Interleukin-6 production in hemorrhagic shock is accompanied
by neutrophil recruitment and lung injury
Christian
Hierholzer1,
Jörg C.
Kalff1,
Laurel
Omert1,
Katsuhiko
Tsukada1,
J. Eric
Loeffert2,
Simon C.
Watkins2,
Timothy R.
Billiar1, and
David J.
Tweardy3,4,5
Departments of 1 Surgery,
2 Cell Biology,
3 Medicine, and
4 Molecular Genetics and
Biochemistry, University of Pittsburgh School of Medicine and
5 University of Pittsburgh Cancer
Institute, University of Pittsburgh Medical Center, Pittsburgh,
Pennsylvania 15213
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ABSTRACT |
Hemorrhagic shock (HS) initiates an inflammatory
cascade that includes the production of cytokines and recruitment of
neutrophils (PMN) and may progress to organ failure, inducing acute
respiratory distress syndrome (ARDS). To examine the hypothesis that
interleukin-6 (IL-6) contributes to PMN infiltration and lung damage in
HS, we examined the lungs of rats subjected to unresuscitated and resuscitated HS for the production of IL-6 and activation of Stat3. Using semiquantitative RT-PCR, we found a striking increase in IL-6
mRNA levels only in resuscitated HS, with peak levels observed 1 h
after initiation of resuscitation. Increased IL-6 protein expression
was localized to bronchial and alveolar cells. Electrophoretic mobility
shift assay of protein extracts from shock lungs exhibited an increase
in Stat3 activation with kinetics similar to IL-6 mRNA. In situ DNA
binding assay determined Stat3 activation predominantly within alveoli.
Intratracheal instillation of IL-6 alone into normal rats resulted in
PMN infiltration into lung interstitium and alveoli, marked elevation
of bronchoalveolar lavage cellularity, and increased wet-to-dry ratio.
These findings indicate that IL-6 production and Stat3 activation occur
early in HS and may contribute to PMN-mediated lung injury, including
ARDS after HS.
cytokines; acute inflammation; signal transducers and activators of
transcription; acute respiratory distress syndrome
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INTRODUCTION |
HEMORRHAGIC SHOCK (HS) initiates a cascade of
inflammatory events after successful resuscitation that may result in
organ impairment, including acute respiratory distress syndrome (ARDS) and increased mortality. We have previously shown that resuscitated HS
in rats results in acute lung injury manifested by infiltration of
polymorphonuclear neutrophils (PMN) into the interstitium and alveoli,
pulmonary edema, and hypoxia (15). Chemokines of the C-X-C class,
especially interleukin (IL)-8, have been demonstrated to be key
mediators of PMN infiltration in HS (20, 28). Recent studies have
demonstrated that IL-6 participates in the recruitment of PMN into
tissue sites by induction of IL-8 (26).
The systemic response to inflammation includes the production of IL-6,
which signals through activation of proteins that serve the dual
function of signal transducers and activators of transcription (STAT)
(18). We previously demonstrated activation of STAT proteins, particularly Stat3, in the lungs of animals subjected to HS and that
levels of Stat3 activity increased with increasing severity of shock
(13).
The current study was designed to determine if IL-6 production occurs
in our rat model of HS where it may contribute to PMN infiltration and
Stat3 activation. We report here that levels of IL-6 mRNA increased
with increased duration of the ischemic phase of resuscitated shock,
similar to our findings regarding Stat3 activity. In addition, levels
of IL-6 mRNA and Stat3 activity were increased above control animals
only in resuscitated HS animals. Furthermore, induction of IL-6 mRNA as
well as the activation of Stat3 occurred simultaneously after
resuscitation, with levels of both peaking 1 h after the onset of
resuscitation. To test the hypothesis that IL-6 protein in the distal
airways of the lung will cause PMN recruitment and lung injury, we
administered IL-6 by intratracheal instillation into the lungs of
anesthetized rats. IL-6 instillation resulted in increased
bronchoalveolar lavage (BAL) fluid cellularity, accumulation of PMN
into widened interstitial and alveolar spaces, and increased wet-to-dry
ratios to levels observed previously in animals subjected to HS. These results suggest that IL-6 is produced in the lung in HS where it
contributes to Stat3 activation, PMN infiltration, and lung injury.
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MATERIALS AND METHODS |
Animals. These studies were approved
by the University of Pittsburgh Institutional Review Board for animal
experimentation and conform to National Institutes of Health guidelines
for the care and use of laboratory animals. Fasted male Sprague-Dawley rats (Charles River Breeding Laboratory, Cambridge, MA) were used for
all phases of these studies.
HS protocols. For initial anesthesia,
penthrane inhalation was used. The animals were intubated orally with a
14-gauge cannula. The right carotid artery and left jugular vein were
cannulated with 21-gauge tubing after surgical preparation and
isolation. The cannulas, syringes, and tubing were flushed with heparin
sodium (1,000 U/ml) before all procedures. Arterial blood pressure was continuously monitored with a Spacelab 514 multimonitor (Spacelabs, Hillsboro, OR). A Harvard small-animal ventilator (Braintree
Scientific, Braintree, MA) was used to administer a 2.5-ml tidal volume
of room air at 72 strokes/min. After vascular cannulation, the animals received intravenous anesthesia (50 mg/kg pentobarbital sodium).
