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Department of Pathology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß, and platelet-derived growth factor was not
affected by the EC-PMN coculture. Neutralizing mAbs to ICAM-1 or
ß2 integrins or a physical segregation of PMNs and ECs
did not reduce EC stimulation. In contrast, cell-free supernatants of
PMNs recapitulated EC activation with an 18-fold up-regulation of EC
IL-6 mRNA. The filtration of PMN supernatant or PMN pretreatment with
metabolic antagonists or membrane cross-linking agents all suppressed
EC activation. By flow cytometry, PMNs released in the supernatant,
heterogeneous membrane-derived microparticles containing discrete
proteins of 28 to 250 kDa as resolved by SDS-PAGE. PMN
microparticle formation was enhanced by inflammatory stimuli, including
formyl peptide and phorbol ester, and was time-dependent, reaching a
plateau after a 1-h incubation from stimulation. Purified PMN
microparticles induced EC IL-6 release in a reaction that was
quantitatively indistinguishable from that observed with unfractionated
PMN supernatant and unaffected by a neutralizing Ab to soluble IL-6R.
These findings demonstrate that membrane microparticles released from
stimulated PMNs are competent inflammatory mediators to produce EC
activation and cytokine gene induction. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we sought to reinvestigate the potential participation of polymorphonuclear leukocytes (PMNs) in EC stimulation as reflected in the release of inflammatory and chemotactic cytokines and the up-regulation of leukocyte-EC adhesion molecules. We found that coculturing PMNs with endothelium induced EC activation and inflammatory gene induction. In addition, this pathway was mediated by membrane microparticles released from activated PMNs acting as competent inflammatory mediators on the endothelium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PMNs were isolated from acid-citrate-dextrose anticoagulated blood drawn after informed consent from normal healthy volunteers by differential centrifugation on a Ficoll-Hypaque gradient and dextran sedimentation as described previously (11). ECs were prepared by collagenase treatment and used between passages 2 and 4.
mAbs
Anti-ICAM-1 mAbs 2D5, 6E6, and 3D6 were generated and characterized for function and epitope recognition in previous studies (12). Neutralizing anti-CD18 mAbs 60.3 and IB4 and anti-CD11a mAb TS1/22 were obtained from American Type Culture Collection (Manassas, VA). Anti-VCAM-1 (E16.15) and anti-E-selectin (H4/18) mAbs were a kind gift of Dr. J. Bender (Yale University). A neutralizing polyclonal anti-IL-6R Ab was purchased from Endogen (Cambridge, MA). Nonbinding mAb 14E11 was used as a negative control.
EC activation by leukocyte-EC cocultures
PMNs were suspended at 3 x 106 cells/ml in
medium 199 (BioWhittaker, Walkersville, MD) supplemented with 20%
heat-inactivated FBS (BioWhittaker), penicillin (100 U/ml)-streptomycin
(100 µg/ml), and L-glutamine (2 mM) (pH 7.4). Cells were
stimulated in the presence or absence of Ca2+ ions (2.5
mM), FMLP (10 µM), Con A (5 µg/ml), or PMA (10 ng/ml) and
added to monolayers of ECs grown to confluency in 96-well plates for
10 h at 37°C. In control experiments, ECs were also directly
stimulated with the same concentrations of the stimuli for 10 h at
37°C. At the end of the incubation under the various conditions
tested, the cell-free supernatants were collected, centrifuged for 10
min at 200 x g, and analyzed for released IL-6, IL-8,
TNF-, IL-1ß, or platelet-derived growth factor by ELISA
(Endogen). In another set of experiments, FMLP (10 µM)-stimulated
PMNs and resting ECs were preincubated with 20 µg/ml of control
nonbinding mAb 14E11 or mAbs to CD18 (60.3), CD11a (TS1/22), or ICAM-1
(2D5, 3D6, or 6E6) for 20 min at 22°C. PMNs were then added to ECs
for 10 h at 37°C before the determination of IL-6 release by
ELISA. In contact inhibition experiments, confluent ECs were grown on
insert Transwell membranes (8 µm, Costar, Cambridge, MA), whereas
FMLP-stimulated PMNs were incubated in the lower Transwell compartment.
After a 10-h culture at 37°C, IL-6 release was determined by ELISA.
In other experiments, cell-free supernatants from FMLP-stimulated PMNs
(3 x 106/ml) or 10 ng/ml of TNF--containing medium
were collected and sterile-filtered through a 0.2-µm pore-sized
membrane of disposable Millex-VV filter units (Millipore, Bedford, MA).
