(Received for publication, September 30, 1996)
From the Institute of Toxicology, Functional receptors for interleukin (IL)-4 and
IL-13 on endothelial cells consist of the 130-kDa IL-4 receptor
Vascular endothelial cells participate actively in a wide variety
of pathophysiological processes, including inflammation and immunity
(1). Inflammatory cells secrete cytokines that bind to endothelial
cells and result in changes of cell surface molecule expression. The
TH1-type proinflammatory cytokines
TNF- Through the sharing of receptor components, TH2-type
cytokines IL-4 and IL-13 exert a similar spectrum of responses on
different leukocytic cells (4). Both interact with human umbilical vein endothelial cells (HUVEC) and induce, like TNF- Recently, we characterized the receptors for IL-4 and IL-13 on human
endothelial cells as a heterodimeric complex lacking expression of the
common Here we describe the effects of proinflammatory cytokines, TNF- The Chinese hamster ovary-derived
human IL-4 together with the human IL4R HUVECs were isolated from human umbilical
cord veins as described previously (24). The cells were seeded on
purified human fibronectin (Winiger AG, CH-5610 Wohlen, Switzerland)
and propagated in Medium 199 enriched with sodium heparin (90 µg/ml;
Novo Industries, Copenhagen, Denmark), endothelial cell growth
supplement (15 µg/ml; Collaborative Research, Inc., St. Waltham, MA)
in the presence of 20% pooled human serum. Final monolayers were used
in their second to fourth passage exhibiting cytoplasmic factor VIII
von Willebrand, which was tested by indirect immunofluorescence with the mAb KG7/30 (BMA Biomedicals AG, Augst, Switzerland). THP1 cells
were cultured in a 1:1 mixture of Dulbecco's modified Eagle medium and
Ham's F-12 nutrient solution (Life Technologies, Inc., Basel,
Switzerland) containing 10% fetal calf serum.
FLAG-labeled human
IL-13 containing the N-terminal octapeptide
N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C was produced by
subcloning the mature peptide coding region for human IL-13 (25) via
PCR into the pFLAG 1 vector (Kodak) and by transfection into the AB1899 strain of Escherichia coli. Bacterial cultures were
harvested, clarified, and supernatants adjusted to PBS and recirculated
overnight at 4 °C through a 5-ml bed volume of M2 resin, eluted with
100 mM glycine-HCl (pH 3.0), neutralized with 1:50 (v/v) 2 M Tris-HCl (pH 8.0), and dialyzed against PBS. Protein was
quantitated by densitometer scanning (Molecular Dynamics Corp.,
Sunnyvale, CA) of 10% SDS-polyacrylamide gels stained with Coomassie
Brilliant Blue (R030-250) using hen egg lysozyme as a standard with
verification by amino acid analysis.
HUVECs were grown to
confluence in 6-well plates (Becton Dickinson, San José, CA).
Reagents were added directly to the medium at indicated concentrations
and for given incubation times. Flow cytometric detection of IL4R Iodination of the cytokines
was performed as described recently (8). The specific activities of
125I-labeled IL-4 and 125I-labeled IL-13 were
60-70 and 90-100 µCi/mg, respectively. Iodination did not lead to a
detectable loss of the biological activity of the cytokines.
Confluent HUVEC monolayers were
briefly trypsinized. The cells were washed three times with cold PBS,
counted at the last washing step, and resuspended in minimum Eagle's
Confluent
HUVEC monolayers were briefly trypsinized, washed three times with cold
PBS, and resuspended in minimum Eagle's Total cellular RNA was isolated by the method of
Chomczynski et al. (27) and quantitated at 260 nm. 5 µg of
total cellular RNA were reverse-transcribed in a final reaction volume
of 30 µl as recently described (8). 1 µl of this reaction was then amplified by PCR. The specific primer pairs for the human IL4R HUVECs were
grown to confluence and either left untreated or stimulated with 1 ng/ml TNF- HUVECs express a functional IL-4·IL-13R complex that
consists of the IL-4R
Endothelial cells are known to express both the p55 and p75 TNFR (37).
