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Molecular Cardiobiology Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06510; and
Genetics Institute, Andover, MA 01810
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain and gp130. Human IL-11 causes rapid (2–10 min)
tyrosine phosphorylation of gp130. IL-11 at 0.1 and 10 ng/ml induces
tyrosine phosphorylation of STAT3 and STAT1, respectively, although
maximal responses require 50 ng/ml. Phospho-STAT3 and phospho-STAT1
levels peak rapidly (2.5 min) and disappear by 60 min. The p42 and p44
mitogen-activated protein kinases (MAPKs) are phosphorylated in
response to 0.3 ng/ml IL-11 with maximal activation at 30 ng/ml IL-11.
Phosphorylation of p42 and p44 MAPKs, which can be prevented by a
mitogen-activated protein/extracellular signal-related kinase kinase-1
inhibitor, peaks by 15–20 min and largely disappears by 40 min. IL-11
does not activate NF-
B nor does it inhibit NF-B activation by
TNF. Similarly, IL-11 neither induces E-selectin or ICAM-1 nor blocks
induction by TNF. Although IL-11 does not alter class I MHC complex
molecule expression, pretreatment with 0.5 ng/ml IL-11 partially
protects HUVECs against lysis by allospecific class I MHC-restricted
cytolytic T lymphocytes or by anti-class I MHC Ab plus heterologous
C. IL-11-induced cytoprotection is protein synthesis dependent and may
depend on mitogen-activated protein/extracellular signal-related kinase
kinase-1. Our results indicate that low (i.e., STAT3- and
MAPK-activating) concentrations of IL-11 confer resistance to
immune-mediated injury in cultured HUVECs without inhibiting
proinflammatory responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to its hemopoietic activities, IL-11 protects against various forms of mucosal injury. These effects are most extensively described in the gastrointestinal tract of rodents where IL-11 protects small intestinal cells from combined radiation, chemotherapy, and ischemia in mice (5, 6, 7); reduces experimental colitis induced by trinitrobenzenesulfonic acid in rats (8); and ameliorates inflammatory bowel disease in mice (6). In these studies, treatment with IL-11 not only decreased mucosal damage but also accelerated healing and improved survival. IL-11 also reduces immune-mediated small bowel injury in acute graft-vs-host disease after murine allogeneic bone marrow transplantation (9).
The protective effects of IL-11 are not restricted to the intestine because IL-11 has also been shown to improve survival after thoracic irradiation (10). Human IL-11, expressed as a transgene in bronchial mucosa, reduces mortality associated with hyperoxia in mice (11). This effect was associated with a reduction in multiple parameters of lung injury including alveolar-capillary protein leak, endothelial cell (EC)4 and epithelial cell membrane injury, lipid peroxidation, pulmonary neutrophil recruitment, IL-12 and TNF production, and DNA fragmentation.
The mechanism(s) by which IL-11 protects mucosae are not completely understood. Both antiinflammatory and direct cytoprotective effects of IL-11 could contribute to the reduction of injury. IL-11 may exert antiinflammatory effects by reducing cytokine production by macrophages (12, 13, 14). It also may promote immune deviation from a TH1-like to a TH2-like phenotype, which may ameliorate some types of immune-mediated injury (9). To date, there have been no reports of direct cytoprotection in cultured cells.
Direct cytoprotection would likely involve the induction of specific
gene products. IL-11 belongs to the IL-6 family of cytokines, all of
which use gp130 as a critical component for signal transduction
(15, 16, 17). IL-11 initiates signaling via binding to a
unique IL-11-receptor- (IL-11R) chain (18, 19). The
IL-11/IL-11R complex is thought to bind to and induce clustering of
gp130, leading to the activation, via transphosphorylation, of
associated Janus kinases (JAKs) (20, 21). Activated JAKs
phosphorylate tyrosine residues within the cytoplasmic region of gp130
which then serve as docking sites for STAT3 and STAT1 proteins
(22, 23). The activated JAKs subsequently phosphorylate
tyrosine residues within the bound STAT proteins, causing the STATs to
dissociate from gp130, dimerize (24), and enter the
nucleus to act as transcriptional activators of target genes (25, 26). STAT dimers may be additionally phosphorylated on serine or
threonine residues by mitogen-activated protein kinases (MAPKs)
(27, 28) that are also activated in response to cytokine
binding to the receptor (29, 30). This additional
phosphorylation may potentiate STAT function as an activator of
transcription.
Immune and inflammatory processes depend on cytokine-mediated
activation of vascular endothelium (e.g., new adhesion molecule
expression via an NF-B-dependent process) (31) and
often injure vascular endothelium, resulting in additional epithelial
injury as a consequence of ischemia which follows endothelial damage.