Filters were rinsed by flushing 2 ml of PBS through the pores and
retrieved in 1 ml of reversed flow. Aliquots of filtered supernatants
or retrieved particles were added to confluent EC monolayers, and the
level of IL-6 secretion was measured after a 10-h incubation as
described previously. In another series of experiments, FMLP-stimulated
PMNs (3 x 106/ml) were incubated in 96-well tissue
culture plates for 2 to 6 h at 37°C. Cells were centrifuged at
200 x g, and the cell-free supernatant (300 µl) was
collected and added to confluent EC monolayers for an additional
10 h of incubation at 37°C before the determination of released
IL-6 and IL-8 as described above. In other experiments, cytokine
release was determined after a 0- to 43-h stimulation of ECs at 37°C.
In another series of experiments, cytokine release from ECs was
determined following heat-denaturation of FMLP-stimulated PMN
supernatants at 100°C for 5 min or pretreatment of the PMN suspension
with 1% paraformaldehyde/PBS for 2 h at 4°C. Alternatively,
FMLP-stimulated PMNs (3 x 106/ml) were incubated with
the metabolic inhibitor 2-deoxy-D-glucose (2DG) (50 mM;
Sigma, St. Louis, MO) alone or in combination with cycloheximide (CX)
(10 µg/ml; Sigma) and sodium azide (0.02%; Fisher Scientific,
Pittsburgh, PA) for 2 h at 37°C. Cell-free supernatants
under the various experimental conditions were collected and analyzed
for induction of EC release of IL-6 following a 10-h incubation as
described above. In another series of experiments, FMLP-stimulated PMN
suspensions or their derived cell-free supernatants were incubated with
an anti-IL-6R neutralizing Ab, anti-CD18 mAb IB4, or control
mAb 14E11 (all at 25 µg/ml) for 20 min at 22°C before addition to
EC monolayers and determination of IL-6 release. In other experiments,
FMLP-stimulated suspensions of PMNs were incubated with confluent EC
monolayers for 10 h at 37°C. After washes, ECs were fixed in
cold (-20°C) methanol for 15 min, washed, and incubated in PBS
containing 5% FBS and 0.05% Tween-20 for 15 min at 22°C. ECs were
then incubated in RPMI 1640 containing 10% FBS and mAbs to
ICAM-1 (2D5, 20 µg/ml), VCAM-1 (E16.15, 1:2), E-selectin (H4/18,
1:10), MHC class I (W6/32, 20 µg/ml), or control nonbinding IgG
(14E11, 20 µg/ml) for 1 h at 22°C. After three washes at
22°C, cells were incubated with peroxidase-conjugated goat
anti-mouse IgG for 30 min at 22°C followed by the addition of
tetramethylbenzidine and H2O2 as substrates and
determination of OD at 
= 450 nm.
Northern hybridization
ECs (passage 2–3) seeded in 75-cm2 cell culture flasks to 90% confluence were incubated in the presence of untreated or 0.2 µm-filtered cell-free supernatant from FMLP-stimulated PMNs (5 x 106/ml) for 10 h at 37°C. At the end of the incubation, ECs were washed, and total RNA was extracted by the RNAzol B method (Tel-Test, Friendswood, TX). Samples (15 µg) were separated by electrophoresis on denaturing agarose formaldehyde gels and transferred to nylon membranes (GeneScreen, New England Nuclear Life Science Products, Boston, MA) by overnight capillary transfer in 20x SSC (1x SSC is 150 mM NaCl and 15 mM sodium citrate (pH 7.0)). Following UV cross-linking, filters were sequentially hybridized with [32P]deoxyCTP (Amersham, Arlington Heights, IL), random-primed (Boehringer Mannheim, La Jolla, CA), labeled IL-6 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes. Hybridization was performed in 5x SSC, 10x Denhardt’s solution, 1% SDS, and 100 µg/ml of salmon sperm DNA for 14 h at 60°C, with washes in 2x SSC and 1% SDS at 60°C. Blots were exposed to a Kodak phosphorimaging screen, and signals were quantified using equipment and software from the same manufacturer. Probe stripping was conducted in 0.5% SDS at 90°C.