A specific role of the p55 TNF receptor was reported for the induction
of ICAM-1, E-selectin, VCAM-1, and CD44. However, the expression of the
TNF- On
mouse sarcoma cells, TNF- By RT-PCR analysis we studied the expression of IL-4R
To investigate whether the subunit
structure of the receptor complex has changed in response to TNF-
Concerning the heterodimeric structure of
the IL-4·IL-13R complex on endothelial cells, we studied the effect
of TNF-
In
previous experiments we studied the binding of iodinated IL-4 and IL-13
to resting HUVECs and analyzed binding of iodinated IL-4 and IL-13 to
resting HUVECs using a one-site binding model (8). These calculations
revealed with ~200 IL-4 receptors/cell and ~700 IL-13
receptors/cell a considerable stoichiometric imbalance of the receptor
subunits. Here we have compared the binding characteristics of resting
and TNF-activated HUVECs and analyzed the binding data by the weighted
least-square curve fitting method regarding one-class or two-class
binding site models (26). Binding of IL-4 in resting and
TNF- Both IL-4 and IL-13 have been shown to signal
through the activation of Stat6 (20, 23). We therefore tested for Stat6 activation by EMSA. In Fig. 6 both cytokines activate
Stat6 to a similar extent. Pretreatment of HUVECs with TNF-
In our previous work, IL-4 was shown to induce a
variable but significant number of HUVECs to express VCAM-1 (8). In
contrast TNF-
It has previously been shown that TNF- IL-4 and IL-13 share a number of biologic activities on different
leukocytic cells (4) and induce markedly similar expression of VCAM-1
on HUVECs in culture (6). Recently, we have characterized on
endothelial cells the receptor subunits for IL-4 and IL-13, which form
a heterodimeric complex consisting of the IL-4R Of importance by flow cytofluorometry and RT-PCR analysis, TNF- Very recently Caput et al. (12) cloned and characterized a
human IL-13-binding protein structurally related to the IL-5R VCAM-1 expression on endothelial cells has been found to be the most
significant function induced by IL-4 and IL-13 (5, 6). Unfortunately,
TNF- Regarding the endothelial barrier it is tempting to speculate that the
proinflammatory TH1-type cytokines, which are
characteristic for an acute inflammation with massive neutrophil
infiltration, provide means for a switch to a chronic inflammatory
state by increasing the expression of TH2-type cytokine
receptors.
We thank Dr. J. Banchereau (Schering Plough,
Dardilly, France) for providing us mAb S697 and the human IL-4. We also
acknowledge the generous supply of IL-1 by Dr. P. T. Lomedico
(Hoffmann-La Roche), TNF by Dr. Z. Nagy (Preclinical Research,
Sandoz Ltd., Basel, Switzerland), and the TNF-
Dupont Merck
Pharmaceutical Co., Stine-Haskell Research Center,
Newark, Delaware 19714, and ** DNAX Research Institute of Cellular and
Molecular Biology, Palo Alto, California 94304
-chain (IL-4R
) and a 65-75-kDa IL-13 binding subunit that are
expressed in a ratio of about 1:3, respectively. The restricted number
of IL-4R
limits subunit heterodimerization and in turn
receptor-mediated signaling. We report here, the effects of tumor
necrosis factor
(TNF-
) on the expression of the receptor
subunits for IL-4 and IL-13. By flow cytofluorometry and
receptor-binding analysis of iodinated IL-4 and IL-13, stimulation with
TNF-
-induced a 2-3-fold increase of the IL-4R
expression. The
up-regulation was also confirmed at the transcriptional level by
reverse transcription-polymerase chain reaction. Radioligand
cross-linking experiments revealed no change in the subunit composition
of the TNF-
-induced receptor complex. Nevertheless, TNF-
stimulation led to increased activation of the IL-4-specific signal
transducers and activators of transcription protein (Stat6) by IL-4 and
IL-13. Thus, TNF-
corrects the subunit imbalance of the endothelial
IL-4·IL-13 receptor complex thereby increasing receptor
heterodimerization and in turn the signaling capability by IL-4 and
IL-13.
1 and IL-1, as well as bacterial
endotoxin, induce the expression of endothelial cell adhesion molecules
like E-selectin (2) intercellular cell adhesion molecule type-1
(ICAM-1) and vascular cell adhesion molecule type-1 (VCAM-1) that are
the basis for the coordinated extravasation of the different types of
leukocytes (for review see Ref. 3).
and IL-1, the expression of VCAM-1 and in turn the adhesion of very late
antigen-4-expressing leukocytes, including eosinophils (5, 6), as well
as their selective transmigration (7, 8). These studies indicate that
IL-4 and IL-13 play an important regulatory role during allergic inflammation.
-chain (
c) (8), which is otherwise shared by
IL-2R, IL-4R, IL-7R, IL-9R, and IL-15R (9, 10). The subunit structure
of the functional receptors for IL-4 and IL-13 on endothelial cells
consists of the 130-kDa IL-4R
-chain (IL-4R
) and a 65-75-kDa
IL-13-binding protein herein called the IL-13R
-chain (IL-13R
). A
mouse and recently a human IL-13R have been cloned and characterized as
new members of the hematopoietin receptor family (11, 12). IL-4 signals
through receptor association and activation of the Janus family of
tyrosine kinases (13) Jak1 and Jak3 in T cells, natural killer cells,
and myeloid cells (14, 15). Hereby Jak1 associates with IL-4R
(16)
and Jak3 with
c (17, 18). Mutations in
c
preventing association with Jak3 result in human X-linked (severe)
combined immunodeficiency and X-linked combined immunodeficiency (17).
