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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3820-3828
Divergent Inducible Expression of P-Selectin and E-Selectin in Mice and
Primates
By
Longbiao Yao,
Hendra Setiadi,
Lijun Xia,
Zoltan Laszik,
Fletcher
B. Taylor, and
Rodger P. McEver
From the W.K. Warren Medical Research Institute, Departments of
Medicine, Biochemistry and Molecular Biology, and Pathology, University
of Oklahoma Health Sciences Center, and Cardiovascular Biology Research
Program, Oklahoma Medical Research Foundation, Oklahoma City, OK.
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ABSTRACT |
We used in vitro and in vivo approaches to examine whether tumor
necrosis factor- (TNF-) and oncostatin M (OSM), cytokines that
bind to distinct classes of receptors, differentially regulate expression of P- and E-selectin in murine and primate endothelial cells. In human umbilical vein endothelial cells, TNF- rapidly increased mRNA for E-selectin but not P-selectin. OSM elicited little
or no change in mRNA for E-selectin, but induced a delayed and
prolonged increase in P-selectin mRNA. TNF- and OSM did not cooperate to further enhance P- or E-selectin mRNA. Intravenous infusion of Escherichia coli, which markedly elevates plasma
lipopolysaccharide and TNF-, increased mRNA for
E-selectin but not P-selectin in baboons. In murine bEnd.3 endothelioma
cells, TNF- and OSM individually and cooperatively increased mRNA
and protein for both P- and E-selectin. Intravenous injection of these
cytokines also individually and cooperatively increased mRNA for P- and
E-selectin in mice. We conclude that the murine P- and E-selectin genes
respond to both TNF- and OSM, whereas the primate P- and E-selectin
genes have much more specialized responses. Such differences should be
considered when extrapolating the functions of P- and E-selectin in
murine models of inflammation to humans.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
LEUKOCYTE EMIGRATION into sites of
inflammation proceeds through a multistep series of adhesive and
signaling events.1 Under shear flow, flowing leukocytes
tether to and roll on the vascular surface, then adhere more firmly,
and finally migrate between endothelial cells into the underlying
tissues. A critical early event is the elaboration of inflammatory
mediators that stimulate endothelial cells to express adhesion
molecules, chemokines, and lipid autacoids.2 The adhesion
molecules include P-selectin and E-selectin, which initiate the rolling
of leukocytes through interactions with cell-surface
glycoconjugates.3-5
The mediators tumor necrosis factor- (TNF-), interleukin-1
(IL-1), or lipopolysaccharide (LPS) induce endothelial cells to
transcribe mRNA for E-selectin, and the translated protein is
transported directly to the cell surface. Transient activation of the
E-selectin gene involves the cooperative binding of nuclear factor  B
(NF-B), activating transcription factor-2 (ATF-2), and other
transcription factors.6 Although the duration of transcriptional activation varies somewhat, TNF-, IL-1, or LPS clearly induce expression of E-selectin in both human and murine endothelial cells in vitro and in vivo.6
Thrombin, histamine, or other secretagogues redistribute P-selectin
within minutes from the membranes of Weibel-Palade bodies to the
surface of both human and murine endothelial cells.7-10 Such early inducible expression does not require new protein synthesis because P-selectin is mobilized from an existing storage compartment. However, this pool of P-selectin is only transiently expressed on the
cell surface, as it is proteolytically released into the circulation11,12 or it is rapidly endocytosed13
and then recycled to Weibel-Palade bodies14 or degraded in
lysosomes.15
Mechanisms for increasing synthesis of P-selectin are suggested by its
presence on the apical surface of endothelial cells in human tissues
with chronic or allergic inflammation.16-18 Indeed, TNF-, IL-1, or LPS increases expression of P-selectin
mRNA and protein in murine endothelial cells in vitro and in
vivo.10,19,20 Transient activation of the murine P-selectin
gene requires the cooperative binding of NF-B, ATF-2, and other
transcription factors.21 However, the binding sites for
NF-B and ATF-2 found in the promoter of the murine P-selectin gene
are not present in the promoter of the human P-selectin
gene.22,23 Furthermore, TNF-, IL-1, or LPS does not
increase P-selectin mRNA in human endothelial cells in
vitro.24,25 It is unknown whether these mediators increase
P-selectin mRNA in humans or in nonhuman primates in vivo, an important
consideration because the regulation of gene expression in cultured
cells may differ from that found in vivo. Confirmation of the inability
of TNF-, IL-1, and LPS to increase P-selectin mRNA in primates in
vivo would suggest that other cytokines must contribute to the
inducible expression of P-selectin mRNA in humans.
