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1 Vascular Biology Program, Peritonitis induced by cecal ligation and
puncture (CLP) produces a systemic inflammatory response that can be
largely mitigated by pretreatment of the animals with
lipopolysaccharide (LPS tolerance). Although cells of
myeloid origin and endothelial cells have been shown to contribute to
the development of LPS tolerance, little is known regarding the
potential role of parenchymal cells in this phenomenon. The major aim
of the present study was to assess whether cardiac parenchymal cells
(myocytes) contribute to the development of LPS tolerance. Six hours
after induction of CLP rats were neutropenic and acidotic, the
myocardium contained a leukocyte infiltrate [myeloperoxidase
(MPO) activity was increased], and myocardial contractile
function was impaired (left ventricular developed pressure was
decreased). In animals that were pretreated with LPS these
manifestations of sepsis were largely reversed. Further studies focused
on the responses of cardiac myocytes to CLP and whether myocytes
contributed to the development of LPS tolerance. Myocytes were isolated
from rat hearts 6 h after induction of CLP. These myocytes
1) exhibited an impaired ability to
shorten in response to pacing, 2)
contained the nuclear transcription factor NF-
intercellular adhesion molecule 1; polymorphonuclear-myocyte
adhesive interactions; nuclear transcription factor- SEPSIS IS A GENERALIZED inflammatory response which
involves organ systems remote from the locus of the initial infectious insult. The release of endotoxin [lipopolysaccharide (LPS)] from bacteria is generally believed to be the initial event in the development of sepsis (25). LPS activates vascular endothelium and
induces the surface expression of adhesion molecules (e.g., proadhesive
phenotype) (5, 9, 17). LPS also activates inflammatory cells of the
myeloid lineage that subsequently amplify the inflammatory response by
releasing various cytokines, such as tumor necrosis factor- The proinflammatory effects of LPS (and cytokines) requires activation
of the nuclear transcription factor NF- Paradoxically, pretreatment of cells or animals with LPS renders them
resistant to a subsequent LPS challenge (LPS tolerance) (33). For
example, LPS-induced TNF- Numerous studies attempting to unravel the mechanisms involved in the
development of tolerance to the proinflammatory effects of LPS have
focused on cells of myeloid origin, i.e., macrophages and monocytes
(33), and, to some extent, endothelial cells (15). There is no
information available on the potential role of parenchymal cells in
tolerance development with respect to inflammation. In the present
study, we used an animal model of peritonitis, cecal ligation and
perforation (CLP), to assess the role of the myocardial parenchyma
(myocytes) in the development of LPS tolerance. To detect critical
early events that occur after CLP and how they are altered during the
development of LPS tolerance, we measured relevant variables 6 h after
induction of CLP. With the use of this model, we show that myocardial
myocytes contribute to the development of LPS tolerance and that the
nuclear transcription factor NF- Animal Studies
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INTRODUCTION
METHODS
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DISCUSSION
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B in their nuclei,
3) increased their surface levels of
intercellular adhesion molecule-1 (ICAM-1), and
4) were hyperadhesive for
neutrophils. All of these events did not occur in myocytes obtained
from animals that were pretreated with LPS before induction of CLP.
These findings indicate that LPS tolerance can be induced in myocytes
with respect to polymorphonuclear leukocyte adhesion, presumably by an
inability of CLP to mobilize NF-B to the myocyte nuclei and,
thereby, preventing an increase in surface levels of
ICAM-1.
B; cecal
ligation and perforation; lipopolysaccharide tolerance
INTRODUCTION
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INTRODUCTION
METHODS
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DISCUSSION
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(TNF-) and interleukin-1
(IL-1) (20, 33). This systemic
inflammatory cascade results in polymorphonuclear leukocytes (PMN)
sequestration in the lungs and adhesion to the endothelium of various
systemic organs, e.g., the heart (3, 18). Subsequent PMN
extravasation can lead to vascular dysfunction (protein leakage and
edema formation) as well as parenchymal cell dysfunction. The
importance of PMN-endothelial cell adhesive interactions in the
development of organ dysfunction is exemplified by the protection
against sepsis- or endotoxemia-induced tissue injury offered by
monoclonal antibodies directed to adhesion molecules on PMN (CD18) (10,
28, 29).
B in both endothelial cells
and cells of myeloid origin (2, 23). In quiescent cells, NF-B
resides in the cytoplasm and is associated with an IB inhibitory protein that prevents its translocation to the nucleus.