In human colon carcinoma cells, expressing a heterodimeric
IL-4·IL-13R complex lacking
c; however, IL-4 and IL-13
activate Jak1, Jak2, and Tyk2 but not Jak3 (19, 20), and in myeloid
cells (21) IL-13 has been reported to activate Jak1 as well. Activated
Jak kinases mediate cellular responses by phosphorylating members of
the STAT family (Signal Transducers and Activators of Transcription), a class of latent cytoplasmatic transcription factors, which translocate into the nucleus and transcriptionally activate target genes (22). IL-4
has been reported to activate Stat6, formerly designated IL-4 Stat
(23), and IL-13 activates in human colon carcinoma cells Stat6 as well
(19, 20).
and
IL-1, on the expression of the receptor subunits for IL-4 and IL-13 on
HUVECs in culture. Stimulation with TNF-
led to a 2-3-fold increase
of the IL-4R
expression, without affecting the number of the
IL-13R
subunits. Concerning the numeric imbalance of the receptor
subunits determined in resting HUVECs (8), endothelial activation
induced an impressive shift toward a 1:1 ratio of both receptor
subunits. The resulting subunit equilibration revealed an increased
binding of both IL-4 and IL-13 to the IL-4·IL-13R complex.
Furthermore, pretreatment of HUVECs with TNF-
resulted in increased
Stat6 activation demonstrating improved signaling capability of the
induced IL-4·IL-13R complex.
Cytokines and Reagents
-chain-specific mAb S697
were a gift of Dr. J. Banchereau (Schering Plough, Dardilly, France).
The S697 antibody is of the mouse IgG1 class and reacts with the
extracellular domain of the human IL-4R
(Zurawski et al.
(35)). The mAb 1299-27-1 directed against VCAM-1 is of the mouse
IgG2a class and was purchased from BMA Biomedicals AG,
Augst, Switzerland. The mAb M2-specific for the flag octapeptide was
from Eastman Kodak Co. Human IL-4 has been biotinylated with
biotin-succinimidyl ester (Amersham, Buckinghamshire, UK). Briefly, 500 µg of IL4 (1 mg/ml) in 0.1 M NaHCO3 (pH 8.5)
was added to 125 µg of biotin-succinimidyl ester (10 mg/ml in
dimethyl sulfoxide (Me2SO)) and incubated for 4 h at
room temperature. Labeling reaction was stopped by adding 20 µl of 1 M NH4Cl and incubating for 10 min at room
temperature. The reaction mixture was then extensively dialyzed against
phosphate-buffered saline (PBS) to remove free biotin and side products
of the reaction. Human IL-1 was generously supplied by Dr. P. T. Lomedico (Hoffman-La Roche Ltd., Basel, Switzerland), human TNF-
by
Dr. Z. Nagy (Preclinical Research, Sandoz Ltd., Basel, Switzerland),
TNF receptor (TNFR)-p55-specific TNF-
mutant
Trp32-Thr86TNF-
and TNFR-p75-specific
TNF-
mutant Asn143-Arg145TNF-
by Dr. H. Loetscher (Pharmaceutical Research, F. Hoffman-La Roche Ltd., Basel,
Switzerland). Lipopolysaccharide (from Salmonella minnesota)
were purchased from Sigma. Herbimycin A, genistein, and staurosporine
(Calbiochem) were diluted in Me2SO and used at indicated
concentrations with a final concentration of Me2SO 1:1000
v/v.
and VCAM-1 expression was essentially performed as described previously
(8). For analysis of IL4 and IL13 binding to cell surface, samples were
incubated with IL-4-biotin or IL-13-FLAG (100 nM) for
2 h at 4 °C followed by several wash steps. IL-13-FLAG-labeled
cells were additionally incubated with mAb M2 anti-flag (10 µg/ml)
for 45 min at 4 °C. Cells were further incubated for 45 min at
4 °C with a 1:30 dilution of goat anti-mouse IgG-phycoerythrin R or
a 1:10 dilution of streptavidin/phycoerythrin R (Sigma). Flow
cytofluorometry was carried out using a 488-nm argon laser FACScan
(Becton Dickinson, San Jose, CA). 10,000 cells/sample were measured,
and fluorescence intensity was determined on a linear scale by the
LYSIS II software (Becton Dickinson). Cells were gated using forward
versus side scatter to exclude dead cells and debris.