Some members of the IL-6 family of cytokines induce proinflammatory
responses in cultured human endothelial cells, including the expression
of selectins. IL-6, oncostatin M (OSM), leukemia inhibitory factor
(LIF), and other IL-6 family members bind to receptors that contain at
least one of the signal-transducing subunit gp130.26 Human
OSM binds to both low affinity and high affinity receptors on human
endothelial cells.27 The high affinity receptor may be a
heterodimer of gp130 and the OSM-specific receptor subunit,
OSMR.28 Signaling through the high affinity receptor induces a marked increase in P-selectin mRNA.24 The
OSM-induced increase in P-selectin mRNA requires new protein synthesis,
is first observed 7 hours after addition of OSM, and persists for at
least 72 hours.24 Signaling through the low affinity
receptor increases E-selectin protein, but to a much lower level than
that induced by TNF-.29 IL-6 does not bind to human
endothelial cells because they lack the IL-6-specific receptor
subunit, IL-6R. However, soluble IL-6R complexed with IL-6 binds
to gp130 on human endothelial cells, propagating signals that include
the synthesis of E-selectin.30,31 It is not known whether
IL-6-related cytokines affect selectin expression in murine
endothelial cells in vitro or in vivo. This issue is important, because
selectin genes in humans may have evolved to respond to different
classes of cytokines.
Because of their small size and relatively low cost, mice are commonly
used to study the functions of adhesion molecules, cytokines, and
chemokines in models of inflammation, atherosclerosis, and vascular
injury.32 Extrapolation of the results from mouse experiments to human biology, however, requires that the molecules of
interest be regulated similarly in both species. The available data
suggest that cytokine induction of selectin expression may differ in
mice and humans to at least some extent. However, specific mediators
have not always been tested in both mice and humans, and in other cases
the comparisons have only been made in vitro. We have directly compared
the effects of TNF- and OSM in inducing expression of P- and
E-selectin in cultured human and murine endothelial cells. To extend
these results to in vivo settings, we have examined the effects of
inflammatory mediators on selectin expression in nonhuman primates and
in mice. Our results suggest that TNF- and OSM function
cooperatively to induce expression of P- and E-selectin in mice but
diverge significantly in their effects on expression of P- and
E-selectin in humans or nonhuman primates.
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MATERIALS AND METHODS |
Reagents and antibodies.
Recombinant human and murine OSM were purchased from R & D Systems, Inc
(Minneapolis, MN). Recombinant human and murine TNF- were obtained
from Boehringer Mannheim Biochemicals (Indianapolis, IN). The murine
anti-human P-selectin monoclonal antibody (MoAb) G1 and the murine
anti-human E-selectin MoAb ES1 were prepared as
described.33,34 The rat anti-murine P-selectin MoAb
RB40.3435 and the rat anti-murine E-selectin MoAb
10E9.610 were kind gifts of Dr Dietmar Vestweber
(University of Muenster, Muenster, Germany). Cycloheximide, actinomycin D, and LPS were from Sigma Chemical Co (St
Louis, MO). Trizol reagent and the Superscript Preamplication System
for first-strand cDNA synthesis were purchased from Life Technologies
Inc (Grand Island, NY). The PCR Mimic construction kit was obtained
from Clontech Laboratories, Inc (Palo Alto, CA). Hi-Lo DNA marker was
obtained from Minnesota Molecular, Inc (Minneapolis, MN). All other
reagents were obtained from Fisher Scientific (Pittsburgh, PA) unless
noted otherwise.
Cell culture.
Human umbilical vein endothelial cells (HUVEC) and murine bEnd.3
endothelioma cells were cultured as described.24 Cytokines or pharmacologic agents, dissolved in fresh medium, were added to
confluent cell monolayers for the time intervals and at the final
concentrations indicated in the text. As a control, fresh medium
lacking the cytokine or agent was added to other cells.
Northern blot analysis.
Northern blot analysis of total RNA from cultured cells was performed
with probes as described,24 except that a radiolabeled linearized HindIII fragment of murine E-selectin cDNA19 was also used as a probe.
Western blots.
Equal numbers of HUVEC were lysed in sodium dodecyl sulfate (SDS)
sample buffer in the absence of reducing agent and boiled for 5 minutes. The samples were subjected to SDS-polyacrylamide gel
electrophoresis (PAGE) in a 7.5% polyacrylamide gel, transferred to an
Immobilon-P membrane (Amersham Corp, Arlington Heights, IL), and probed
with 1 µg/mL anti-E-selectin MoAb ES1. Bound MoAb was detected using
a horseradish peroxidase-conjugated goat anti-mouse antibody with
enhanced chemiluminescence (Amersham).
Binding of 125I-labeled MoAbs to bEnd.3 cells.
Binding of 125I-labeled anti-murine P-selectin or
E-selectin MoAbs to fixed, permeabilized bEnd.3 cell monolayers was
performed exactly as described for binding of 125I-labeled
anti-human P-selectin MoAb to HUVEC.24
Animal experiments.
Female 6- to 16-week-old Balb/C mice were purchased from Charles River
Laboratories (Wilmington, MA). Each mouse received a tail-vein
injection of 50 µL of buffer (phosphate-buffered saline, pH 7.4 plus
1% human serum albumin) as control, or 50 µL of buffer containing 25 ng murine OSM, 3000 U murine TNF-, or both OSM and TNF-. In other
experiments mice were injected with buffer containing 25 ng human OSM.