Two HS protocols were performed: HS without resuscitation and HS with
resuscitation as previously described (13). Briefly, the unresuscitated
HS protocol was carried out to the predefined end points of
compensated, decompensated, and irreversible shock. After the initial
bleed of 2.25 ml/100 g body wt, mean arterial pressure (MAP) dropped
sharply and then slowly stabilized. In compensated shock (1 h of
ischemia), additional blood was withdrawn to maintain MAP at 40 mmHg for 1 h, and no shed blood was returned (0% shed blood return,
SBR). In decompensated shock (2.5 h of ischemia), the MAP was
maintained at 40 mmHg for 2.5 h and required 35% of the shed blood to
be returned (35% SBR). In irreversible shock (3.5 h of
ischemia), MAP was maintained at 40 mmHg for 3.5 h and required
70% of the shed blood to be returned (70% SBR). Animals in the
unresuscitated HS protocol were killed at the end of the ischemic
phase. Time-matched sham control animals underwent all preparations and
monitoring procedures but were not bled. Animals were randomly
subjected to either the shock or sham protocol. There were three to
five animals in each shock and sham group.
In the resuscitated HS protocol, animal preparation and hemorrhage were
carried out as in the unresuscitated protocol to the defined end
points: for the compensated shock (1 h of ischemia) groups to
compensation end point with 0% SBR and for the decompensated shock
(2.5 h of ischemia) groups to midcompensation phase with 35%
SBR. Once the animals had attained these points in the protocol, they
were resuscitated, and the MAP was normalized using shed blood plus two
times the shed blood volume in lactated Ringer solution. Once the
animal had recovered, all cannulas were removed, and the incision was
closed. Rats were killed from 1 to 8 h after the initiation of
resuscitation. Time-matched sham animals again served as controls, and
there were four rats in each group at each time point. The irreversible
shock group (70% SBR) was excluded from the resuscitation protocol
because animals subjected to 70% SBR could not be resuscitated.
IL-6 instillation into the lungs of normal
animals. Recombinant human IL-6 was obtained from
Genzyme (Cambridge, MA). The specific activity was 8.1 × 107 U/mg as determined by the
manufacturer. Endotoxin levels were 0.102 ng/µg as determined using a
kinetic-chromogenic method. IL-6 was administered by intratracheal
injection as previously described (14). In brief, male Sprague-Dawley
rats were subjected to instillation of IL-6 or saline fluid by
intratracheal injection. Animals were intubated orally. To prepare a
surfactant-containing vehicle for IL-6 instillation, 20 ml of saline
were used to lavage the lung of a normal rat. The lavage fluid was
centrifuged (2,500 g, 5 min) to remove
cells and debris. IL-6 doses (0, 10, 30, 100, or 300 ng) were diluted
in 1 ml of cell-free lavage fluid and injected into the trachea of
anesthetized animals followed by three strokes of air with a 3-ml
syringe. After instillation and recovery from the anesthesia, all rats
were active and alert in their cages. Animals were killed at 2, 4, 8, 12, 24, and 48 h after IL-6 instillation.
Isolation of lungs and cells and measurement of lung
injury. The rats subjected to the HS protocols were
killed at the completion of each experiment. After the carcasses were
flushed with cold (4°C) isotonic saline solution, the left lung was
removed and used for wet-to-dry determination and PMN counts. The right
lung was immediately frozen in liquid nitrogen and stored at
80°C. The right lung then was used for total cellular RNA
extraction using the method of Chomczynski and Sacchi (7) and for
protein extraction from frozen sections using high-salt buffer for use in electrophoretic mobility shift assay (EMSA) as described (12). In
rats subjected to the instillation of IL-6, median sternotomy and
preparation of the trachea were performed, and the left pulmonary hilus
was isolated and ligated. The left lung was excised and removed for
wet-to-dry ratio. The right lung was fixed by inflation with
formaldehyde solution (4%). BAL was performed by injecting and
retracting 3 ml of sterile saline 10 times into the airways. Cells were
counted using a hemocytometer. A differential count was performed on
Wright-stained cytospins.
For histopathological examination, the lungs of animals were sectioned
and stained for myeloperoxidase (MPO; see Ref. 11) and with hematoxylin
and eosin (H+E) using standard procedures. The stained slides were
examined at ×400 magnification. Ten randomly chosen fields of
each lung specimen were blindly scored for number of intensely staining
MPO-positive PMN as described (14).
RT-PCR amplification. Total RNA (2.5 µg) was subjected to first-strand cDNA synthesis using oligo(dT)
primer and Moloney murine leukemia virus reverse transcriptase (15).