Results are expressed as mean fluorescence intensities (MFI) after
subtraction of unspecific binding of a irrelevant isotype-matched
control antibody or as percentage immunopositive stained cells.
medium containing 1% bovine serum albumin. Aliquots of 200 µl
containing 4 × 106 HUVECs were incubated on ice for
1 h with 0.5 nM 125I-labeled IL-4 and 3 nM 125I-labeled IL-13. For competition, an
excess of 1 µM of unlabeled ligand was added 20 min prior
to the iodinated cytokines. The affinity cross-linking procedure was
performed as described recently (8), and the samples were analyzed by
SDS-polyacrylamide gel electrophoresis under reducing conditions using
3-10% (w/v) polyacrylamide gels. Autoradiography was performed at
70 °C exposing x-ray Hyperfilms (Amersham Int.) for 2-3
weeks.
medium containing 1%
bovine serum albumin. The binding studies were performed as described
previously (8). Nonspecific binding was determined by incubating the
same number of cells with a 1000-fold excess of unlabeled IL-4 or IL-13
at 4 °C for 30 min. Binding data were analyzed with the computerized
weighted least-square curve fitting software described by Munson and
Rodbard (26).
and
the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were custom
synthesized and quality checked by capillary electrophoresis by
MWG-Biotech (85560 Ebersberg, Germany). The human GAPDH primers spanning a 480-bp fragment and the human IL4-receptor
-chain primers
spanning a fragment of 571 bp were as described (8, 28). The
presumptive human IL-13R transcript was amplified using primers
corresponding to nucleotides 25-43 of Hs074281 and nucleotides 302-323 of Hs334240 spanning a fragment of 692 bp. PCR reactions were
carried out in a final volume of 25 µl in 500-µl microtubes (Perkin-Elmer), and each sample was overlaid with 25 µl of paraffin oil. The mixtures contained 1 µl of RT reaction in 10 mM
Tris-HCl (pH 9.0), 50 mM KCl, 1.6 mM
MgCl2 (IL-4R
and IL-13R
), 0.8 mM MgCl2 (GAPDH), 0.01% gelatin, 0.1% Triton X-100, 0.2 mM of each dNTP, 0.8 µM of each primer, and
0.5 units of Super Taq polymerase (P. H. Stehelin & Cie AG,
Basel, Switzerland). Human GAPDH was amplified using 35 cycles at
94 °C (for 1 min), at 62 °C (for 1 min), and at 72 °C (for 1 min), IL-4R
using 35 cycles at 94 °C (for 30 s) and at
53 °C (for 30 s), and IL-13R using 35 cycles at 94 °C (for 1 min), at 50 °C (for 2 min), and at 72 °C (for 2 min). The
identity of the RT-PCR products of IL-4R
and the human homolog of
the mouse IL-13R (11) was confirmed by sequencing. To control for
genomic DNA amplification, PCR analyses were done without reversed
transcription. In all cases no products of corresponding size could be
amplified. Twenty microliters of each RT-PCR reaction were run on a
1.5% agarose gel containing 0.2 µg/ml ethidium bromide in 1 × TAE buffer. pBR322 DNA completely digested by AluI (MBI Fermentas, Vilnius, Lithuania) was used as DNA molecular weight marker.
The gels were photographed under UV illumination.
for 18 h before activation with the indicated
concentrations of IL-4 or IL-13 for 15 min. Nuclear extracts were
prepared with a modified Nonidet P-40 method as follows. Cells were
lysed in low salt buffer (20 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM NaVO4, 1 mM EDTA, 1 mM EGTA, 0.2% Nonidet P-40, 10%
glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 2 µg/ml aprotinin, 1 µg/ml
leupeptin, 1 µg/ml pepstatin) for 10 min on ice. Lysates were
centrifuged in a microcentrifuge at 13,000 × g for 1 min at 4 °C. Supernatants were frozen on dry ice and stored at
70 °C as cytoplasmic extracts. The pellets were resuspended in
high salt buffer (420 mM NaCl, 20 mg/ml Hepes (pH 7.9), 10 mM KCl, 0.1 mM NaVO4, 1 mM EDTA, 1 mM EGTA, 20% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml
pepstatin) and incubated on ice for 30 min. These nuclear extracts were
cleared by centrifugation at 13,000 × g in a
microcentrifuge for 5 min. The supernatants were frozen on dry ice and
stored at
70 °C until used. 2 µl of nuclear extracts were used
for EMSA analysis. EMSA was done as described previously (29) using an
oligonucleotide probe derived from the C
gene promotor (30, 31) with
the core sequence TTCCCAAGAA. For supershifts, extracts were mixed with
1 µl of a 1:10 dilution of specific antisera against Stat1 (32),
Stat2 (33), Stat3, Stat4 (34), Stat5, and Stat6. Stat5 antiserum was
raised in rabbits against amino acids 687-794 of ovine Stat5. Polyclonal rabbit antiserum against Stat6 was raised in rabbits against
amino acids 633-837 of mouse Stat6.