Mice were killed 1 hour, 4 hours, or 24 hours after the injection, and
organs were then immediately collected for RNA isolation or immunohistochemistry.
Healthy adolescent baboons (Papio c. anubis) were
purchased from a breeding colony maintained by the University of
Oklahoma Health Sciences Center, Oklahoma City, OK. A dose
of live Escherichia coli known to induce lethal septic shock
was infused intravenously over a 2-hour period into a baboon as
described.36-38 A control animal received no E coli
but was otherwise treated identically. After 4 hours (2 hours after the
end of the E coli infusion), the experimental and control
animals were killed with a lethal intravenous infusion of sodium
pentobarbitol, and the organs were then immediately collected for RNA isolation.
The protocols for mice and baboons were approved by the appropriate
Animal Use Committees of the University of Oklahoma Health Sciences
Center, the Veterans Administration Hospital, or the Oklahoma Medical
Research Foundation, Oklahoma City, OK.
Quantitative reverse transcriptase-polymerase chain reaction
(RT-PCR).
Total RNA was isolated from murine or baboon organs with the Trizol
reagent. First-strand cDNA synthesis was performed with the Superscript
Preamplification System as specified by the manufacturer. Briefly, RNA
(5 µg) was incubated with 0.5 µg oligo-dT for 10 minutes at 70°C
and then chilled on ice for 1 minute. A PCR buffer that included final
concentrations of 0.2 mmol/L MgCl2, 1.0 mmol/L dNTP mix,
and 0.01 mol/L DTT was added to each RNA/primer mix and incubated for 5 minutes at 42°C. Then 200 U of Superscript II reverse transcriptase
was added to give a final volume of 20 µL, and the mixture was
incubated for 50 minutes at 42°C, for 15 minutes at 70°C, and then
chilled on ice. RNase H (1 µL containing 2 U) was added and incubated
for 20 minutes at 37°C before PCR was conducted.
Quantitative PCR was performed with the PCR Mimic construction kit as
specified by the manufacturer. Briefly, PCR mimics were constructed
with 2 rounds of PCR amplification. Each composite primer consisted of
target gene sequence (from P-selectin, E-selectin, or -actin)
attached to 1 of 2 20-nucleotide sequences designed to hybridize to
opposite strands of a mimic DNA fragment (a 574-bp Bam HI/Eco RI
fragment of the v-Erb gene). A dilution of the first PCR
reaction was reamplified using only gene-specific primers. The PCR
mimic was then purified on a ChromaSpin+TE-100 column. The purified
mimic was adjusted to a concentration of 100 attomoles/µL. For
quantitative PCR, 10-fold dilutions of mimic were added to a series of
tubes containing a fixed concentration of first-strand cDNA and a pair
of gene-specific primers. The PCR conditions were as follows: 25 cycles
at 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1.5 minutes. The amplified products were electrophoresed in a 1.5% agarose
gel, and the gel was stained with ethidium bromide. The concentration
of mRNA for P-selectin, E-selectin, or actin was estimated from the
lane in which the quantity of the amplified target cDNA was similar to
that of the mimic cDNA. For each such lane, the original concentration
of mimic DNA (in attomoles/µL) was used to assign a semi-quantitative
measurement to the concentration of mRNA as follows:  < 10 5, + = 104, ++ = 103,
+++ = 102, ++++ = 101.
The gene-specific primers to amplify baboon P- and E-selectin were
derived from the published sequences for the human cDNAs for P- and
E-selectin.39,40 The size of the amplified products exactly
matched the predicted size of the human orthologues. The identity of
the amplified baboon cDNA fragments was confirmed by DNA sequencing;
the nucleotide sequence of the baboon P-selectin cDNA fragment was 99%
identical to that of the human P-selectin cDNA, and the nucleotide
sequence of the baboon E-selectin cDNA was 97% identical to that of
the human E-selectin cDNA. For the experiments shown in Fig 2 and Table
1, the primers for P-selectin were derived from the experimentally
determined baboon sequence. The baboon E-selectin product was amplified
using the primers derived from the human E-selectin sequence. The
gene-specific primers for murine P- and E-selectin were derived from
the published sequences for the murine cDNAs for P- and
E-selectin.19 The sizes of the amplified products exactly
matched those predicted. The identity of the amplified fragments was
further confirmed by restriction digestions with appropriate enzymes.
Human and murine -actin mimic and primers were provided by the
manufacturer. The P- and E-selectin primers were as follows: Murine
P-selectin sense primer: 5'-CTATACCTGCTCCTGCTACCCAGGC-3' (nt 549-573),
antisense primer: 5'-TTCACTCCACTGACCAGAGCCAGTG-3' (nt 951-937); murine
E-selectin sense primer: 5'-CCTCTGACAGAGGAAGCTCAGAACT-3' (nt 401-425),
antisense primer: 5'-TCCACTCTCCAGAGGACGTACACCG-3' (nt 830-806); baboon
P-selectin sense primer: 5'-GACATGTCCTGCAGCAAACAAGGAGAGTGC-3' (nt
531-560), antisense primer 5'-CCGGTCCAACTAATGCAAATCCCTCTTCAC-3' (nt
942-913); human E-selectin sense primer: 5'-CAGCAAGAAGAAGCTTGCCCTATG-3' (nt 506-530), antisense primer 5'-TTGTGTTCCATGGGAAGCTTCCAGG-3' (nt
915-891).
