, NF-kappa B and AP-1 activation, and IL-8 gene expression in human leukocytes PNAS, October 1, 2002; 99(20): 13266 - 13271. [Abstract] [Full Text] [PDF]
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 J. E. Moore, T. C. B. McMullen, I. L. Campbell, R. Rohan, Y. Kaji, N. A. Afshari, T. Usui, D. B. Archer, and A. P. Adamis The Inflammatory Milieu Associated with Conjunctivalized Cornea and Its Alteration with IL-1 RA Gene Therapy Invest. Ophthalmol. Vis. Sci., September 1, 2002; 43(9): 2905 - 2915. [Abstract] [Full Text] [PDF]
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 C. Bonnans, I. Vachier, C. Chavis, P. Godard, J. Bousquet, and P. Chanez Lipoxins Are Potential Endogenous Antiinflammatory Mediators in Asthma Am. J. Respir. Crit. Care Med., June 1, 2002; 165(11): 1531 - 1535. [Abstract] [Full Text] [PDF]
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F. Bureau, C. Desmet, D. Melotte, F. Jaspar, C. Volanti, A. Vanderplasschen, P.-P. Pastoret, J. Piette, and P. Lekeux A Proinflammatory Role for the Cyclopentenone Prostaglandins at Low Micromolar Concentrations: Oxidative Stress-Induced Extracellular Signal-Regulated Kinase Activation Without NF-{kappa}B Inhibition J. Immunol., May 15, 2002; 168(10): 5318 - 5325. [Abstract] [Full Text] [PDF]
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 I. M. Fierro, J. L. Kutok, and C. N. Serhan Novel Lipid Mediator Regulators of Endothelial Cell Proliferation and Migration: Aspirin-Triggered-15R-Lipoxin A4 and Lipoxin A4 J. Pharmacol. Exp. Ther., February 1, 2002; 300(2): 385 - 392. [Abstract] [Full Text] [PDF]
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J. Goh, A. W. Baird, C. O'Keane, R. W. G. Watson, D. Cottell, G. Bernasconi, N. A. Petasis, C. Godson, H. R. Brady, and P. MacMathuna Lipoxin A4 and Aspirin-Triggered 15-Epi-Lipoxin A4 Antagonize TNF-{alpha}-Stimulated Neutrophil-Enterocyte Interactions In Vitro and Attenuate TNF-{alpha}-Induced Chemokine Release and Colonocyte Apoptosis in Human Intestinal Mucosa Ex Vivo J. Immunol., September 1, 2001; 167(5): 2772 - 2780. [Abstract] [Full Text] [PDF]
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B. McMahon, C. Stenson, F. McPhillips, A. Fanning, H. R. Brady, and C. Godson Lipoxin A4 Antagonizes the Mitogenic Effects of Leukotriene D4 in Human Renal Mesangial Cells. DIFFERENTIAL ACTIVATION OF MAP KINASES THROUGH DISTINCT RECEPTORS J. Biol. Chem., September 1, 2000; 275(36): 27566 - 27575. [Abstract] [Full Text] [PDF]
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), LXA4
(10-9 M, 
), or IL-1ß (500 pg/ml, 
) alone or
IL-1ß in presence of the indicated LXA4 concentrations
(
). IL-6 (A), IL-8 (B), and MMP-3
(C) were measured in supernatants by ELISA.
Insets, Samples were kept for 24 h in the
presence of IL-1ß (
) were
compared with IL-1ß-stimulated samples (
). Student’s t test analysis for the
IL-1ß-stimulated samples was assessed comparing treatments in
presence or absence of LXA4. Results with P values of
<0.05 (*) have been indicated. B, Vehicle,
lanes 1, 5, 9, 13, and 17; IL-1ß,
lanes 2, 6, 10, 14, and 18; IL-1ß +
LXA4, lanes 3, 7, 11, 15, and
19; LXA4 alone, lanes 4, 8, 12,
16, and 20. C, Histogram
indicates densitometric results (after ß-actin normalization) for
IL-6 mRNA signals obtained from the RT-PCR shown in B.
Vehicle (