TNF- Up-regulates the Expression of IL-4R
on HUVEC
Monolayers
and a 65-75-kDa IL-13-binding protein, the
IL-13R
, and lacks the common
c (8). By flow
cytofluorometry we studied the expression of the IL-4R
with the
non-blocking mAb S697 (35). Untreated HUVECs in culture constitutively
expressed the IL-4R
, although the amount varied considerably among
the different cell batches (MFI, 171 ± 15, mean ± S.E. of
20 experiments). After activation with TNF-
at a concentration of 1 ng/ml for 24 h, we observed significant increase of the IL-4R
expression (MFI, 251 ± 17, mean ± S.E. of 20 experiments;
p < 0.001). Similar up-regulation was obtained with 10 ng/ml IL-1 or lipopolysaccharide at a concentration of 1 µg/ml (data
not shown). Increased expression of IL-4R
was detectable at a
TNF-
concentration of 0.1 ng/ml and was optimal at 1 ng/ml. Higher
concentrations were less efficient, probably due to TNF-
-mediated
toxicity (Fig. 1A). After 6 h the
IL-4R
expression was significantly enhanced and linearly increased
up to 24 h (Fig. 1B). Prolonged incubation led to
decreased IL-4R
expression, probably due to desensitization (data
not shown). Of importance, TNF-
stimulation did not induce
c (data not shown). Moreover, the IL-4R
was not
inducible in synovial and dermal fibroblasts as well as in the human
mast cell line 1 expressing the
c-containing
heterotrimeric form of the IL-4·IL-13R (36) (data not shown). This is
indicative for a specific regulatory mechanism in endothelial
cells.
Fig. 1.
Dose dependence and kinetics of IL-4R
induction by TNF-
. HUVEC monolayers were incubated in medium
with increasing concentrations of TNF-
for 24 h (A)
or with TNF-
at 1 ng/ml for the indicated periods (B).
Flow cytofluorometry for cell surface expression of IL-4R
was
performed using the specific mAb S697 as primary antibody. Results are
from one representative experiment using the same batch of endothelial
cells in A and B, respectively. Data are
expressed as corrected MFI.
[View Larger Version of this Image (26K GIF file)]
2/
1 integrin was shown to be mediated through both the p55 and p75 TNFR (37, 38). To investigate which TNFR
controls up-regulation of the IL-4R
, HUVECs were stimulated for
24 h either with wild-type TNF-
(1 ng/ml) or with receptor type-specific agonists (39), binding exclusively to p55 or p75 TNFRs.
The p55 TNFR selective mutant of TNF-
(p55 TNF-
) binds with the
same affinity as wild-type TNF-
(39). At a concentration of 5 ng/ml,
it fully up-regulates the expression of IL-4R
in our experiments
(Fig. 2A). Conversely, the p75 TNFR selective mutant (p75 TNF-
) (Fig. 2B) with a 5-10-fold lower
affinity for p75 TNFR did not exhibit any inducing effect at
concentrations of 5 or 10 ng/ml (Fig. 2B), and even at 50 ng/ml (data not shown).
Fig. 2.
Up-regulation of IL-4R is mediated through
the p55 TNFR and involves the activation of a protein tyrosine
kinase. HUVEC were stimulated in culture with wild-type TNF-
or
Trp32- Thr86TNF-
(p55 TNF-
)
(A) or with Asn143-Arg145TNF-
(p75 TNF-
) (B) at the indicated concentrations for
24 h. C, HUVEC were incubated for 2 h with either
Me2SO (1:1,000 v/v) (resting) or herbimycin A before
stimulation with TNF-
for 24 h. Flow cytofluorometry for cell
surface expression of IL-4R
was performed using the specific mAb
S697 as primary antibody. Values represent corrected MFI from one
representative experiment.
[View Larger Version of this Image (25K GIF file)]
is known to signal through the activation of various kinases,
like cAMP-dependent protein kinase (40), protein kinase C
(41), and a pp60src-like tyrosine kinase (42). To investigate
the TNF-
-dependent signaling pathway leading to enhanced
expression of IL-4R
, HUVECs were pretreated with kinase inhibitors
either specific for tyrosine kinases as herbimycin A and genistein
(data not shown) or for protein kinase C as staurosporine (data not
shown) for 2 h at 37 °C and coincubated with TNF-
(1 ng/ml)
for an additional period of 24 h. Only treatment with herbimycin
A, a specific pp60src tyrosine kinase inhibitor, at a
concentration of 1 µM resulted in complete inhibition
of the IL-4R
up-regulation (Fig. 2C).
Stimulates the Expression of IL-4R
mRNA
has been reported to increase IL-4
receptor-specific mRNA levels with maximal transcript up-regulation after 4 h of TNF-
treatment, although the requirement for
protein synthesis could not be clearly determined (43).
-specific
mRNA of resting HUVECs and HUVECs previously stimulated with TNF-
for various periods (Fig. 1B). With appropriate
primers (see "Materials and Methods"), which amplify a 571-bp
fragment encoding the IL-4R
, specific transcripts were detectable in
resting HUVEC and were clearly increased after TNF-
stimulation.
However, a 692-bp transcript with close homology to the mouse IL-13R
(11) was constitutively expressed in resting HUVEC and was not
increased after TNF-
stimulation (Fig. 3). In
addition, we were unable to detect any message for
c
(data not shown), as previously reported for resting HUVECs (8).
Fig. 3.