Microparticle analysis
PMN-derived microparticles were quantitated by flow cytometry using a fluorescent lipid intercalating dye, PKH26-GL (Sigma). This aliphatic chromophore partitions into lipid bilayers and confers a red fluorescence. PMNs (5 x 106 cells/ml) were labeled with PKH26-GL (4 µM) according to the manufacturer’s specifications. The labeled cells were incubated in the presence or absence of CaCl2 (2.5 mM), serum, and 10 µM FMLP. At various time intervals between 3 min and 6 h at 37°C, the cell-containing supernatant, cell-free supernatant containing PMN-derived vesicles, and 0.2-µm pore-filtered cell-free supernatant were isolated and analyzed by flow cytometry. In other experiments, increasing concentrations of FMLP (0–10 µM) were used to stimulate PMN suspensions in medium alone for 1 h before analysis of microparticle release by flow cytometry. Samples were analyzed for forward and side scatter parameters using a FACS (Becton Dickinson, Mountain View, CA). Each sample was analyzed for a total of 25,000 events or a 10-s interval. A gate was chosen to include particles distinctly positive for red fluorescence. In parallel experiments, membrane microparticles purified from the supernatant of FMLP-stimulated PMNs by ultracentrifugation at 60,000 rpm for 2 h were lysed in 1% Triton X-100 and 0.05% SDS plus protease inhibitors. Extracts were separated by electrophoresis on a 6% SDS polyacrylamide gel with visualization of protein bands by Coomassie blue staining under nonreducing conditions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 10 h-coculture of FMLP (10 µM)-stimulated PMNs with ECs
resulted in a dramatic increase in the release of cytokines IL-6 and
IL-8 (Fig. 1
A). Under these
experimental conditions, IL-6 release (888 ± 71 pg/ml) increased
by 35-fold over values observed with either cell types alone; IL-8
release (45.2 ± 14.5 ng/ml) increased by 6.4- and 173-fold over
values observed with PMNs or ECs alone, respectively (Fig. 1A). The potential requirement of PMN stimulation and/or
divalent ion in EC activation was investigated. First, optimal
induction of EC release of IL-6 by PMN coculture was observed in the
presence of Ca2+ ions (Table I), and serum (see below). Second, PMN
stimulation with inflammatory agonists, including the chemoattractant
FMLP, phorbol ester (PMA), or Con A, all resulted in increased EC
release of IL-6 as compared with unstimulated cultures or ECs directly
stimulated with FMLP, PMA, or Con A under the same experimental
conditions (Table I). The addition of PMNs to EC monolayers was also
associated with an up-regulation of inducible leukocyte-EC adhesion
molecules, ICAM-1, VCAM-1, and E-selectin (Fig. 1B). As
determined by ELISA, the magnitude of this response was quantitatively
indistinguishable from that observed in control incubation reactions
with TNF--activated ECs (Fig. 1B). In contrast, the
release of IL-1ß, platelet-derived growth factor, or TNF-
or the modulation of MHC class I molecules was not affected under the
same experimental conditions of PMN-EC coculture (Fig. 1, A
and B).
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A). Similarly, a physical
separation of ECs and PMNs in Transwell chambers only partially reduced
the IL-6 generation by ECs observed in coculture experiments (Fig. 2B). At variance with these conditions, cell-free
supernatants of FMLP-stimulated PMNs induced EC release of IL-6 in a
reaction that was quantitatively indistinguishable from that obtained
in coculture experiments (Fig. 3). This
response was abrogated by heat denaturation of the PMN supernatant or
by prior treatment of the PMN suspension with paraformaldehyde or with
metabolic inhibitor 2DG alone or in combination with sodium azide and
CX (Fig. 3). Filtration of the cell-free supernatant from
FMLP-stimulated PMNs through a 0.2-µm filter or ultrafiltration with
a 100-kDa cut-off range completely abolished the stimulatory effect of
this supernatant on EC release of IL-6 (Fig. 3). In control
experiments, filtration of a TNF--containing supernatant did not
reduce EC release of IL-6 (Fig. 3). Finally, cell-free supernatants of
FMLP-stimulated PMNs induced EC release of IL-6 and IL-8 in a
time-dependent manner, reaching a maximum secretion at 10 to 18 h
poststimulation (Fig. 4, A and
B).