Primers for amplification of rat IL-6 cDNA were based on rat cDNA and
were designed with the assistance of a PCR primer design program, PCR
Plan (Intelligenetics, Mountain View, CA). The 5' primer sequence
was ACAGCGATGATGCACTGTCAG. The 3' primer sequence was
ATGGTCTTGGTCCTTAGCCAC. The primers amplified a product of ~339 bp in
length. Restriction enzyme digestion with two enzymes,
Hinf I and
EcoR I, confirmed the identity of the
fragment. Semiquantitative RT-PCR was performed as described (12).
Briefly,
-32P end-labeled
5' primer was used, and PCR conditions were as follows: denaturation at 94°C for 1 min; annealing at 57°C for 1 min;
and polymerization at 72°C for 2 min in a Perkin-Elmer 480 thermocycler. The optimized cycle number was identified at 30 cycles.
Rat peritoneal macrophages elicited with thioglycolate and RAW cells
stimulated in vitro with lipopolysaccharide (LPS) served as a positive
control for rat IL-6 mRNA. The negative control for each set of PCR
reactions contained water instead of DNA template. Fifteen microliters
of the PCR reaction were separated on a 10% polyacrylamide gel. After gel drying and exposure to a PhosphorImager screen (Molecular Dynamics,
Sunnyvale, CA), the relative radioactivity of the bands was determined
by volume integration using laser scanning densitometry. Each gel
contained the same positive control, which permitted normalization of
samples and comparison between gels.
EMSA. EMSA was performed using whole
tissue extracts of lung sections from the experimental groups as
described (12). Binding reactions were performed using 20 µg of
extracted protein and radiolabeled DNA-binding element. The activation
of Stat3 was assessed using the high-affinity serum-inducible element
(hSIE) duplex oligonucleotide that preferentially binds Stat3 and Stat1 (30). EMSA was performed on a 4% polyacrylamide gel as described (6).
The level of Stat3 activation was quantitated using PhosphorImager analysis of gel shift band intensities. Where indicated, EMSA binding
reactions were incubated with antibodies specific for Stat3
or
Stat3
. The Stat3
-specific antibody was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA) and was generated in rabbits against the
COOH-terminal 20 of 21 amino acid residues of murine Stat3.
Stat3
-specific antibody was generated at Charles River
Pharmaservices (Southbridge, MA) by immunizing chickens with the
COOH-terminal 10 amino acid residues of human Stat3
conjugated to
thyroglobulin (12).
In situ hSIE binding assay.
Five-micrometer sections were cut on a cryostat microtome and mounted
on positively charged "Superfrost" slides (Superfrost, Fisher,
PA). Specimens were washed three times in 1× PBS and incubated in
1× PBS and DNase (1 µl/50 ml; Ambion, Austin, TX) for 30 min.
After 30 min of incubation in 2% paraformaldehyde at room temperature,
specimens were washed three times with 1× PBS and then incubated
with the 35S-labeled hSIE duplex
oligonucleotide for 60 min at 30°C and washed again three times in
1× PBS. Specimens were then dehydrated and submerged in Kodak
photographic emulsion (1:1 dilution with deionized H2O). Specimens were exposed to
Kodak film for 5 days, developed in D-19 (Kodak) for 20 min, and
visualized with a Nikon FXA photomicroscope.
Immunohistochemistry. Frozen lung
sections (5 µm) mounted on positively charged Superfrost slides were
washed with 0.05 M PBS and PBS containing 1% bovine serum albumin
(BSA) each three times, incubated in goat serum for 15 min, blotted,
and incubated for 2 h at room temperature with mouse anti-human IL-6
monoclonal antibody, which cross-reacts with rat IL-6 (1:50 dilution;
Endogen, Cambridge, MA) or with a nonspecific isotype-matched control. Specimens were washed three times with BSA, incubated in the
fluorescent-labeled secondary indocarbocyanine-conjugated goat
anti-mouse IgG antibody (1:250 dilution; Jackson ImmunoResearch
Laboratories, West Grove, PA) for 1 h at room temperature, washed three
times in BSA, incubated in Hertz stain for 30 s, and washed three times
in BSA. After air drying, specimens were covered with coverslips and
Gel-mount (Biomeda) and inspected by fluorescent microscopy (Nikon
FXA photomicroscope).
Statistics. Unless otherwise
indicated, data are presented as means ± SE. Comparisons of means
were performed using ANOVA followed by comparison of individual pairs
of means using the Scheffé's test. Both tests are contained
within the StatView 4.1 program (Abacus Concepts, Berkeley, CA).
 |
RESULTS |
IL-6 mRNA and protein are increased in the lungs of
rats subjected to HS. We have previously reported that
rats subjected to our HS protocol demonstrated evidence of acute lung
damage with increased PMN infiltration and elevated wet-to-dry ratio (15). To determine if the proinflammatory cytokine IL-6 was expressed
locally in the lung in HS where it may contribute to the PMN
recruitment and lung damage, we determined IL-6 mRNA levels and IL-6
protein expression in the lungs of rats subjected to HS. Using RT-PCR
with end-labeled 5' primer, we performed semiquantitative RT-PCR
to measure levels of IL-6 mRNA in the lungs of normal rats and rats
subjected to the HS or sham protocol (Fig.