Immunohistochemistry.
Frozen sections of murine brain, skin, heart, mediastinal large veins
and elastic arteries, lungs, spleen, kidneys, liver, and esophagus were
fixed in 4% buffered paraformaldehyde for 10 minutes at 4°C.
Immunohistochemical staining was performed using a standardized
streptavidin-biotin-peroxidase method. All incubations were at room
temperature and were separated by washes with PBS. The sections were
treated with 1.25% hydrogen peroxide to block endogenous peroxidase
activity and with PBS containing 10% rabbit serum to reduce
nonspecific antibody binding. The sections were next treated for 20 minutes with 10 µg/mL of anti-P-selectin MoAb RB40.34 or
anti-E-selectin MoAb 10E9.6, or as a negative control, with buffer
containing no primary antibody. The sections were then incubated with
10 µg/mL biotin-conjugated rabbit anti-rat IgG (Dako Corporation,
Carpinteria, CA) for 20 minutes and finally with a
streptavidin-peroxidase reagent (Dako) for 30 minutes. Diaminobenzidine
(Sigma) was used as chromagen, and hematoxylin was used for nuclear counterstaining.
| |
RESULTS |
Stimulation of HUVEC with TNF- plus OSM does not
cooperatively increase mRNA for P- and E-selectin.
We used Northern blot analysis to examine the effects of human OSM and
TNF- on expression of P- and E-selectin in HUVEC. We also measured
the mRNA levels of CHO-B, a control, ubiquitously expressed transcript
that does not change its levels when cells are
stimulated.41 As observed previously,24 OSM
induced a delayed and sustained increase in P-selectin mRNA levels that reached maximum within 10 to 24 hours (Fig
1A). In contrast, stimulation with TNF-
did not change or even slightly decreased P-selectin mRNA levels.
Costimulation of HUVEC with TNF- and OSM did not cooperatively
increase P-selectin mRNA and, in fact, often blunted the OSM-induced
increase in P-selectin mRNA levels. As documented previously,40 stimulation of HUVEC with TNF- markedly
increased E-selectin mRNA within 3 hours, followed by a gradual decline over 10 to 24 hours. Stimulation with OSM, in contrast, had little effect on E-selectin mRNA levels, although a small accumulation of
transcripts was sometimes observed after 24 hours. Costimulation with
TNF- and OSM increased E-selectin mRNA to levels observed after
TNF- alone, although a slight further increase was sometimes observed after 24 hours. These data show that TNF- and OSM do not
cooperatively increase mRNA for P- or E-selectin in HUVEC. OSM is the
major inducer of P-selectin mRNA, whereas TNF- is the major inducer
of E-selectin mRNA.
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| Fig 1.
Stimulation of HUVEC with TNF- plus OSM does not
cooperatively increase mRNA for P- and E-selectin. (A) Confluent
monolayers of HUVEC were treated with 10 ng/mL human OSM, 100 U/mL
human TNF-, or a combination of both cytokines. After the indicated
time, total RNA was isolated, and 20 µg of RNA was electrophoresed
and then transferred to a membrane for Northern blot analysis. The same
membrane was sequentially hybridized with the indicated cDNA probes.
The mobilities of the hybridized transcripts corresponded to published
values. (B) HUVEC were treated with 10 ng/mL OSM in the presence or
absence of 10 µg/mL cycloheximide. After the indicated time, total
RNA was isolated and analyzed by Northern blotting. (C) HUVEC were
treated with 1 µg/mL OSM or 100 U/mL TNF- in the presence or
absence of 5 µg/mL actinomycin D. After 4 hours, the cells were lysed
and analyzed by Western blotting with a MoAb to E-selectin.
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The lag period required for OSM to increase P-selectin mRNA differs
from the ability of TNF- to rapidly increase mRNA for E-selectin, a
characteristic of immediate early genes. When pretreated with
cycloheximide, HUVEC did not increase P-selectin mRNA levels in
response to OSM (Fig 1B). In contrast, these cells expressed higher
levels of E-selectin mRNA, presumably because they could not synthesize
new IB- to retain heterodimeric NF-B complexes in the
cytoplasm.42 These results show that, to fully activate the
P-selectin gene, OSM must first induce the synthesis of one or more
proteins in HUVEC.