Reverse transcriptase-PCR analysis of
IL-4R mRNA in presence of TNF-
. Total cDNA was
synthesized from 5 µg of total cellular RNA harvested from HUVECs
stimulated in culture with TNF-
for the times indicated
(h). PCR amplification was performed as described under
"Material and Methods." Specific primer pairs were used to detect
IL-4R
mRNA, the presumptive IL-13R
mRNA and GAPDH
mRNA expression for equal amplification. PCR products were analyzed
by electrophoresis through ethidium bromide-stained agarose gels. The
gels were photographed under UV illumination. The results are from one
representative experiment using the same batch of endothelial
cells.
[View Larger Version of this Image (62K GIF file)]
Does Not Change the Subunit Composition of the
IL-4·IL-13R Complex
treatment, iodinated IL-4 and IL-13 were cross-linked by the
succinimidyl suberate method to their binding sites on the surface of
endothelial cells (Fig. 4). Autoradiographies of
SDS-polyacrylamide gel electrophoresis from resting and TNF-treated
HUVECs revealed for both cytokines exactly the same pattern of
interacting subunits as recently reported (8). In detail, the majority
of the iodinated IL-4 was cross-linked to the 130-kDa IL-4R
, whereas
a minor band was detected at 65-75 kDa. However, iodinated IL-13 bound
predominantly to a 65-75-kDa protein, and only a trace amount of
radioactivity was detected at the 130-kDa level. Total displacement of
the iodinated IL-4 and IL-13 by an excess of the respective unlabeled
cytokine (1 µmol/L) indicated the specificity of the interaction
(data not shown). From these experiments we conclude that stimulation
of endothelial cells does not modify the subunit composition of the IL-4·IL-13R complex.
Fig. 4.
Radioligand affinity cross-linking of IL-4
and IL-13 to HUVECs. Confluent HUVECs either resting () or
activated for 24 h with 1 ng/ml TNF-a (+) were detached and
labeled with 0.5 nmol/liter 125I-labeled IL-4 (lanes
1 and 2) or 3 nmol/liter 125I-labeled IL-13
(lanes 3 and 4) before cross-linking was
performed using 2.5 mmol/liter disuccinimidyl suberate. The lysates
were analyzed under reducing conditions on a gradient (3-10%) of
SDS-polyacrylamide gel electrophoresis and exposed to x-ray films for 3 weeks at
70 °C. Net molecular masses of the receptors were
calculated by subtracting 19 kDa for bound IL-4 or 15 kDa for bound
IL-13.
[View Larger Version of this Image (53K GIF file)]
Increases the Binding of Biotinylated IL-4 and
Flagged-IL-13 to HUVECs
stimulation on binding of both IL-4 and IL-13. Flow
cytofluorometric analysis was performed with biotinylated IL-4 and
flagged IL-13. Resting HUVECs showed detectable binding of biotinylated
IL-4 and flagged IL-13 both at 100 nM and after activation
with TNF-
for 24 h, the binding of both ligands was 2-3-fold
increased (Fig. 5A). In all experiments,
binding of biotinylated IL-4 or FLAG-labeled IL-13 could be effectively
competed by addition of excess unlabeled IL-4 or IL-13 (data not
shown).
Fig. 5.
Binding of IL-4 and IL-13 to resting and
TNF--activated HUVECs. A, flow cytofluorometry for cell
surface binding of IL-4-biotin (left) or IL-13-FLAG
(right). HUVEC monolayers were stimulated with TNF-
(1 ng/ml) or medium alone (Resting; gray filled histograms) for
24 h. Data are expressed as corrected MFI from one representative
experiment. B, binding of radiolabeled IL-4 and IL-13 to
HUVECs. Receptor binding analysis of radiolabeled IL-4 (closed
circles) and IL-13 (open circles) was performed with resting HUVECs (Resting) or HUVECs stimulated with 1 ng/ml
TNF-
for 24 h (TNF-
). Data were analyzed with the
computerized weighted least-square curve fitting software described by
Munson and Rodbard (26).
[View Larger Version of this Image (25K GIF file)]
-activated HUVECs fitted with high confidence to the one-class
binding site model (p < 0.02). In resting HUVECs, IL-4 bound to 228 ± 82 receptors per cell expressing a
Kd of 33.0 ± 13.0 pM. After TNF
activation there was 2.7-fold increase of the number of receptors/cell,
whereas the Kd value remained unchanged (Fig.
5B). Binding of IL-13 to resting HUVECs fitted the two-class
rather than the one-class binding site model (p < 0.02) that suggests 142 ± 24 receptors/cell with a
Kd 1 of 7.4 ± 1.7 pM.
According to the two-class binding site theory a second receptor
population is predicted with a Kd 2 of
4.9 ± 3.0 nM and 575 ± 144 sites/cell (Fig.