Therefore, we investigated whether IL-11 could act on cultured HUVECs
to inhibit proinflammatory functions or to induce cytoprotection. We
report here that HUVECs express functional IL-11 receptors and respond
to IL-11 by activating both STAT and MAPK signaling pathways.
Pretreatment of HUVECs with low dose IL-11 can protect HUVECs from
immune-mediated injury, measured as cytolytic CTL- or Ab plus
C-mediated lysis, but did not inhibit TNF-induced activation of NF-B
or expression of leukocyte adhesion molecules.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human IL-11, neutralizing mouse mAb to IL-11, and
three different mouse mAbs to IL-11R-chain were provided by Genetics
Institute (Andover, MA). Recombinant human oncostatin M and recombinant
human IFN-
were purchased from R&D Systems (Minneapolis, MN).
Recombinant human TNF was a gift from Biogen (Cambridge, MA).
Fibroblast growth factor-1, commonly called endothelial cell growth
supplement (ECGS), was obtained from Collaborative Research/Becton
Dickinson (Bedford, MA) and used in conjunction with porcine intestinal
heparin (Sigma, St. Louis, MO). The pharmacological inhibitor of
mitogen-activated protein/extracellular signal-related kinase kinase
(MEK-1) (PD98059) was obtained from Calbiochem (La Jolla, CA), and the
protein synthesis inhibitor cycloheximide was obtained from Sigma.
Rabbit polyclonal Abs reactive with STAT1, phosphotyrosine-STAT1,
STAT3, phosphotyrosine-STAT3, p42, and p44 MAPK and
phosphothreonine/phosphotyrosine p42 and p44 MAPK were purchased from
New England Biolabs (Beverly, MA). Rabbit polyclonal Ab to gp130 and
mouse mAb to phosphotyrosine were purchased from Upstate Biotechnology
(Lake Placid, NY). Rabbit polyclonal Ab to I-B was obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-class I MHC mAb (W6/32)
was prepared as ascites in our laboratory from a clone provided by Dr.
Jack Strominger (Harvard University, Cambridge, MA), and
FITC-conjugated anti-class I MHC mAb (W6/32) was purchased from
Serotech (Oxford, U.K.). Mouse anti-E-selectin mAb (clone H14/18)
and nonbinding (K16/16) Ab were made as ascites in our laboratory.
Mouse anti-ICAM-1 mAb (2D5) was a gift from Dr. Dario Altieri (Yale
University, New Haven, CT). FITC-conjugated polyclonal goat
anti-mouse Ab was purchased from Boehringer Mannheim (Indianapolis,
IN). Baby rabbit C was purchased from Pel-Freez (Brown Dear,
WI).
Cell isolation and culture
Human EC were isolated from umbilical veins as previously described (32, 33) and cultured on gelatin (J. T. Baker, Phillipsburg, NJ)-coated tissue culture plastic at 37°C in 5% CO2, humidified air in Medium 199 containing 20% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Life Technologies, Grand Island, NY), 50 µg/ml ECGS and 100 µg/ml porcine intestinal heparin. K562 and CACO-2 cells were obtained from the American Type Culture Collection (Rockville, MD; accession number for K562, CCL-243) and cultured at 37°C in 5% CO2-humidified air in RPMI 1640 (Life Technologies) containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.