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By Northern hybridization, incubating resting EC monolayers with
cell-free supernatants of FMLP-stimulated PMNs resulted in an
18-fold induction of IL-6 mRNA as compared with background levels of
untreated ECs (Fig. 5). Consistent with
the data presented above, filtration of the PMN supernatant
significantly reduced the increase in IL-6 mRNA expression under the
same experimental conditions (Fig. 5). In control hybridization
studies, PMN-derived supernatants failed to modulate the mRNA levels of
GAPDH under the same experimental conditions (Fig. 5).
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, top left quadrant and
inset). PMN stimulation with FMLP resulted in an 4-fold increase in
the population with smaller forward and side scatter parameters
(Fig. 6, top right quadrant), which is consistent with its
potential identity with released membrane microparticles. This
population was heterogeneous in size and was maintained in cell-free
PMN supernatants, whereas the larger population corresponding to the
cellular fraction was entirely depleted under these experimental
conditions (Fig. 6, lower left quadrant). However,
filtration of cell-free PMN supernatant through a 0.2-µm filter
resulted in removal of the microparticle population by flow cytometry
(Fig. 6, lower right quadrant). Microparticle release of PMN
suspensions in medium alone occurred in an FMLP concentration-dependent
manner, reaching a plateau at 100 nM of added stimulus (Fig. 7A). In time-course
experiments, microparticle release occurred in a time-dependent
reaction, reaching a plateau at 1 h after stimulation, with no
further increases for 
6 h incubation (Fig. 7B). Consistent
with the data presented above, optimal microparticle release required
the presence of serum and was enhanced by FMLP stimulation of PMNs
(Fig. 7B).
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85-kDa component and
fainter bands ranging in relative molecular mass between 28 and
250 kDa (Fig. 8). In EC activation
experiments, the addition of purified PMN microparticles to EC
monolayers resulted in prominent IL-6 release in a reaction that was
quantitatively indistinguishable from that observed in coculture
conditions or with the unfractionated, cell-free, PMN-derived
supernatant (Fig. 9). Under these
experimental conditions, preincubating FMLP-stimulated PMNs with
neutralizing mAbs to ß2 integrins or to soluble IL-6R
failed to decrease EC release of IL-6 stimulated by PMN microparticles
(Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The possibility that cellular-derived microparticles could contribute to vascular cell responses has been postulated earlier. In the model of the platelet, activation by disparate stimuli including thrombin and the complement membrane attack complex C5b-9 has been characterized previously for its ability to induce membrane microvesiculation and particle release (13, 14). This process was shown to require a calpain-dependent dissociation of membrane proteins from the submembrane cytoskeleton (15) and potential intracellular signaling by the activity of one or more platelet protein kinases (i.e., myosin light chain kinase and Ca2+-calmodulin complex) (14). Functionally, released platelet microparticles contributed a favorable, negatively charged, phosphatidylserine environment for prothrombinase complex assembly and amplification of a procoagulant response. In this context, blood samples from patients with a generalized activation of coagulation were shown to contain platelet-derived microparticles, thus suggesting their potential role in disseminated amplification of coagulation in vivo (16). Alternatively, the expression of functional ß3 integrins on platelet-derived microparticles suggested their potential involvement in adhesion and intracellular signaling mechanisms (17). Recently, a similar paradigm of microvesiculation has been extended to the leukocyte, with the demonstration that endotoxin-stimulated monocytes released heterogeneous phosphatidylserine-containing membrane microparticles, potentially contributing to prothrombinase activity and integrin-dependent adhesion reactions (9, 10).
In expanding these earlier observations, we now show that
PMN-derived microparticles not only provide a negatively charged
microenvironment for potential amplification of the coagulation cascade
but also act as potent proinflammatory agonists competent to initiate a
broad pathway of signal transduction and gene expression in ECs. This
finding was reflected in the dramatic de novo up-regulation of
endothelial IL-6 mRNA, which was associated with prominent release of
IL-6 and IL-8, and in the induction of multiple EC-leukocyte adhesion
molecules. Consistent with this paradigm, depletion of the
microparticle fraction from PMN supernatants abrogated the inflammatory
response of the endothelium, whereas prevention of cell-to-cell contact
or neutralizing Abs to ICAM-1 or ß2 integrins was
ineffective. Although the mechanism of the observed leukocyte
vesiculation remains to be elucidated, previous studies have suggested
a role for cytoskeletal rearrangements (18) and/or a protein synthesis-
and energy-dependent response to stimulation in this process (9).