5B). In contrast, the IL-13 binding data derived from
TNF-activated HUVECs only fitted the one-class binding site model with
856 ± 171 sites/cell and Kd value of 35 ± 7.7 pM. These data indicate that TNF-
significantly
up-regulates the number of IL-4R
but not that of the IL-13R
subunits. The resulting corrected subunit stoichiometry and in turn
increased heterodimerization of the receptor complex may explain the
enhanced number of high affinity IL-13Rs.
results
in enhanced Stat6 activation. Supershifting experiments with antibodies against the various STATs identified the IL-4- and IL-13-induced shift
as Stat6-specific shift.
Fig. 6.
TNF treatment enhances the IL-4 and
IL-13-induced IL-4-STAT (STAT-6) activation. HUVECs were left
untreated (lanes 3-9) or treated with 1 ng/ml TNF for
18 h (lanes 11-16) before they were stimulated for 15 min with IL-4 or IL-13 at indicated concentrations. Both the IL-4- and
the IL-13-induced shift complex can be supershifted with antisera
against Stat6 (lanes 19, 20, 22, and 23).
Supershift controls in lanes 18 and 21 are from
TNF-activated HUVEC costimulated with IL-4 or IL-13, respectively.
Resting and IL-4-activated THP-1 were used as a control (lanes
1 and 2).
[View Larger Version of this Image (86K GIF file)]
Increases IL-4-induced VCAM-1
Expression
caused strong VCAM-1 expression in all HUVECs. Of
interest to this study, VCAM-1 induction in response to TNF-
combined with IL-4 or IL-13 was more than additive (8). In Fig.
7, we primed HUVEC monolayers with TNF-
for 30 min
before VCAM-1 expression was determined in the presence or absence of
IL-4. Priming with TNF-
led to an enhanced IL4R
expression (data
not shown). After 16 h, non-primed HUVECs partially expressed
VCAM-1 in response to IL-4, whereas TNF-
priming caused only
marginal VCAM-1 induction. However, TNF-
priming in combination with
IL-4 revealed an additive increase of the VCAM-1-expressing HUVEC
population.
Fig. 7.
Induction of VCAM-1 expression on
HUVECs. HUVEC monolayers were either pulse-stimulated with TNF-
(1 ng/ml) for 30 min or incubated with IL-4 at 20 ng/ml for 16 h
alone or in combination. Control monolayers were kept in medium alone
(resting). Flow cytofluorometry for VCAM-1 expression was
determined at the end of the 16-h incubation time. Corrected MFI and
the immunopositive population (given in percent) within the margin (M1)
are indicated from one representative experiment using the same batch
of endothelial cells.
[View Larger Version of this Image (31K GIF file)]
has the ability to
induce the expression of the IL-4 receptors on a mouse sarcoma cell
line (43). In our study with HUVEC we demonstrate similar induction
kinetics of the IL-4R
, however at a much lower concentration of
TNF-
. Furthermore, this observation could be extended to other proinflammatory stimuli such as lipopolysaccharide and IL-1. In stimulation experiments with TNF-
mutants selective for the 55- or
75-kDa TNF-
receptors, specific p55 TNFR-mediated effects were
reported to cause endothelial activation (38) such as the induction of
E-selectin, ICAM-1, and VCAM-1. Solely, the induction of
2/
1 integrin was shown to be mediated
through both TNF receptors (37). In the present study, the
up-regulation of the IL-4R
was clearly under the control of the p55
TNFR, further confirming that the p55 TNFR is a major mediator of
endothelial activation. Our data also support the
TNF-
-dependent activation of a protein tyrosine kinase,
as shown with the specific pp60src-tyrosine kinase inhibitor
herbimycin A (42).
and a 75-kDa
IL-13-binding protein and lack the expression of the
c (8). In that study, high concentrations of cold IL-4
displaced radiolabeled IL-13 from its binding to HUVECs and cold
IL-13 competed for binding of radioactive IL-4. Such cross-competition
suggests that IL-4 and IL-13 share the subunits of a heterodimeric
receptor complex. The cross-linking experiments in this study
show that TNF treatment did not modify the subunit composition of the
receptor complex.
treatment did not reveal any induction of the
c.
Nevertheless, binding of biotinylated IL-4 and flagged IL-13 to
TNF-
-activated HUVECs was significantly enhanced. Therefore,
receptor binding assays with radiolabeled IL-4 and IL-13 were performed
to further analyze the qualitative changes in the IL-4·IL-13R
complex. Previous receptor binding studies with resting endothelial
cells, calculated according to a one-class binding site model, revealed
a striking imbalance of the receptor subunits with a 2-3-fold excess
of the IL-13R subunit (8). The use of a two-binding site model revealed for both cytokines ~200 high affinity receptors and a second receptor population for IL-13 with considerably lower affinity. TNF pretreatment significantly increased the number of IL-4 binding sites to about 600, whereas the Kd value did not change. Thus, TNF
stimulation equilibrates the subunit ratio by solely up-regulating the
number of IL-4R
. The resulting 1:1 stoichiometry enhances receptor
subunit dimerization causing increased numbers of high affinity
IL-13Rs, as IL-13 binds now to a single class of receptors with a
similar high affinity as for IL-4. Recently, Leonard et al.