RNase protection assay
RNA was harvested from HUVECs, K562 cells, and CACO-2 cells
using an RNeasy kit (Qiagen, Santa Clarita, CA), and 4 µg of each RNA
were incubated with a 32P-labeled probe cocktail
against human-IL-11R-chain and GAPDH as loading control (Riboquant
kit and custom template, PharMingen, San Diego, CA). Hybridization
reactions were incubated overnight at 56°C and then digested with
RNAs A/T1 and proteinase K. Protected fragments were precipitated and
separated using a 6% acrylamide Tris-borate EDTA (TBE)-urea gel and
then visualized by autoradiography. Autoradiograms were scanned with a
laser densitometer (Molecular Dynamics, Sunnyvale, CA)
Immunoprecipitation and immunoblotting
To analyze protein by immunoblotting of cultured cell lysates, cells (ECs and K562) were washed twice with ice-cold PBS containing 1 mM sodium orthovanadate and 1 mM sodium fluoride and were then lysed in 100 µl cold RIPA lysis buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 10 µg/ml leupeptin, 1 mM sodium orthovanadate). Cell lysates were clarified by centrifugation at 10,000 x g for 15 min, and protein concentrations of the supernatant were determined by using a Bio-Rad assay kit (Bio-Rad, Hercules, CA). Lysates were prepared for SDS-PAGE by adding an equal volume of 2x SDS-PAGE sample buffer (100 mM Tris-Cl (pH 6.8), 200 mM DTT, 4% SDS, 0.2% bromphenol blue, 20% glycerol) and heating the mixture in a boiling water bath for 3 min. Aliquots (10 µg) of cell lysate were resolved by SDS-PAGE using 8% acrylamide gels and a Tris-glycine electrophoresis buffer system (25 mM Tris, 250 mM glycine, 0.1% SDS, pH 8.3), and separated proteins were transferred to a polyvinylidene difluoride membrane by electrophoresis (Immobilon P, Millipore, Bedford, MA). Membranes were incubated with blocking solution containing 5% nonfat dry milk in Tris-buffered saline-Tween (TBST) (10 mM Tris-HCl (pH 8.0), 0.150 mM NaCl, 0.05% Tween 20) at room temperature for 30 min followed by incubation with TBST containing the indicated Ab overnight at 4°C. Membranes were washed and incubated with a suitable HRP-conjugated detecting reagent (Jackson ImmunoResearch, West Grove, PA), and HRP activity was detected using an enhanced chemiluminescence (ECL) kit according to the manufacturer’s instructions (Pierce, Rockford, IL). Autoradiograms were scanned by laser densitometry.
For immunoprecipitation before immunoblotting of gp130, 500 µg of total cell lysate were precleared by incubation with 4 µg rabbit IgG for 90 min on rotator at room temperature, followed by addition of 50 µl GammaBind G-Sepharose beads (Pharmacia, Piscataway, NJ) with continual incubation on a rotator at room temperature for an additional 90 min. The beads were removed from the precleared lysates by centrifugation. To form specific immune complexes, 4 µg anti-gp130 Ab were added to the precleared lysate which was then incubated for 90 min on a rotator at room temperature. To collect specific immune complexes, 50 µl GammaBind G-Sepharose beads were added, and the sample was incubated on a rotator at 4°C overnight at which time beads were collected by centrifugation at 13,000 x g for 1 min. The beads containing immune complexes were washed five times with PBS. The immune complexes were solubilized from the beads by addition of 1x SDS-PAGE sample buffer and heated in a boiling water bath for 5 min. Aliquots were resolved by SDS-PAGE and immunoblotted for total gp130 and for phosphotyrosine residues, as described above.
Indirect immunofluorescence and FACS analysis
After treatment with cytokines, HUVECs were washed with HBSS and incubated for 1 min with trypsin-EDTA. Detached cells were collected and washed twice with ice-cold PBS containing 1% BSA and were incubated with specific primary mAb (either anti-E-selectin, anti-ICAM-1, or FITC-conjugated anti-class I MHC) for 30 min at 4°C. Replicate aliquots were incubated with nonbinding isotype control mAb. Labeled cells were washed twice with PBS-1% BSA and were fixed with 2% paraformaldehyde before being analyzed. In the case of E-selectin and ICAM-1, cells were incubated with a FITC-conjugated goat anti-mouse Ab for 30 min on ice followed by washing twice with PBS-1% BSA before fixation. After fixation, cells were analyzed by FACS using a FACSort and Lysis II software (Becton Dickinson, San Jose, CA). Corrected mean fluorescence values was calculated as follows. For each treatment, the mean fluorescence value for the isotype-matched nonbinding control Ab was subtracted from the mean fluorescence value for the specific Ab.
Transfection and promoter reporter gene assays
Transient transfection of HUVECs were performed using a
DEAE-dextran protocol as described previously (34). Cells
were transfected with both a B-luciferase promoter reporter gene,
which contains two B sites from Ig enhancer (35),
and a constitutively active ß-galactosidase expression construct
(Promega, Madison, WI). Cell lysates were assayed for luciferase and
ß-galactosidase activities using the Promega reporter assay system
(Promega). Luciferase activity was measured using a Berthold
(Schwarzwald, Germany) model LB9501 luminometer, and ß-galactosidase
activity was determined by measuring absorbance at 420 nm using a
spectrophotometer (Dynatech Laboratories, Easton, MA). Luciferase
values in relative light units were normalized to ß-galactosidase
units to control for transfection efficiency.