Consistent with this view, PMN microparticle release required divalent
cations and serum and was significantly enhanced by stimulation
with various inflammatory agonists in a rapid reaction that was
completed 1 h after stimulation. By flow cytometry and initial
biochemical characterization, PMN microparticles were heterogeneous in
size and of limited complexity, containing a prominent 85-kDa band
and additional proteins ranging in relative molecular mass between 28
and 250 kDa. It is unlikely that the stimulatory effect on the
endothelium described here reflects endotoxin contamination of the
culture medium of leukocyte microparticles. First, comparable
experimental conditions in the absence of PMN supernatant were not
associated with endothelial cytokine release. Second, ultrafiltration
and heat treatment of PMN supernatant completely abrogated this pathway
of EC activation, whereas endotoxin stimulation of ECs was unaffected.
The molecular basis of EC stimulation by PMN-derived microparticles is
currently unknown. A potential working hypothesis for this pathway may
involve a direct physical association of membrane microparticles with
the EC surface followed by transmembrane signal transduction and de
novo gene expression in ECs. Regardless of the underlying mechanism(s),
the pathway described here appears entirely unrelated to the process of
retrograde activation of endothelium by PMN-released soluble IL-6R
described by Modur et al. (19). In this context, the same neutralizing
Ab to IL-6R used by Modur et al. failed to reduce EC release of IL-6
stimulated by PMN supernatants.
The in vivo existence of the leukocyte-derived microparticles described here has not yet been investigated. However, we hypothesize that this pathway of leukocyte stimulation of endothelium may have potentially significant implications for general inflammatory responses and the pathogenesis of vascular injury in vivo (20). Microparticle release from activated leukocytes may provide a mechanism to amplify the local concentration of inflammatory and chemotactic cytokines and inducible adhesion molecules, facilitating intercellular communication and cross-signaling pathways between leukocytes and ECs (1). Alternatively, this process may also contribute in the exacerbation of aberrant leukocyte activation, prothrombin activation (9), and intercellular adhesion and migration during the initial phases of vascular injury and the atherosclerotic disease (20).
In summary, we have described an alternative mechanism of leukocyte-EC cross-talk that is of potential relevance for the earliest aspects of inflammation and vascular cell responses. Elucidation of the complex signal transduction pathway initiated by leukocyte microparticles in endothelium should provide important insights into the potential impact of this mechanism for inflammatory conditions in vivo.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dario C. Altieri, Department of Pathology, Boyer Center for Molecular Medicine Room 436B, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06536. E-mail address:
3 Abbreviations used in this paper: EC, endothelial cell; PMN, polymorphonuclear leukocyte; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 2DG, 2-deoxy-D-glucose; CX, cycloheximide.
Received for publication February 25, 1998. Accepted for publication June 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Clin. Invest. 100:2752.[Medline]
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 F. Sabatier, P. Darmon, B. Hugel, V. Combes, M. Sanmarco, J.-G. Velut, D. Arnoux, P. Charpiot, J.-M. Freyssinet, C. Oliver, et al. Type 1 And Type 2 Diabetic Patients Display Different Patterns of Cellular Microparticles Diabetes, September 1, 2002; 51(9): 2840 - 2845. [Abstract] [Full Text] [PDF]
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C. M. Boulanger, A. Scoazec, T. Ebrahimian, P. Henry, E. Mathieu, A. Tedgui, and Z. Mallat Circulating Microparticles From Patients With Myocardial Infarction Cause Endothelial Dysfunction Circulation, November 27, 2001; 104(22): 2649 - 2652. [Abstract] [Full Text] [PDF]
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A. Stucki, A.-S. Rivier, M. Gikic, N. Monai, M. Schapira, and O. Spertini Endothelial cell activation by myeloblasts: molecular mechanisms of leukostasis and leukemic cell dissemination Blood, April 1, 2001; 97(7): 2121 - 2129. [Abstract] [Full Text] [PDF]
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Y. Cadroy, D. Dupouy, B. Boneu, and H. Plaisancie Polymorphonuclear Leukocytes Modulate Tissue Factor Production by Mononuclear Cells: Role of Reactive Oxygen Species J. Immunol., April 1, 2000; 164(7): 3822 - 3828. [Abstract] [Full Text] [PDF]
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M. Mesri and D. C. Altieri Leukocyte Microparticles Stimulate Endothelial Cell Cytokine Release and Tissue Factor Induction in a JNK1 Signaling Pathway J. Biol. Chem., August 13, 1999; 274(33): 23111 - 23118. [Abstract] [Full Text] [PDF]
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