1). Lungs of normal rats demonstrated a low
level of an amplified IL-6 product. Sham animals demonstrated a 5.1- to
11.2-fold increase in amplified IL-6 product over normal animals
(P < 0.01). The amount of amplified IL-6 fragment was further increased 15.4- to 26.2-fold in the shock
animals over normal animals (P < 0.001) and 2.4- to 3-fold compared with sham control animals
(P
0.01) for each group examined (compensated/4 h, compensated/8 h, decompensated/4 h, decompensated/8 h). Comparison of compensated shock groups (1 h of ischemia)
with decompensated shock groups (2.5 h of ischemia) at
identical time points of death after resuscitation to determine the
influence of the duration of the ischemic phase of shock on IL-6 mRNA
levels revealed a 43% increase in IL-6 mRNA levels after 2.5 h of
ischemia and resuscitation (decompensated shock) and death
after 4 h versus 1 h of ischemia and resuscitation (compensated
shock) and death after 4 h (P = 0.01).
Similarly, we observed a 69% increase in IL-6 mRNA levels in animals
subjected to 2.5 h of ischemia and resuscitation (decompensated
shock) and death after 8 h versus animals subjected to 1 h of
ischemia and resuscitation (compensated shock) and death after
8 h (P = 0.03). Comparisons made to
determine the influence of the duration of the resuscitation phase on
IL-6 mRNA levels revealed no difference in IL-6 mRNA levels 4 h after resuscitation compared with 8 h after resuscitation in either group.

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Fig. 1.
Semiquantitative RT-PCR of interleukin (IL)-6 RNA from lungs of rats
subjected to resuscitated hemorrhagic shock (HS). Reactions were
performed using total RNA (2.5 µg) from the lungs of normal animals
or animals subjected to compensated or decompensated HS and killed 4 or
8 h after resuscitation. RT-PCR products from normal animals (NL), sham
control animals (Sham), and shock animals (Shock) were separated on
polyacrylamide gels (A). Gels were
dried, and the bands were identified using a PhosphorImaging screen.
The position of the IL-6 amplified product (339 bp) is shown on
right.
B: radioactive signal within the
339-bp amplified fragment was quantitated by PhosphorImager analysis,
and the mean ± SE for normal animals (NL; open bar), sham control
animals (open bars), and shock animals (filled bars) was plotted.
Differences between each shock and sham group were significant
(P 0.01 for each group). Levels of
amplified IL-6 were increased 43% at 4 h after resuscitation in
animals subjected to decompensated vs. compensated
(* P = 0.01) and 69% at 8 h
after resuscitation in animals subjected to decompensated vs.
compensated HS (# P = 0.03).
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To establish that increased IL-6 mRNA production in the lungs in HS is
accompanied by increased IL-6 protein, we performed immunohistochemistry (Fig. 2). IL-6 protein
was increased in shock animals compared with sham animals, with the
most prominent staining occurring in cells lining the bronchioles. IL-6
protein was also observed in alveolar macrophages, but staining was
less intense. The lungs of normal animals did not demonstrate specific
staining for IL-6 protein (data not shown).

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Fig. 2.
Immunofluorescent staining of rat lungs for IL-6. Frozen sections of a
representative lung from a shock animal (HS 100×, HS 400×,
and HS-IgG 400×) and a sham animal (Sham 400×) were
incubated with IL-6 monoclonal antibody (HS 100×, HS 400×,
and Sham 400×) or with an isotype control antibody (HS-IgG
400×) followed by incubation with indocarbocyanine-conjugated
secondary antibody. Sections were visualized using an immunofluorescent
microscope and photographed at the indicated magnification.
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Increased expression of lung IL-6 in HS requires both
the ischemic and resuscitation phases and occurs rapidly after
resuscitation. To determine whether or not
resuscitation from HS was required for the induction of IL-6 mRNA and,
if so, to examine the kinetics of IL-6 mRNA production during the
resuscitation phase, we measured levels of IL-6 mRNA at the end of the
ischemic phase and at 1, 2, 3, and 4 h after resuscitation (Fig.
3, A and
B). IL-6 mRNA was not increased in
shock animals at the end of the ischemic phase compared with sham
animals; however, levels of IL-6 mRNA were increased over sham animals
after resuscitation at all time points examined, with the peak
occurring 1 h after resuscitation and demonstrating a 5.9-fold increase
over sham levels (P
0.04). To
confirm the failure of ischemia alone to induce IL-6 production above sham levels, we examined the lungs of animals for IL-6 mRNA production in two additional HS protocols in which animals were subjected to unresuscitated compensated and unresuscitated irreversible HS. With semiquantitative IL-6 RT-PCR, there was no difference in IL-6
mRNA levels between shock and sham animals regardless of the severity
of shock (Fig. 3C). Even in animals
subjected to irreversible HS, an ischemic injury that would have had a
100% lethality, levels of IL-6 mRNA were not elevated over levels
found in sham animals.