C-mediated lysis
Target HUVECs were grown in 96-well plates to confluence and incubated with 20 µM calcein-AM (Molecular Probes, Eugene, OR) in Medium 199 and 5 mM HEPES for 30 min at 37°C. The medium was replaced by complete EC growth medium, and cells were rested overnight. IL-11 in media or media alone were added for an addition of 6 h, after which cells were washed twice with Medium 199 containing 5% FBS, 5 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Where indicated, 20 µg/ml cycloheximide were included during the incubation with IL-11. To initiate cytotoxicity, cells were incubated with anti-class I MHC mAb (W6/32) for 30 min at 37°C. Baby rabbit C was then added at the indicated dilutions. To measure the extent of lysis after 1 h, supernatant was removed and transferred into a flat-bottom 96-well plate and released calcein was quantitated using a fluorescence multiwell plate reader (Cytofluor 2, PerSeptive Biosystems, Cambridge, MA; excitation wavelength, 485 nm; emission wavelength, 530 nm). Replicate wells were incubated with lysis buffer (50 mM sodium borate, 0.1% Triton X-100, pH 9.0) to determine maximum release or without C treatment to determine spontaneous release. Percent specific lysis was calculated as [(sample release - spontaneous release)/(maximal release - spontaneous release)] x 100%. Spontaneous release was generally <25%.
Generation of allospecific CTLs and CTL killing assay
Alloreactive class I-restricted CD8+ T cells clones were produced as described elsewhere from peripheral blood CD8+ T cells (36, 37). To determine percent lysis, target HUVECs were loaded with calcein as described above and incubated overnight. IL-11 in media or media alone were added either overnight or 6 h before assay, after which cells were washed twice with Medium 199 containing 5% FBS, 5 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin. Where indicated, the MEK-1 inhibitor PD98059 or vehicle control was added 90 min before adding IL-11. Effector CTL cells were added in a total volume of 150 µl/well at varying E:T ratios. In some experiments, 1 µg/ml PHA (Sigma) was added to potentiate killing by lectin bridging. Replicate wells were incubated with lysis buffer (50 mM sodium borate, 0.1% Triton X-100, pH 9.0) to determine maximum release or with media alone to determine spontaneous release. After 4 h incubation at 37°C, released calcein was measured as described above. Percent specific lysis was calculated as described above. Spontaneous release was generally <25%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The functional receptor for IL-11 is a heterodimer composed of a
signal transduction subunit, gp130, and a specific ligand-binding
receptor component called IL-11R (18). HUVECs have
previously been reported to express gp130 and respond to leptin
(38) and oncostatin M (39, 40, 41, 42), two cytokines
that utilize the gp130 signal transducer. We used an RNase protection
assay to assess the presence of IL-11R mRNA in HUVECs. A human
myeloid cell line, K562, was used as a positive control because these
cells express IL-11R (43). As shown in Fig. 1
A, both HUVECs and K562 cells
express mRNA for IL-11R. In contrast, IL-11R mRNA was not
detected in CACO-2 cells which are unresponsive to IL-11.
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-chain protein in HUVECs we
performed an immunoblot using three newly generated anti-IL-11R
mAbs. As shown in Fig. 1
B, we detected a single major
immunoreactive species in K562 cells and in HUVECs with apparent
molecular mass of 
80 kDa. The same results were obtained with the
other two mAbs (data not shown). The RNase protection assay and
immunoblot indicated that HUVECs constitutively express IL-11R mRNA
and protein, respectively. IL-11 causes tyrosine phosphorylation of gp130 in HUVECs
Binding of IL-11 to IL-11R is necessary, although not
sufficient for signal transduction. Previous studies have indicated
that IL-11 utilizes JAK-induced tyrosine phosphorylation of gp130 to
initiate signal transduction (18, 20). HUVECs express
gp130 and use it to transduce signals in response to an oncostatin M
(40, 44, 45) or leptin (38), two other IL-6
families of cytokines. We were unable to detect IL-11-induced tyrosine
phosphorylation of gp130 by immunoblot of HUVECs lysates, although
oncostatin M response could be detected by this approach (data not
shown). To increase the sensitivity of the assay, we immunoprecipitated
gp130 before immunoblotting with phosphotyrosine Ab. In this
experiment, HUVECs were stimulated with either IL-11 or oncostatin M
for 2 or 10 min, and lysed. The lysates were immunoprecipitated with
gp130 Ab, and the anti-gp130 immunoprecipitates were subjected to
SDS-PAGE followed by immunoblotting with Abs to phosphotyrosine (Fig. 2). In the absence of stimulation, HUVECs
express no detectable tyrosine phosphorylation of gp130. However,
tyrosine phosphorylation of gp130 was reproducibly detected after 2 or
10 min of stimulation with IL-11. Oncostatin M, used as a positive
control, was a more potent inducer of gp130 phosphorylation than
IL-11.