LPS, TNF-, IL-1, as well as other cytokines induce a series of
cellular events that result in the disassociation of NF-B and IB,
thus allowing NF-B to move into the nucleus. Activation and
translocation of NF-B to the nucleus results in the transactivation
of a variety of genes that contribute to the systemic inflammatory
response. In general, myeloid cells synthesize and secrete cytokines
(e.g., TNF-, IL-1), whereas endothelial cells increase their
surface expression of adhesion molecules [intercellular adhesion
molecule 1 (ICAM-1), E-selectin]. These responses facilitate
PMN-endothelial cell adhesive interactions, which subsequently lead to
dysfunction of the affected organs.
secretion from a monocytic cell line can
be substantially reduced by pretreating the cells with LPS (32). In
rats, LPS-induced mortality and PMN accumulation within the heart can
be largely ameliorated by pretreating the animals for several days with
sublethal doses of LPS (3). LPS tolerance has also been demonstrated in
humans (1). A pretreatment regimen with a neutral LPS derivative
(monophosphoryl lipid A) decreased the systemic response (fever and
tachycardia) to a subsequent LPS challenge. The growing body of
evidence indicating that LPS tolerance can be induced in cells,
animals, and humans has prompted the suggestion that "LPS tolerance
may be exploited for prophylaxis of severe sepsis in patients at
risk" (33).
B plays an important role.
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CLP was then carried out on day 3 in
both groups as previously described (13). Briefly, the animals were
anesthetized 6 h after the LPS intervention on day
3 with halothane. Subsequently, the internal carotid
artery and external jugular vein were cannulated under sterile
conditions. These lines were tunneled subcutaneously to the back of the
neck where they were attached to a swivel device. Animals then
underwent CLP. After laparotomy, a ligature was placed around the cecum
immediately distal to the ileocecal valve. With the use of unipolar
diathermy, the cecum was then opened along a segment of 0.5 cm at the
antimesenteric border of the gut. After recovery from anesthesia, the
following infusions were commenced: normal saline at 300-400
ml · kg
1 · day1
and fentanyl at 400 µg · kg1 · day1.
Heparin (400 U · kg1 · day1)
was administered to assure the patency of intravascular lines. Water
and laboratory chow were available ad libitum. Sham-operated rats were
used as controls for CLP. These animals were instrumented in a similar
fashion to the endotoxin or saline pretreated group but did not undergo
laparotomy. This animal study was reviewed and approved by the
University of Western Ontario Committee on Animal Care.
Systemic variables. Mean arterial pressure was measured via the arterial catheter which was connected to a pressure transducer (Inflow, Baxter, Toronto, ON) and recorded with a multichannel, amplifier-recording system (Hewlett-Packard 78353A). Six hours after the CLP induction, arterial blood samples were withdrawn for complete cell count and analysis of blood gases and lactate.
Myocardial inflammation. As an index of PMN infiltration, myeloperoxidase (MPO) activity in the heart tissue was determined, as previously described (8). Briefly, after euthanasia, hearts were excised and placed in phosphate buffer, pH 7.4, at 10% wt/vol and homogenized. One milliliter of the homogenates was then adjusted to a total volume of 10 ml with phosphate buffer and centrifuged at 6,000 g for 20 min at 4°C. The pellet was rehomogenized and sonicated for 10 s in 1 ml of 50 mM acetic acid (pH 6.0) containing 0.5% CETOH detergent. Twenty microliters of the prepared samples were used in reactions for MPO activity determined spectrophotometrically (650 nm) by measuring hydrogen peroxide-dependent oxidation of 3,3',5,5'-tetramethylbenzidine. The results were expressed as absorbance per gram of tissue.
Myocardial function.
Heart contractile function was assessed using the Langendorff isolated
heart preparation (27). Briefly, after anesthesia and heparinization,
the heart was rapidly excised, the ascending aorta was cannulated, and
retrograde perfusion (10 ml/min) was initiated with Krebs-Henseleit
solution containing (in mM) 120 NaCl, 4.8 KCl, 1.2 KH2PO4,
1.2 MgSO4, 1.25 CaCl2, 25 NaHCO3 and 11 glucose; 37°C
saturated with 95% O2-5%
CO2 gas mixture. To assess
contractile function, a water-filled latex balloon was inserted through
the left atrium into the left ventricle and connected to a pressure
transducer. This balloon was then adjusted to a left ventricular
end-diastolic pressure (LVEDP) of 5 mmHg. The heart was paced at 300 beats/min and allowed to equilibrate for 40 min. Left ventricular
developed pressure (LVDP) and its first derivatives
(+dP/dt and
dP/dt) were monitored and
recorded on a chart recorder (Gould 8188, Gould). After baseline
measurements, LVDP and LV volume-LVEDP preload relationships (LVEDP
from 5 to 20 mmHg) were obtained.