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Fig. 3.
Semiquantitative RT-PCR of IL-6 mRNA from lungs of rats subjected to
unresuscitated and resuscitated HS. RT-PCR reactions were performed
using total RNA (2.5 µg) from the lungs of animals subjected to the
decompensated (2.5 h of ischemia) HS or sham protocol without
resuscitation (0 h) or 1 (R1), 2 (R2), 3 (R3), or 4 (R4) h after
resuscitation. Reaction products were separated on polyacrylamide gels.
Gels were dried and exposed to a PhosphorImaging screen and developed
using a PhosphorImager (A). The
location of the amplified IL-6 fragment (339 bp) is indicated on
right. In
B, the radioactive signal within the
amplified fragment was quantitated using scanner laser densitometry and
ImageQuant software, and the mean ± SE of the sham groups (open
bars) and shock groups (filled bars) was plotted. The differences
between each resuscitated shock and sham group were significant
(P 0.01 for each comparison). There
was no difference between the mean of the shock and sham groups without
resuscitation (0 h; P = 0.6). The mean
of the shock groups at 1, 2, 3, and 4 h was significantly increased
over the unresuscitated (0 h) group (P 0.04). In C, RT-PCR reactions were
performed using total RNA (2.5 µg) from the lungs of animals
subjected to compensated (C; 1 h of ischemia) or irreversible
(I; 3.5 h of ischemia) shock protocol without resuscitation
(filled bars) or to the corresponding sham procedure (open bars).
Reaction products were separated on polyacrylamide gels and quantitated
as above. Values shown are means ± SE of each group
(n = 4). There were no differences
between shock and sham groups within either set
(P = 0.7).
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Stat3 activation in HS requires the resuscitation
phase and exhibits similar kinetics to IL-6 induction.
IL-6 signals through activation of Stat3. We previously demonstrated
activation of STAT proteins, particularly Stat3, in the lungs of
animals subjected to resuscitated HS (13). It is not known if the
resuscitation phase is necessary for the activation of Stat3 or the
time course of the activation of Stat3 after resuscitation. Protein
extracts of lungs from shock animals killed at the end of the 2.5-h
ischemic phase (decompensated shock) did not demonstrate increased
Stat3 activity compared with sham animals with the use of EMSA gel
shift (Fig. 4,
A and
B). In contrast, we found that Stat3
activity was increased after resuscitation at all time points examined,
peaking 1 h after resuscitation with a level of activity 9.2-fold
greater than sham controls (P
0.02). Similar to IL-6 mRNA levels, there was no difference in Stat3
activity between shock and sham animals in unresuscitated compensated
(1 h of ischemia) and irreversible (3.5 h of ischemia)
HS (Fig. 4C).

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Fig. 4.
Stat3 activation in the lung after unresuscitated and resuscitated HS.
In A, protein extracts were obtained
from frozen sections of lungs from animals subjected to decompensated
(2.5 h of ischemia) HS without resuscitation (R0) or
decompensated HS followed by 1 (R1), 2 (R2), 3 (R3), or 4 (R4) h of
resuscitation or to the corresponding sham procedure. Extracts (20 µg) were used in electrophoretic mobility shift assays (EMSA) with
the radiolabeled high-affinity serum-inducible element (hSIE) duplex
oligonucleotide. The positions of the serum-inducible factor (SIF)-A
(Stat3 homodimer), SIF-B (Stat1/Stat3 heterodimer), and SIF-C (Stat1
homodimer) complexes are indicated on
right. In
B, the SIF-A band was quantitated by
PhosphorImager analysis, and the mean ± SE for shock animals
(filled bars) and sham control animals (open bars) was plotted.
Differences between each resuscitated shock and sham group were
significant (P < 0.01 for each
comparison). There was no difference between the mean of the shock and
sham groups without resuscitation (0 h;
P = 0.4). The mean of the shock groups
at 1, 2, 3, and 4 h was significantly increased over the unresuscitated
(0 h) group (P < 0.02). In
C, EMSA was performed using extracts
from the lungs of rats subjected to compensated (C; 1 h of
ischemia) or irreversible (I; 3.5 h of ischemia) HS
(filled bars) without resuscitation or to the corresponding sham
procedure (open bars). The SIF-A band was quantitated by PhosphorImager
analysis as above, and the mean ± SE for each group was plotted.
There was no difference between the mean of the shock and sham groups
in any of these unresuscitated animals
(P = 0.5, 0.7, and 0.8). In
D, extracts of lungs from a
representative shock animal were incubated with antibodies specific for
Stat3 or Stat3 or with both antibodies. The positions of the
SIF-A, -B, and -C complexes, the supershifted complex, and the residual
SIF-A complex after supershift of Stat3 and Stat3 (Stat3 ) are
indicated.