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Cytokines that signal through gp130 typically cause JAK-mediated
tyrosine phosphorylation of STAT3 and, to a lesser extent, of STAT1
(22, 28). Therefore, we used immunoblotting to determine
whether IL-11 induces tyrosine phosphorylation of STAT3 and STAT1 in
HUVECs. In these experiments, the specific anti-phospho-STAT Abs we
used only detect phosphorylation that occurs on the specific tyrosine
residues involved in JAK-mediated activation of STAT dimerization, and
thus immunoreactivity can be used to infer STAT activation, although
this cannot be directly tested without knowledge (and analysis) of a
STAT3-dependent gene. No tyrosine phosphorylation of either STAT3 or
STAT1 was detected in unstimulated HUVECs by immunoblot analysis using
Ab to phospho-STAT3 or phospho-STAT1 (Fig. 3, control). After 10 min of cytokine
treatment, tyrosine phosphorylation of STAT3 was induced by 0.1 ng/ml
IL-11 and increased, in a dose-dependent manner, to maximal levels at
50 ng/ml IL-11 (Fig. 3). IL-11 induced tyrosine phosphorylation of
STAT1 only at concentrations of >10 ng/ml (Fig. 3). Tyrosine
phosphorylation of STAT1 was increased at higher concentrations of
IL-11, peaking at 50 ng/ml IL-11 (Fig. 3). These experiments suggest
that the threshold concentration for an IL-11 effect on STAT1
phosphorylation was >100-fold higher than for phosphorylation of
STAT3, although maximal phosphorylation occurred at similar
concentrations. Im- munoblot analysis of the same samples with
anti-STAT3 and anti-STAT1 Abs (Fig. 3) indicated that the total
levels of STAT3 and STAT1 proteins, respectively, were not changed by
IL-11 treatment (i.e., phosphorylated plus nonphosphorylated). In
parallel control experiments, oncostatin M induced detectable tyrosine
phosphorylation of STAT3 and STAT1 at 60 pg/ml and 1.5 ng/ml,
respectively (Fig. 3). These data show that IL-11 is a less potent
inducer of STAT3 and STAT1 phosphorylation than is oncostatin M.
Moreover, even maximal concentrations of IL-11 failed to induce levels
of STAT phosphorylation observed at optimal oncostatin M levels. In
other words, IL-11 is also less efficacious than oncostatin as an
activator of the JAK-STAT signaling pathway in HUVECs. Nevertheless,
IL-11 clearly does activate gp130/JAK/STAT3 and STAT1 pathways in this
cell type.
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). Similarly, phospho-STAT1 was detected
after 2.5 min stimu- lation with IL-11 and disappeared at 40 min
(Fig. 4). Replicate gels probed for STAT3 (data not shown) and STAT1
(data not shown) indicated that there were no changes in expression of
these proteins after stimulation with IL-11 over the same time
interval. Interestingly, this kinetic study indicated that IL-11 acted
more rapidly than oncostatin M, which did not induce detectable STAT
phosphorylation until 5 min.
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Ab have been
identified to date, so we could not assess the role of the receptor in
this response. IL-11 activates p42 and p44 MAPK in a dose- and time-dependent manner in HUVECs
In addition to activation of STAT3 and STAT1, the MAPK signaling
pathway has also been reported to be activated by cytokines that signal
through gp130 (15). Specifically, IL-11 has been reported
to activate both p42 and p44 MAPK in K562 cells, in monocytic leukemia
U937 cells, and in 3T3-L1 adipocytes (29, 30). To assess
the potential involvement of MAPK signaling pathway in the IL-11 signal
transduction in HUVECs, we determined whether IL-11 increases
threonine/tyrosine phosphorylation of p42 and p44 using Ab specific for
the phosphorylated forms of these enzymes. These MAPKs are continuously
activated in our cultured HUVECs through the actions of serum and
growth factors necessary for cell culture. Therefore, to assess an
effect of IL-11 on MAPK activation, we first made the cells quiescent
by removing growth factors and lowering serum levels to 1%. Under
these conditions, HUVECs contain minimal phospho-p42/p44 (Fig. 5, control). Addition of 0.3 ng/ml IL-11
caused detectable phosphorylation of p42/p44 above background, and the
level of phosphorylation increased with increasing concentration of
IL-11 (Fig. 5A). Lower concentrations of IL-11 (<0.3 ng/ml)
did not cause significant phosphorylation of p42/p44 (data not shown).