The HUVEC in Fig 1A and 1B were stimulated with OSM at 10 ng/mL (0.45 nmol/L), a concentration that saturates the high affinity receptors for
OSM on HUVEC27 and causes maximal accumulation of
P-selectin mRNA.24 OSM at a higher concentration (1 µg/mL; 45 nmol/L) binds to low affinity receptors and increases
E-selectin protein, although to a much lower level than does
TNF-.29 Treatment of HUVEC with 45 nmol/L OSM only
slightly increased E-selectin mRNA levels over that observed with 0.45 nmol/L OSM in Fig 1A (data not shown). As observed
previously,29 stimulation of HUVEC with 45 nmol/L OSM for 4 hours did increase E-selectin protein as detected by immunoblotting,
but the increase was only 10% of that observed after stimulation with
TNF- (Fig 1C). Pretreatment of HUVEC with actinomycin D blocked the
increase in E-selectin protein, suggesting that OSM induced E-selectin
expression at the transcriptional level. These data indicate that high
concentrations of OSM do activate the E-selectin gene, but to a much
lesser extent than does TNF-.
Intravenous infusion of E coli increases mRNA for E-selectin
but not P-selectin in baboon tissues.
TNF-, IL-1, or LPS increases mRNA for E-selectin but not for
P-selectin in HUVEC.6,24 (See also Fig 1A.) This strongly suggests that these mediators, which function by activating NF-B heterodimers and ATF-2-containing dimers, do not activate the P-selectin gene in human endothelial cells. However, transcriptional regulation of genes in vitro and in vivo may differ, because the environment of cultured cells has been drastically altered from the
normal environment. We therefore used a well-characterized, nonhuman
primate model to test whether LPS increases mRNA for P- and E-selectin
in vivo. A baboon was given an intravenous infusion of E coli
at a dose that consistently induces death through septic shock within
12 to 36 hours.38 The injected E coli produce high circulating levels of LPS, which initiates signals that elevate plasma
levels of TNF- within 1 to 2 hours.36 After 4 hours the
baboon was killed, and RNA was isolated from multiple organs; as a
control, RNA was isolated from the organs of a baboon that did not
receive E coli. The levels of mRNA for P-selectin, E-selectin, or -actin were measured by a competitive RT-PCR method in which serial dilutions of a DNA mimic containing flanking sequences matching
the target DNA were added to first-strand cDNA prepared from a fixed
concentration of tissue RNA. The amount of mimic that allows
amplification of equal quantitities of mimic and target DNA provides a
measure of the level of target DNA, and thus mRNA, in the sample.
Figure 2 shows representative agarose gel
electrophoresis of RT-PCR products from RNA of the heart. The levels of
P-selectin mRNA, like those of the control -actin mRNA, were
identical in the control baboon and in the baboon that received E
coli. In contrast, mRNA levels for E-selectin were significantly
higher in the baboon that received E coli than in the control
baboon. Similar results were obtained when mRNA from other organs was measured (Table 1). The E coli
infusion markedly increased mRNA for E-selectin in all organs, whereas
it did not change or even decreased mRNA for P-selectin. These data
show that circulating LPS and TNF- in concentrations that
significantly increase E-selectin mRNA do not increase P-selectin mRNA
in a nonhuman primate. Thus, the results obtained in vivo support the
observations made in cultured human endothelial cells.
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| Fig 2.
Intravenous infusion of E coli increases mRNA for
E-selectin but not P-selectin in baboon tissues. A lethal dose of E
coli was infused intravenously into a baboon. A control baboon
received no E coli but was otherwise treated identically. After
4 hours, the animals were killed, and the organs were immediately
collected for RNA isolation. The levels of mRNA for P-selectin,
E-selectin, and -actin were measured by a competitive RT-PCR method
in which serial dilutions of a DNA mimic containing flanking sequences
matching the target DNA were added to first-strand cDNA prepared from a
fixed concentration of tissue RNA. The amount of mimic that allows
amplification of equal quantitities of mimic and target DNA provides a
measure of the level of target DNA, and thus mRNA, in the sample. Shown
is representative agarose gel electrophoresis of the PCR products from
baboon heart. In this example, mimic cDNA at a concentration of
103 attomoles/µL allowed equal amplification of mimic
and P-selectin cDNA in both the control baboon and the baboon that
received E coli. Mimic cDNA at a concentration of
101 attomoles/µL allowed equal amplification of mimic
and E-selectin cDNA in the E coli baboon, whereas no E-selectin
cDNA was amplified in the control baboon even at the lowest
concentration of mimic (105 attomoles/µL). Mimic cDNA
at a concentration of 101 attomoles/µL allowed equal
amplification of mimic and -actin cDNA in both the control baboon
and the E coli baboon. Identical results were obtained with
another pair of baboons, one of which received E coli.
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Stimulation of murine bEnd.3 cells with TNF- plus OSM
cooperatively increases mRNA for P- and E-selectin.
To determine whether OSM affected expression of P- or E-selectin in
murine endothelial cells, we treated bEnd.3 cells with 25 ng/mL (1.125 nmol/L) murine OSM for various intervals. OSM markedly increased the
levels of P-selectin mRNA (Fig 3A). Unlike the delayed accumulation of P-selectin mRNA in HUVEC after OSM stimulation, P-selectin mRNA in bEnd.3 cells increased rapidly, reaching maximum within 1 to 3 hours. Levels declined within 10 hours
but remained elevated at least 48 hours after addition of OSM. Unlike
the little or no change in E-selectin mRNA in HUVEC after OSM
stimulation, E-selectin mRNA in bEnd.3 cells increased rapidly,
reaching maximum within 1 hour. Levels then declined but remained
slightly elevated at least 48 hours after addition of OSM.