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Three distinct isoforms of Stat3 have been identified, Stat3
,
Stat3
, and Stat3
. Stat3
(92 kDa) is the predominant isoform expressed in most cells (2). Stat3
(83 kDa) arises from alternative splicing of the Stat3 gene transcript, resulting in a 50-nucleotide deletion at the 3' end of the open reading frame of Stat3
(6, 27). The third isoform, Stat3
, is a 72-kDa protein identified in
granulocyte colony-stimulating factor-activated mature neutrophils and
is derived from Stat3
by posttranslational modification involving proteolysis (A. Chakraborty and D. J. Tweardy, unpublished
observation). Stat3
and Stat3
each are supershifted
by specific antibodies; no antibody is available that can supershift
only Stat3
. We added antibody specific to either Stat3
or
Stat3
or both antibodies to binding reactions containing
radiolabeled hSIE duplex oligonucleotide to determine which of these
Stat3 isoforms are activated in the lungs of shock animals. Addition of
either Stat3
-specific antibody or Stat3
-specific antibody each
supershifted a portion of the serum-inducible factor
(SIF)-A complex. Addition of both antibodies together
supershifted a greater portion than either alone but did not supershift
all of the SIF-A complex (Fig. 4D).
The remaining SIF-A complex has the mobility of Stat3
(Fig.
4D). Thus all isoforms of Stat3 were
activated in the lung in HS. The activation of Stat3
corresponds to
our finding of PMN recruitment into the lung after resuscitated HS.
To determine the cellular site of Stat3 activation in the lungs after
HS, we performed an in situ binding assay using frozen lung sections
and 35S-radiolabeled hSIE duplex
oligonucleotide (Fig. 5). Examination of
the shock lung specimens after incubation with the hSIE probe was most
notable for binding of labeled probe within alveolar cells. Specimens
from sham animals did not demonstrate binding of the probe.

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Fig. 5.
In situ hSIE binding assay of shock and sham lung. Frozen sections of
uninflated lung from a representative decompensated shock animal (2.5 h
ischemia followed by resuscitation and death at 4 h;
B) or the corresponding sham animal
(A) were incubated with
35S-labeled hSIE duplex
oligonucleotide and developed as described in MATERIALS AND
METHODS. Photographs of bright-field views (magnification
×400) are shown. Prominent binding of radiolabeled hSIE is
indicated by arrows in B.
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Intratracheal instillation of IL-6 into the lungs
resulted in PMN infiltration and lung damage. IL-6 has
been recently shown to cause tissue accumulation of PMN through the
induction of chemokines such as IL-8 (26). To determine whether or not
IL-6 protein alone is sufficient to cause PMN accumulation in the lungs
of rats, we instilled IL-6 protein (300 ng) by syringe injection through an intratracheal tube. BAL was performed on IL-6-treated animals, and BAL cellularity was determined at 2, 4, 8, 12, 24, and 48 h (Fig. 6). The BAL cellularity was
increased at all time points examined and peaked at 4 h, achieving a
5.6-fold increase compared with control saline-treated animals
(P = 0.03). Examination of
Wright-Giemsa stain of BAL cells at 4 h after IL-6 instillation revealed that 45 ± 8% of the isolated cells were PMN.

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Fig. 6.
Effect of IL-6 instillation (300 ng) on bronchoalveolar lavage (BAL)
cellularity. BAL was performed at the time points indicated after
instillation of IL-6 (n = 4; filled
bars) or saline control (n = 4; open
bars). Cells in the BAL fluid were counted, and the means ± SE were
plotted. Animals that received IL-6 showed a significant increase in
cell number in the BAL fluid compared with animals that received saline
at 4, 8, 12, 24, and 48 h (* P 0.03).
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To examine IL-6-induced PMN recruitment into the lung in greater
detail, lungs of rats were sectioned and stained for MPO and with H+E.
Examination of MPO-stained sections of lungs of rats that received IL-6
revealed many MPO-positive cells compared with animals receiving the
saline control (Fig. 7,
A and
C). To quantitatively assess the
increase in PMN recruitment into the interstitium and alveoli of lungs
of animals receiving IL-6 or saline control, MPO-stained slides from
lung cross sections were examined at ×400 magnification at 2, 4, 8, 12, 24, and 48 h after IL-6 instillation. Ten random fields of each
lung specimen were scored blindly for number of intensely staining
MPO-positive PMN. The scores were pooled, and the means ± SE were
calculated (Fig. 8A).
Lungs of IL-6-treated animals demonstrated an increase in MPO-positive
PMN in both the interstitium and alveoli at 4, 8, 12, 24, and 48 h
compared with saline-treated animals, with the peak of PMN accumulation
occurring from 4 to 12 h. To determine the minimum amount of IL-6
necessary to result in lung PMN accumulation, animals were examined 4 h
after receiving 10, 30, 100, and 300 ng of IL-6 by tracheal
instillation (Fig. 8B). There was a
linear increase in MPO-positive cells
(r = 0.74;
P < 0.03) with increasing doses of
IL-6. Instillation of as little as 30 ng of IL-6 increased PMN
infiltration by 7.1-fold over animals receiving saline control (P = 0.01).