In time course experiments, phospho-p42/p44 was detected by 10–15 min
but decreased at 30 min and largely disappeared after 40 min of
stimulation with IL-11 (Fig. 5B). However, significant
levels of phospho-p42/p44 were still detected after 40 min of
stimulation with oncostatin M (1 ng/ml) (Fig. 5B). No change
in the total p42/p44 protein levels were observed on stimulation with
IL-11 over this time period (Fig. 5). These data suggest that IL-11 can
activate MAPK signaling in HUVECs. In growth factor responses, the
threonine/tyrosine phosphorylation of p42 and p44 MAPK is mediated by
MEK-1. Both IL-11- and ECGS-induced phosphorylation of p42/p44 MAPK are
comparably reduced by the MEK-1 inhibitor PD98059 in a dose-dependent
manner (Fig. 6).
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B signaling
pathway in HUVECs
Many proinflammatory cytokines activate the transcription factor
NF-B (46). The activation of NF-B has also been
associated with cytoprotection against TNF-induced apoptosis (47, 48). On the other hand, part of the antiinflammatory actions of
IL-11 have been attributed to inhibition of NF-B activation in
LPS-treated macrophages (12). TNF is the prototypic
activator of the NF-B pathway in HUVECs, causing rapid degradation
of I-B proteins (49, 50). We initially assessed the
effects of IL-11 on NF-B activation in the presence or absence of
TNF by measuring the disappearance of I-B by immunoblotting. As
shown in Fig. 7A, IL-11 by
itself, at either 10 or 100 ng/ml, did not cause degradation of
I-B. Moreover, pretreatment of HUVECs cultures for 4 h with
various doses of IL-11 did not inhibit I-B degradation induced by
TNF (Fig. 7B). Consistent with these biochemical
data, IL-11 at various doses did not increase the transcription
of a transiently transfected B-promoter-reporter gene construct
(Fig. 8). Moreover, pretreatment with
various doses of IL-11 did not inhibit activation of the
B-promoter-reporter gene induced by a submaximal dose of TNF (3
U/ml) (Fig. 8).
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B-dependent responses in HUVECs include the
up-regulation of leukocyte adhesion molecules such as E-selectin and
ICAM-1 (31). To assess whether IL-11 modulates
inflammatory functions of ECs, we examined the effects of IL-11 on
E-selectin and ICAM-1 expression on HUVECs using indirect
immunofluorescence. IL-11 had no direct effect on E-selectin or ICAM-1
expression (Table I). In contrast, a
submaximal concentration of TNF (1 U/ml) significantly induced both
E-selectin and ICAM-1 expression at 5 h (Table I). Neither 6
h pretreatment (data not shown) nor cotreatment (Table I) with IL-11
inhibited E-selectin or ICAM-1 expression induced by TNF.
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We next assessed whether IL-11 could protect HUVECs from
immune-mediated injury in two assays: killing by class I MHC-restricted
allospecific CTL clones; and killing by anti-class I MHC mAb plus
C. Pretreatment with IL-11 at 0.5 ng/ml was capable of partially
protecting HUVECs from lysis by CTL, but pretreatment with a higher
concentration of IL-11 (50 ng/ml) failed to do so (Fig. 9). Preincubation of the IL-11 (0.5
ng/ml) with anti-IL-11 mAb, but not with control K16/16 Ab,
completely inhibited the cytoprotective effect of IL-11 in this assay
(data not shown). Cytoprotection is not mediated by reduction in the
expression of target molecule (i.e., class I MHC) because expression of
these molecule is not altered (Table II).
IL-11 pretreatment also reduced cytolysis by CTL that has been
potentiated by PHA lectin bridging (Table III). Pretreatment of HUVEC targets with
the MEK-1 inhibitor PD98059 also reduced CTL-mediated killing. However,
in this case, no further protection could be conferred by IL-11. These
data support a link between IL-11 cytoprotection and p42/p44 MAPK
activation, but this should be interpreted with caution because of the
independent actions of the MEK-1 inhibitor.
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A). The action of IL-11
quantitated by dye release at 1 h seems to be truly protective
because it correlated with cell survival 24 h after mAb plus C
treatment determined by a Hoechst staining assay (data not shown). The
protective effect of IL-11 pretreatment is dependent on protein
synthesis because it was not observed when cycloheximide was present
during the pretreatment period (Fig. 10B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA
and protein as well as gp130 and that stimulation of HUVECs with IL-11
induced rapid phosphorylation of gp130, STAT3, STAT1, and p42/p44
MAPKs. We also found that IL-11 pretreatment can induce HUVECs to
become partially resistant to injury mediated by CTL or mAb plus C.