Quantification of selectin transcripts, normalized for the level of
CHO-B mRNA, indicated that OSM increased P-selectin mRNA up to 8-fold
(Fig 3B) and E-selectin mRNA up to 14-fold (Fig 3C). OSM increased P-
and E-selectin mRNA in a concentration-dependent manner, with maximal
effects achieved at 25 ng/mL (data not shown). The effect of murine OSM
was not because of contamination with LPS because boiled OSM did not
increase P- or E-selectin transcripts. In contrast, added LPS increased
both P- and E-selectin mRNA (Fig 3A). Stimulation of bEnd.3 cells with
25 ng/mL human OSM increased P- and E-selectin mRNA exactly like that
observed with murine OSM (data not shown).
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| Fig 3.
Stimulation of murine bEnd.3 cells with OSM increases
mRNA for both P- and E-selectin. (A) Confluent monolayers of bEnd.3
cells were incubated in the presence or absence of 25 ng/mL murine OSM.
To ensure that the effects of OSM were not caused by contaminating LPS,
some cells were treated with OSM that was boiled to inactive the
cytokine, or with 10 ng/mL of exogenously added LPS. After the
indicated time, total RNA was isolated and analyzed by Northern
blotting. (B) and (C) The OSM-induced increase in P- and E-selectin
mRNA in each lane was quantified by densitometric scanning. The level
of P- and E-selectin mRNA in each lane was normalized according to the
level of CHO-B mRNA, which was not affected by OSM. The data are
representative of 4 independent experiments.
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Previous studies showed that TNF- increases mRNA expression for P-
and E-selectin in murine endothelioma cells.10,19 We treated bEnd.3 cells with murine TNF-, OSM, or both cytokines. The
combination of TNF- and OSM increased mRNA for both selectins to
much higher levels than those observed after treatment with either
individual cytokine (Fig 4A). The increase
was most prominent after 3 hours but was still observed at least 24 hours after addition of the mediators. These data show that, unlike
their lack of cooperativity in HUVEC, TNF- and OSM cooperatively
increase mRNA for both P- and E-selectin in bEnd.3 cells. TNF- and
OSM also cooperatively increased protein for both P- and E-selectin in
bEnd.3 cells (data not shown).
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| Fig 4.
Stimulation of bEnd.3 cells with TNF- plus OSM
cooperatively increases mRNA for P- and E-selectin. (A) bEnd.3 cells
were treated with 25 ng/mL murine OSM, 100 U/mL murine TNF-, or a
combination of both cytokines. After the indicated time, total RNA was
isolated and analyzed by Northern blotting. (B) bEnd.3 cells were
treated with fresh medium in the presence or absence of 25 ng/mL murine
OSM, 100 U/mL murine TNF-, or a combination of both cytokines, in
the presence or absence of 10 µg/mL cycloheximide or 5 µg/mL
actinomycin D (Act. D). After 3 hours, total RNA was isolated and
analyzed by Northern blotting.
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The rapid increase in mRNA for P- and E-selectin in response to TNF-
or OSM is characteristic of immediate early genes, which do not require
new protein synthesis for their activation. Consistent with this
notion, pretreatment of bEnd.3 cells with cycloheximide did not block
the induction of P- and E-selectin mRNA by either or both cytokines
(Fig 4B). In fact, cycloheximide potentiated the ability of TNF- or
OSM to increase selectin transcripts, perhaps because it prevented the
synthesis of labile protein inhibitors of transcription. Pretreatment
of bEnd.3 cells with actinomycin D blocked the increase in P- and
E-selectin mRNA, suggesting that the cytokines acted at the level of
mRNA transcription.
Intravenous injection of TNF- and OSM cooperatively
increases mRNA for P- and E-selectin in murine tissues.
The experiments with bEnd.3 cells showed that TNF- and OSM
independently increase mRNA for murine P- and E-selectin, and act
cooperatively to further increase selectin mRNA expression. To
determine whether these in vitro experiments predicted in vivo responses, mice were given an intravenous injection of 25 ng of murine
OSM, 3,000 U of murine TNF-, or a combination of both cytokines.