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Fig. 7.
Photomicrographs of sections of inflated and formaldehyde-fixed lungs.
Lungs from saline-treated control animals
(A and
B) or animals treated with IL-6 (300 ng; C and
D) were stained for myeloperoxidase
(MPO; A and
C) or with hematoxylin and eosin
(H+E; B and
D) and examined at ×400
magnification.
|
|

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Fig. 8.
Effect of IL-6 instillation on accumulation of MPO-positive
polymorphonuclear neutrophils (PMN) in interstitium and alveolar space.
In A, IL-6 (300 ng) was instilled into
the lungs of animals (n = 4), and the
animals were killed at the time points indicated. Lungs were inflated,
fixed in formaldehyde, sectioned, and stained for MPO. MPO-stained
slides were examined at ×400 magnification. Ten random fields of
each lung specimen were blindly scored for number of intensely
MPO-positive PMN. Scores were pooled, and the means ± SE
were plotted. Means were increased compared with normal lung at 4, 8, 12, and 24 h (* P 0.03). In
B, IL-6 was instilled into the lungs
of animals (n = 4) at the doses
indicated. Lungs were harvested at 4 h, sectioned, and stained for MPO.
MPO-positive PMN were quantitated and plotted as above. There was a
linear increase in MPO-positive cells with increasing dose
(r = 0.74;
P < 0.03) 4 h after IL-6
instillation. There was a significant increase in MPO-positive PMN in
the interstitial and alveolar spaces after administration of 30, 100, and 300 ng IL-6 (P 0.01).
|
|
PMN migration into alveolar spaces is not always accompanied by injury
of the alveolar capillary wall in vivo and by pulmonary edema (8, 10,
19). To determine if IL-6-mediated PMN infiltration caused lung damage,
we examined the lungs histologically and measured lung wet-to-dry
ratio. The accumulation of PMN was accompanied by histological signs of
lung injury with widened interstitium and interstitial and alveolar
edema (Fig. 7). The wet-to-dry ratio increased in the IL-6-treated
animals at 4, 8, 12, and 24 h compared with control animals (Fig.
9) and peaked at 4 h, achieving a 45% increase over normal lung (P = 0.03).
To determine the minimum amount of IL-6 necessary to cause an increase
in the wet-to-dry ratio, animals were examined 4 h after receiving 10, 30, 100, and 300 ng of IL-6 by tracheal instillation (Fig.
8B). We observed a linear increase
in the wet-to-dry ratio (r = 0.82, P = 0.001) with increasing doses of
IL-6. Instillation of as little as 30 ng of IL-6 increased the
wet-to-dry ratio by 11% compared with animals receiving the saline
control (P = 0.04).

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Fig. 9.
Effect of IL-6 instillation on lung wet-to-dry ratio. In
A, IL-6 (300 ng) was intratracheally
instilled into the lung of rats (n = 5; filled bars) by syringe injection. Control rats
(n = 6; open bars) received saline
control alone. Animals were killed at the times indicated, and the left
lung was removed for determination of wet-to-dry ratio. Data presented
are means ± SE. The wet-to-dry ratio was significantly increased in
the IL-6-treated animals compared with control animals at 4, 8, 12, and
24 h (* P = 0.03, P = 0.04, P = 0.02, and
P = 0.03, respectively). In
B, IL-6 at the doses indicated was
instilled into the lungs of animals
(n = 5) as described above.
Animals were killed at 4 h, and the left lung was removed for
determination of wet-to-dry ratio. The wet-to-dry ratio increased with
increasing IL-6 dose (r = 0.82;
P = 0.001). A significant increase in
lung wet-to-dry ratio was detected after administration of an IL-6 dose
at 30, 100, and 300 ng (* P 0.03).
|
|
 |
DISCUSSION |
In this study, we demonstrated that IL-6 is locally produced in the
lungs of rats subjected to resuscitated HS. Levels of IL-6 mRNA
increased with increasing duration of the ischemic phase of
resuscitated shock. IL-6 protein was located in bronchoepithelial cells
and in alveolar macrophages. Both the ischemic and resuscitation phases
were required to increase IL-6 mRNA levels above those of sham control
animals. We found increased activation of Stat3 compared with sham
animals only in the lungs of rats subjected to resuscitated HS. IL-6
mRNA expression and Stat3 activation demonstrated similarly rapid
kinetics, with levels of both peaking 1 h after the onset of
resuscitation. Intratracheal instillation of IL-6 into the lungs of
normal rats caused PMN infiltration and lung damage characterized by
pulmonary edema. Our results indicate that IL-6 alone can induce lung
injury and suggest that the production of IL-6 may contribute to ARDS
after HS.