Both p42/p44 MAPK phosphorylation and protection from CTL were reduced
by the MEK-1 inhibitor PD98059. However, this drug independently
reduced killing; therefore, this connection must be regarded with
caution. In contrast, IL-11 does not inhibit proinflammatory responses
of HUVECs to TNF, such as NF-B activation or adhesion molecule
expression. These data provide the first in vitro evidence of a direct
cytoprotective action of IL-11 and do not support an antiinflammatory
effect of this cytokine on endothelium. A similar lack of
antiinflammatory effect of IL-11 was observed when LPS rather than TNF
was used as stimulus for endothelial activation (K.M. and J.S.P.,
unpublished observations).
Functional receptor complexes for the IL-6 family of cytokines
including IL-11, oncostatin M, and IL-6 share gp130 as a component
critical for signal transduction (16, 17). IL-11 binds to
IL-11R on the cell surface, and the IL-11/IL-11R complex then
associates with gp130, causing it to cluster. This is essentially the
same mechanism of action that has been observed for IL-6/IL-6R
signaling. Oncostatin M differs from IL-11 and IL-6 in that it directly
binds to gp130 and signals through either gp130/leukemia-inhibitory
factor receptor ß or gp130/oncostatin M receptor heterodimers
(51, 52). Because gp130 is ubiquitously expressed, the
responsiveness of cells to a particular cytokine of the IL-6 family is
determined by the relative expression of other receptor components.
Among receptors for IL-6 cytokines, endothelial cells lack IL-6R
(53) but express receptors for leptin (38)
and oncostatin M (40, 44, 45). EC respond to oncostatin M
with activation of MAPK activity (39), IL-6 secretion
(40), P-selectin (42), ICAM-1, and E-selectin
synthesis (41), and increased growth (39). In
the present study, we showed that, compared with IL-11, oncostatin M is
a much stronger inducer of gp130, STAT3, STAT1, and MAPK
phosphorylation in HUVECs. Oncostatin M but not IL-11 appears to
activate proinflammatory functions of HUVECs (41, 42, 44).
Thus, not all gp130-signaling cytokines induce the same biological
responses in EC, and some differences may relate to signal strength. We
have not been able to observe cytoprotection using oncostatin M (our
unpublished observation), but this may be an issue of finding a
suitable concentration.
The present study raises the question of how IL-11 treatment results in an injury-resistant state. The concentrations of cytokine that are protective correlate with those that activate STAT3 and p42/p44 MAPK. Because cytoprotection depends on new protein synthesis, it is reasonable to suppose that STAT3 and/or MAPK lead to transcription of a cytoprotective protein. These two signals may act coordinately; i.e., MAPK may phosphorylate STAT3 to increase its transcription factor activity. However, we have not rigorously established a causal link between these signals and cytoprotection. There are no available agents to block STAT3 signaling. The best inhibitor of MAPK activation, the MEK-1 inhibitor PD98059, does block IL-11 effects but seems to have independent actions unrelated to IL-11 signaling. It is also unclear why higher concentrations of IL-11 are not protective. Such concentrations activate STAT1, and it has been observed that STAT1 may heterodimerize with STAT3, preventing STAT3 homodimerization. Again, we cannot causally connect STAT1 activation to loss of cytoprotection. Transgenic and knockout mouse studies may be helpful in testing these hypotheses.
We have provided the first evidence of a direct cytoprotective effect of IL-11 in vitro. The responsive target cell type, namely vascular endothelium, could well be a primary site for injury in many of the processes for which IL-11 is cytoprotective. Although our focus has been on immune-mediated injury, a primary membrane action, as we have proposed to explain our data, could extend to nonimmune models of cell injury as well.
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2 Current address: Medinische Universitaetsklinik, Bruderholzspital, CH-4101 Bruderholz/Basel, Switzerland.
3 Address correspondence and reprint requests to Dr. Jordan S. Pober, Boyer Center for Molecular Medicine, Room 454, 295 Congress Avenue, New Haven, CT 06510. E-mail address:
4 Abbreviations used in this paper: EC, endothelial cell(s); ECGS, endothelial cell growth supplement; IL-11R, IL-11-receptor -chain; JAK, Janus kinase; MAPKs, mitogen-activated protein kinases; MEK, mitogen-activated protein/extracellular signal-related kinase kinase; ECL, enhanced chemiluminescence.<./>
Received for publication June 4, 1999. Accepted for publication January 27, 2000.