Control mice received an injection of buffer alone. After 1 hour, the
mice were killed, and RNA was isolated from multiple organs; the short
1-hour time interval was selected to maximize the probability that any
observed changes in mRNA levels were because of direct actions of
TNF- or OSM on the endothelium rather than to induced expression of
mediators in other cells that then stimulated the endothelium. The
levels of mRNA for murine P-selectin, E-selectin, and -actin were
measured by the competitive RT-PCR method used to measure baboon mRNA
levels. Figure 5 shows representative
agarose gel electrophoresis of RT-PCR products from RNA of murine
kidney. The cytokines did not increase mRNA for -actin. In contrast,
injection of either OSM (Fig 5A) or TNF- (Fig 5B) significantly
increased mRNA levels for both P- and E-selectin. Injection of both
cytokines increased mRNA levels for P- and E-selectin to even higher
levels (Fig 5C). Similar results were obtained in other murine organs
(Table 2). Immunohistochemical analysis
revealed that combined infusion of TNF- and OSM increased expression
of P- and E-selectin protein only in endothelial cells, primarily in
small veins and postcapillary venules of multiple organs (data not
shown). Injection of 25 ng human OSM for 1 hour increased P- and
E-selectin mRNA to levels identical to those observed after injection
of murine OSM (data not shown). Unlike the results obtained after 1 hour, no increase in P- or E-selectin mRNA was observed 24 hours after
injection of murine OSM and TNF-, either alone or in combination
(data not shown). These results show that TNF- and OSM cooperatively
increase mRNA and protein for P- and E-selectin in murine tissues.
Thus, the results obtained in vivo support the observations made in
cultured murine endothelioma cells.
View larger version (83K):
[in this window]
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| Fig 5.
Intravenous injection of TNF- and OSM cooperatively
increases mRNA for P- and E-selectin in murine tissues. Each mouse
received an intravenous injection of buffer (Control), or of buffer
containing 25 ng murine OSM, 3000 U murine TNF-, or a combination of
both cytokines. After 1 hour, the mice were killed and the organs were
immediately collected for RNA isolation. The levels of mRNA for
P-selectin, E-selectin, and -actin were measured by a competitive
RT-PCR method as in Fig 2, except that the primers contained the
respective murine cDNA sequences. Shown is representative agarose gel
electrophoresis of the PCR products from mouse kidney. The data are
representative of 4 independent experiments for control mice and mice
receiving OSM, and 2 independent experiments for mice receiving TNF-
or the combination of both cytokines.
|
|
| |
DISCUSSION |
We used both in vitro and in vivo approaches to examine how TNF- and
OSM, cytokines that bind to distinct classes of receptors, affect the
expression of P- and E-selectin in murine and primate endothelial
cells. Our results indicate that the murine P- and E-selectin genes
respond similarly to both cytokines. TNF- or OSM markedly increased
mRNA for both selectins, and a combination of both cytokines elicited
additive or synergistic increases in P- and E-selectin mRNA and
protein. In marked contrast, the human and nonhuman primate P- and
E-selectin genes have diverged in their responsiveness to specific
cytokines. OSM, but not TNF-, increased P-selectin mRNA. TNF- was
the major inducer of E-selectin mRNA, whereas OSM even at high
concentrations was a much weaker agonist. Notably, a combination of
TNF- and OSM did not cooperatively increase human P- or E-selectin mRNA.
TNF- or OSM rapidly increased P- and E-selectin mRNA in murine
bEnd.3 cells. The early induction and the lack of requirement for new
protein synthesis are characteristic of the signal-mediated activation
of immediate early genes. The relatively slow decline in transcript
levels could reflect long mRNA half-lives and/or a biphasic response of
the gene to differentially mobilized transcription factors. That
cycloheximide potentiated the cytokine-mediated accumulation of mRNA
suggests that short-lived proteins repress transcription of the genes
for both P- and E-selectin. The ability of TNF- and OSM to
cooperatively increase selectin mRNA levels suggests that each cytokine
activates gene expression through distinct signaling pathways that
function additively or synergistically.
The ability of TNF- and OSM to individually or cooperatively
increase murine mRNA for P- and E-selectin in vivo supports the results
obtained in vitro, and indicates that the signaling events observed in
bEnd.3 cells are not unique to this cell line. The induction of P- and
E-selectin mRNA only 1 hour after intravenous injection of TNF- or
OSM strongly suggests that each cytokine acted directly at the level of
the endothelial cell. The immunohistochemical results indicate that
inducible expression of P- and E-selectin protein was limited to
endothelial cells. The lack of detectable increase in P- and E-selectin
mRNA 24 hours after injection suggests that murine endothelial cells
must be continuously exposed to cytokine, as performed in the in vitro
experiments, to maintain elevated transcript levels. The dose of OSM
injected, 25 ng, was low but was calculated to be sufficient to bind to
high affinity receptors. In mice, murine OSM binds only to the OSM
receptor, which consists of gp130 associated with the OSMR subunit,
and human OSM binds only to the LIF receptor, which consists of gp130 associated with the LIFR subunit.43-45 Because murine or
human OSM increased P- and E-selectin mRNA in bEnd.3 cells and in
murine tissues in vivo, signaling through either the OSM receptor or the LIF receptor is sufficient to activate the murine P- and E-selectin genes. Signaling through the LIF receptor may account for the ability
of subcutaneously injected human OSM to induce an acute inflammatory
response in mice.29
The experiments in HUVEC suggest that the genes for P- and E-selectin
in humans, unlike the corresponding genes in mice, have developed
specialized responses to cytokine signals. TNF- retained the ability
to rapidly activate the human E-selectin gene but lost the ability to
activate the human P-selectin gene, confirming previous
studies.24,25,40 OSM had little or no ability to increase
E-selectin mRNA levels, and did not cooperate with TNF- to further
increase E-selectin mRNA. In contrast, OSM markedly increased mRNA for
P-selectin in a delayed and prolonged fashion that required new protein synthesis.