We previously observed that the lungs of animals subjected to 2.5 h of
ischemia followed by resuscitation and death at 8 h demonstrated the greatest increase in PMN infiltration and wet-to-dry ratio compared with sham animals (15). PMN accumulation (48 ± 4.9 MPO-positive PMN/×400 field) and wet-to-dry ratio (6.2 ± 0.3)
in these animals were similar to PMN accumulation (61 ± 12 and 73 ± 11) and wet-to-dry ratio (6.05 ± 0.25 and 5.69 ± 0.28) observed in animals 4 and 12 h, respectively, after IL-6 instillation (300 ng).
The pathogenesis of ARDS is thought to involve a diffuse alveolar
capillary injury. Activated neutrophils are the major cellular elements
that mediate acute inflammation and have been implicated in the
pathogenesis of the microvascular injury that occurs in ARDS (29). The
results from our study indicate that the presence of IL-6 alone can
lead to pulmonary recruitment of PMN and pulmonary edema. Thus
increased IL-6 production in the lung of patients suffering from HS may
be an additional mechanism that contributes to PMN-induced lung injury
and possibly ARDS.
IL-6 is a multifunctional cytokine that is part of the acute
inflammatory response. IL-6 stimulates neutrophilia and thrombopoiesis and induces the synthesis of acute-phase proteins (16, 17, 25).
Sustained elevations of IL-6 in the plasma and BAL of patients suffering from ARDS have been demonstrated and negatively correlated with disease outcome and patient survival (5, 21). Although the acute
phase of ARDS involves PMN recruitment and PMN-mediated tissue injury,
there has been little evidence to suggest that IL-6 contributes
directly to these processes. In a previous study, overexpression of
IL-6 in the pancreas of transgenic mice promoted local inflammation
(4). However, our study is the first to demonstrate that the presence
of IL-6 in the lung alone is sufficient to promote PMN infiltration and
pulmonary edema.
In contrast to our findings of acute inflammation after IL-6
instillation, several studies have shown that IL-6 has
anti-inflammatory effects. Exposure of cells to IL-6 or intraperitoneal
injection of IL-6 was demonstrated to inhibit tumor necrosis factor-
and IL-1
production and to protect against LPS toxicity in vitro (1)
and in vivo, respectively (3). IL-6 also decreased neutrophil infiltration into the lung in a mouse model of chronic pneumonitis elicited by fungal exposure (9). In addition, intraperitoneal injection
of IL-6 antibody and intratracheal administration of IL-6 (5,000 units)
decreased BAL cellularity and peripheral blood neutrophilia. However,
these models of LPS toxicity and chronic inflammation differ
substantially in either the nature or duration of the insult from the
acute inflammation occurring in our models of HS and IL-6 instillation.
Recent studies have demonstrated that IL-6 contributes to tissue
recruitment of PMN by induction of chemokines (26); however, little is
known about the mechanisms of IL-6-mediated local chemokine production.
The promoter region of IP-10, a member of the C-X-C subgroup of
chemokines, contains an interferon-stimulated response element capable of binding STAT proteins (23). Our
findings in HS that IL-6 is produced in the lung and Stat3 is locally
activated raise the possibility that IL-6-activated Stat3 may
contribute to local chemokine production through transcriptional
activation of chemokine genes such as IL-8.
The amount of IL-6 instilled into the lung of rats in our studies
ranged from 10 to 300 ng. We observed a significant increase in lung
wet-to-dry ratio and PMN infiltration after instillation of 30, 100, and 300 ng. In patients with ARDS, levels of IL-6 in BAL fluid exceeded
20 ng/ml, and IL-6 levels in plasma exceeded 1.5 ng/ml (22). Levels of
IL-6 in lung tissue in humans or animals have not been reported. Where
simultaneous lung tissue and plasma levels of a cytokine have been
determined, such as IL-1
levels in steroid-induced
Pneumocystis carinii pneumonia, a
lung-to-plasma gradient greater than 60 was found (24). If such a
gradient occurred for IL-6, the lung tissue levels of IL-6 in patients with ARDS would exceed 90 ng/ml. Consequently, the doses used in our
studies may very well reflect lung tissue levels of IL-6 achievable in
acute inflammatory states such as early ARDS.
To determine if IL-6 administered into the lungs results in systemic
PMN activation and PMN-mediated damage to other organs such as the
liver, we measured serum levels of liver enzymes, including alanine
aminotransferase, aspartate aminotransferase, lactate
dehydrogenase, and alkaline phosphatase. Liver enzymes were within normal limits in all IL-6 instillation groups (data not
shown). In addition, histological examination of representative cross
sections of liver did not demonstrate increased PMN infiltration (data
not shown). Taken together, these results indicate that IL-6 delivered
intratracheally leads to lung injury as a result of its local effects
on PMN recruitment and activation and not as a consequence of a
systemic effect of IL-6 on PMN.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
GM-53789 and CA-72261 and by the Deutsche Forschungsgemeinschaft HI
614/1-1 and Ka 1270/1-1.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: D. J. Tweardy, W1052 Biomedical Science
Tower, Univ. of Pittsburgh Cancer Institute, 200 Lothrop St.,
Pittsburgh, PA 15213.
Received 30 January 1998; accepted in final form 13 May 1998.
 |
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