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 E. Dimitriadis, C. Stoikos, Y.-L. Tan, and L. A. Salamonsen Interleukin 11 Signaling Components Signal Transducer and Activator of Transcription 3 (STAT3) and Suppressor of Cytokine Signaling 3 (SOCS3) Regulate Human Endometrial Stromal Cell Differentiation Endocrinology, August 1, 2006; 147(8): 3809 - 3817. [Abstract] [Full Text] [PDF]
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J. F. Grehan, B. K. Levay-Young, J. L. Fogelson, V. Francois-Bongarcon, B. A. Benson, and A. P. Dalmasso IL-4 and IL-13 Induce Protection of Porcine Endothelial Cells from Killing by Human Complement and from Apoptosis through Activation of a Phosphatidylinositide 3-Kinase/Akt Pathway J. Immunol., August 1, 2005; 175(3): 1903 - 1910. [Abstract] [Full Text] [PDF]
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 N. Torpey, S. E. Maher, A. L. M. Bothwell, and J. S. Pober Interferon {alpha} but Not Interleukin 12 Activates STAT4 Signaling in Human Vascular Endothelial Cells J. Biol. Chem., June 18, 2004; 279(25): 26789 - 26796. [Abstract] [Full Text] [PDF]
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S. Kiessling, G. Muller-Newen, S. N. Leeb, M. Hausmann, H. C. Rath, J. Strater, T. Spottl, K. Schlottmann, J. Grossmann, F. A. Montero-Julian, et al. Functional Expression of the Interleukin-11 Receptor {alpha}-Chain and Evidence of Antiapoptotic Effects in Human Colonic Epithelial Cells J. Biol. Chem., March 12, 2004; 279(11): 10304 - 10315. [Abstract] [Full Text] [PDF]
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 A. B. Waxman, K. Mahboubi, R. G. Knickelbein, L. L. Mantell, N. Manzo, J. S. Pober, and J. A. Elias Interleukin-11 and Interleukin-6 Protect Cultured Human Endothelial Cells from H2O2-Induced Cell Death Am. J. Respir. Cell Mol. Biol., October 1, 2003; 29(4): 513 - 522. [Abstract] [Full Text] [PDF]
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K. Mahboubi, N. C. Kirkiles-Smith, J. Karras, and J. S. Pober Desensitization of Signaling by Oncostatin M in Human Vascular Cells Involves Cytoplasmic Tyr Residue 759 in gp130 but Is Not Mediated by Either Src Homology 2 Domain-containing Tyrosine Phosphatase 2 or Suppressor of Cytokine Signaling 3 J. Biol. Chem., June 27, 2003; 278(27): 25014 - 25023. [Abstract] [Full Text] [PDF]
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 J. H. Von der Thusen, J. Kuiper, T. J. C. Van Berkel, and E. A. L. Biessen Interleukins in Atherosclerosis: Molecular Pathways and Therapeutic Potential Pharmacol. Rev., March 1, 2003; 55(1): 133 - 166. [Abstract] [Full Text] [PDF]
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 M. A. Zimmerman, C. H. Selzman, L. L. Reznikov, C. D. Raeburn, K. Barsness, R. C. McIntyre Jr., C. R. Hamiel, and A. H. Harken Interleukin-11 attenuates human vascular smooth muscle cell proliferation Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H175 - H180. [Abstract] [Full Text] [PDF]
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K. Mahboubi and J. S. Pober Activation of Signal Transducer and Activator of Transcription 1 (STAT1) Is Not Sufficient for the Induction of STAT1-dependent Genes in Endothelial Cells. COMPARISON OF INTERFERON-gamma AND ONCOSTATIN M J. Biol. Chem., March 1, 2002; 277(10): 8012 - 8021. [Abstract] [Full Text] [PDF]
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M. S. Kluger, S. L. Shiao, A. L. M. Bothwell, and J. S. Pober Cutting Edge: Internalization of Transduced E-Selectin by Cultured Human Endothelial Cells: Comparison of Dermal Microvascular and Umbilical Vein Cells and Identification of a Phosphoserine-Type Di-leucine Motif J. Immunol., March 1, 2002; 168(5): 2091 - 2095. [Abstract] [Full Text] [PDF]
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 T. Lahiri, J. D. Laporte, P. E. Moore, R. A. Panettieri Jr., and S. A. Shore Interleukin-6 family cytokines: signaling and effects in human airway smooth muscle cells Am J Physiol Lung Cell Mol Physiol, June 1, 2001; 280(6): L1225 - L1232. [Abstract] [Full Text] [PDF]
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 C. V. Denis, K. Kwack, S. Saffaripour, S. Maganti, P. Andre, R. G. Schaub, and D. D. Wagner Interleukin 11 significantly increases plasma von Willebrand factor and factor VIII in wild type and von Willebrand disease mouse models Blood, January 15, 2001; 97(2): 465 - 472. [Abstract] [Full Text] [PDF]
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