To extend the HUVEC studies to an in vivo setting, we examined the
effects of infusion of a uniformly lethal dose of E coli on the
expression of P- and E-selectin mRNA in baboons. The infused E
coli produce high circulating levels of LPS, which, like TNF-, augments E-selectin but not P-selectin mRNA in HUVEC.24
Previous immunohistochemical analysis showed that E coli
induces expression of E-selectin protein in venular endothelium within
2 hours after injection into baboons.37 After 4 hours, we
observed consistent induction of E-selectin mRNA in multiple baboon
tissues, but found no increase in the constitutively expressed levels
of P-selectin mRNA. The ability of LPS and TNF- to increase mRNA for
E-selectin, but not P-selectin, in a nonhuman primate supports the
results obtained in vitro and indicates that the signaling events
observed in HUVECs are not unique to these cultured human endothelial
cells. We did not examine whether infusion of OSM altered selectin mRNA expression in baboons. High circulating levels of OSM have been reported in patients with septic shock.46 If OSM is
generated after infusion of E coli in baboons, it might elevate
P-selectin mRNA, but perhaps only after a lag period of 7 to 10 hours
as observed in HUVEC.24 A similar lag might be required for
signaling after binding of circulating IL-6/sIL-6R complexes to
gp130 on endothelial cells.30,31
The different inducible responses of murine and human selectin genes
extend to at least one other cytokine, IL-4. Like OSM, IL-4 increases
P-selectin mRNA in both bEnd.3 cells and HUVEC.24 Also like
OSM, IL-4 rapidly increases P-selectin mRNA in bEnd.3 cells, whereas it
increases P-selectin mRNA in HUVEC in a delayed and prolonged fashion
that requires new protein synthesis.24 Murine IL-4 rapidly
increases E-selectin mRNA in bEnd.3 cells, and a combination
of IL-4 and TNF- further augments E-selectin mRNA (L.Y. and R.P.M.,
unpublished observations, October 1997). In contrast,
human IL-4 does not activate E-selectin expression in HUVEC, and, in
fact, inhibits the ability of TNF- to increase E-selectin mRNA. IL-4
signals in part by activating Stat6.47,48 IL-4 prevents
E-selectin expression in HUVEC by activating Stat6, which binds to a
DNA element that overlaps a B element in the human E-selectin
promoter, thereby competitively inhibiting binding of
NF-B.49 Notably, the Stat6 recognition sequence is not
conserved in the murine E-selectin promoter.50 There are 2 Stat6 elements in the human P-selectin promoter, which are in different
locations than a single Stat6 element in the murine P-selectin
promoter.22 Binding of activated Stat6 to the 2 elements in
the human P-selectin gene contributes to IL-4-inducible
expression.51
As in murine cells, TNF-, IL-1, or LPS rapidly increases mRNA for
P- and E-selectin in rat,52 bovine,19,53 and
canine54 endothelial cells. Thus, in the mammals studied,
only the P-selectin gene in primates has lost its responsiveness to
these mediators. It is not known whether the specialized responses of
P- and E-selectin to OSM or IL-4 are limited to primates. What are the
biological implications of these divergent responses across species? In
mice, the ability of TNF- and OSM to cooperatively stimulate both P- and E-selectin expression may ensure overlapping or redundant functions
of these proteins during acute and chronic inflammatory responses. This
may explain why targeted deletion of both the P- and E-selectin genes
is required to significantly alter the phenotype in some murine models
of inflammation or atherosclerosis.55-61 In humans,
cytokines may have much more specific effects on expression of the P-
or E-selectin gene. This specialized response to cytokines may allow P-
and E-selectin to make more distinct contributions to leukocyte
recruitment, depending on the nature of the inflammatory stimulus.
| |
ACKNOWLEDGMENT |
We thank Junliang Pan for helpful discussions, Gary Farrell and Michael
McDaniel for valuable technical assistance, Kelsey Kennedy for
assistance with the figures, Dietmar Vestweber for providing MoAbs, and
James Morrissey and James Jarvis for critical reading of the manuscript.
| |
FOOTNOTES |
Submitted May 7, 1999; accepted July 21, 1999.
Supported by Grant No. HL 54502 from the National Institutes of Health,
Bethesda, MD. H.S. is the recipient of a postdoctoral fellowship award
from the Oklahoma Affiliate of the American Heart Association.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Rodger P. McEver, MD, W.K. Warren
Medical Research Institute, University of Oklahoma Health Sciences
Center, 825 NE 13th St, Oklahoma City, OK 73104;
e-mail: rodger-mcever{at}ouhsc.edu.
| |
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ABSTRACT
INTRODUCTION