(44) proposed a model for the IL-4R in which IL-4 may bind to IL-4R
with low affinity before either the
c or the IL-13R are
recruited. The resulting heterodimeric complex is proposed to form a
high affinity receptor. Complex formation of a primary binding subunit,
the
-chain, with a secondary affinity triggering subunit would
explain the observed cross-competition of both cytokines. Recently,
Hilton et al. (11) have cloned the mouse IL-13R
. Its
expression in various cells allowed the step by step validation of the
above model. Here we first demonstrate the efficiency of the Leonard model on the basis of a regulatory mechanism in differentiated human
cells.
-chain. Single chains of the receptor, when transfected in COS-7 cells, are shown to bind IL-13 with high affinity. In cotransfection experiments with IL-4R
heterodimerization and the related
cross-competition were minimal. However, in HUVECs heterodimerization
of IL-4R and IL-13R and ligand cross-competition were prominent,
whereas single chain IL-13 receptors bound IL-13 with low affinity.
These compelling differences suggest that a different kind of IL-13R is
expressed in HUVECs. Indeed, by RT-PCR a transcript with close homology to the mouse IL-13R (11) could be amplified from HUVEC mRNA preparations. Sequence analysis of a related full-length cDNA clone
revealed that the RT-PCR product represents a transcript with close
homology to the above-mentioned mouse
IL-13R.2 The fact that the IL-13R
message was not induced by TNF-
supports the concept of a regulatory
induction of the IL-4R
.
is a prominent inducer of VCAM-1 (45). However, VCAM-1
induction was more than additive when HUVECs were pretreated with a
combination of TNF-
and IL-4 or IL-13 (8). Moreover, short time
priming with TNF-
, which results in suboptimal VCAM-1 induction but
fully enhanced IL-4R
expression, increased in turn IL-4-mediated
VCAM-1 expression (Fig. 7). Combined effects of TNF-
and IL-4 on
VCAM-1 expression have been reported to be due to activation of VCAM-1
gene transcription by TNF-
and stabilization of the resultant VCAM-1
transcripts by IL-4 (46). IL-4 was found to exert its transcriptional
effects on the VCAM-1 gene by an NF-
B-independent mechanism, which
does not exclude the transcriptional contribution of other upstream
cis-acting sequences nor the influence of
posttranscriptional mechanisms (47). Many activities of IL-4 are shown
to be mediated by the activation of STAT transcription factors (for
review refer to Ref. 48). In hematopoietic cells, IL-4 specifically
activates Stat6. Recently, Palmer-Crocker et al. (49)
reported that in HUVECs IL-4 and IL-13 activate the JAK2 tyrosine
kinase and Stat6. We studied Stat6 activation in order to discriminate
between TNF-
and IL-4·IL-13-mediated effects. Our data support
dose-dependent Stat6 activation in response to IL-4 and
IL-13. Moreover, TNF-
treatment clearly enhanced the IL-4- and
IL-13-mediated Stat6 activation. These data provide convincing evidence
that the TNF-
-induced IL-4·IL-13R complex has an improved
signaling capability. Since STATs are transcriptional activators,
increased Stat6 activation may enhance transcription of different IL-4
and IL-13 target genes including VCAM-1. However, no data are yet
available reporting a Stat6 consensus sequence in the VCAM-1
promoter.
*
This work was supported by the Swiss National Science
Foundation Grant 3100-40796.94 (to R. M.) and ETH Grant 41-2522.5 (to H.-P. E.). The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Contributed equally to this report.
Supported by the Stiftung Prof. Max Cloëtta, Zurich,
Switzerland. To whom correspondence should be addressed: Institute of Toxicology, Federal Institute of Technology, Schorenstrasse 16, CH-8603
Schwerzenbach, Switzerland. Tel.: 41 52 3782666; Fax: 41 52 3783138.
1
The abbreviations used are: TNF-, tumor
necrosis factor
; IL, interleukin; ICAM-1, intercellular cell
adhesion molecule type-1; VCAM-1, vascular cell adhesion molecule
type-1; HUVEC, human umbilical vein endothelial cells; Jak, Janus
family tyrosine kinase; STAT, signal transducers and activators of
transcription; IL-4R
and IL-13R
, interleukin 4 and interleukin 13 receptor
-chain;
c, common
-chain; RT-PCR, reverse
transcription-polymerase chain reaction; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; MFI, mean fluorescence
intensity; EMSA, electrophoretic mobility shift assays; PBS,
phosphate-buffered saline; Me2SO, dimethyl sulfoxide; mAb,
monoclonal antibody; bp, base pair.
2
J. F. Gauchat, manuscript in preparation.
mutant by Dr. H. Loetscher (Pharmaceutical Research, F. Hoffmann-La Roche Ltd., Basel,
Switzerland).
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.