Myocyte Studies
Ventricular myocytes were isolated as previously described (7). The heart was perfused through the aorta for 5 min with Ca2+-free buffer (buffer A) containing (in mM) 120 NaCl, 1 MgCl2, 5.4 KCl, 0.33 NaH2PO4, 10 HEPES, and 10 glucose; pH 7.4. Subsequently, the buffer was changed to one containing 2 mg/ml collagenase (Sigma, type 1) and 0.1 mg/ml protease (Sigma, protease type XIV) and the heart perfused for 12 min in a recirculating manner. The protease-containing buffer was washed out for 2 min with buffer A containing 0.2 mM CaCl2. The heart was removed from the perfusion cannula and chopped with scissors in 15 ml of buffer containing 0.2 CaCl2 and 25 mM KCl. The tissue was incubated at 37°C for 15 min, filtered through a 210-mesh nylon screen and the filtrate was centrifuged gently for 45 s. The buffer was aspirated off and the cells were resuspended in 50 ml of buffer containing 0.5 mM CaCl2 for 10 min. The cells were centrifuged again and the buffer aspirated off. The final cell pellet was suspended in buffer A containing 1 mM CaCl2. The cells were diluted to a concentration of ~100,000 cells/ml.Myocyte function. The isolated myocytes were transferred to a Biophysica chamber (Biophysica Technologies, Baltimore MD) and seeded on the thermoregulated (36°C) stage of a Zeiss Axiovert 35 inverted micoscope. The cells were continuously superfused (bath volume 1 ml, flow rate 1 ml/min) with HEPES solution containing 1.8 mM CaCl2 and field-stimulated with bipolar platinum electrodes with pulses of 5 ms duration at a frequency of 0.5 Hz using a Grass SD6 stimulator. Voltage was set at 4 V above threshold (12-25 V). Myocytes were allowed to stabilize for 30 min before pacing. Myocyte contractile activity was assessed by measuring their percent shortening. The cells were illuminated using a red filter (>600 nm) and the images were analyzed using a video edge-detector system (Colorado Video, Boulder, CO). The field was first calibrated at two distances with a graduated microscopy slide and contractions of each myocyte were measured along the long axis. Each estimate of myocyte shortening represents the average of five consecutive contractions.
Myocyte-PMN adhesive interactions. Rat PMNs were isolated from rat blood using a two-component step gradient centrifugation approach using NIM-2 (Cardinal Association, Santa Fe). This procedure yields a PMN population that is 95% viable (trypan blue exclusion) and 90% pure (acetic acid-crystal staining).
Myocytes were plated in six-well plates and allowed to stabilize for 30 min (37°C) and then PMN added at a ratio of 1 myocyte to 10 PMNs. After 15 min of coincubation, PMN-myocyte adhesive interactions were assessed microscopically under static conditions with occasional shaking to differentiate adherent PMN from those making passive contact with myocytes. The number of PMN attached to myocytes were counted on at least 25 randomly chosen myocytes. Three experimental approaches were used in interacting myocytes with PMN: 1) myocytes and PMN from CLP animals, 2) myocytes from sham animals and PMN from CLP animals, and 3) myocytes from CLP animals and PMN from sham animals.Myocyte ICAM-1 expression. Isolated rat heart myocytes were fixed in 3% paraformaldehyde for 30 min at room temperature. After being washed with PBS, myocytes were treated with mouse anti-rat ICAM-1 antibody (1A29, 20 µg/ml) in the presence of 2% BSA for 1 h at room temperature. Subsequently, myocytes were washed and incubated with a biotinilated horse anti-mouse antibody (Pierce) (dilution 1:300 in 2% BSA-PBS solution). The ICAM-1 antibody binding efficiency was assessed using ImmunoPure peroxidase ABC staining kit (Pierce) with 3,3',5,5'-tetramethylbenzidine as a substrate. Cells (5 × 103) in total volume of 200 µl were used for the experiments. After incubation of myocytes for 5 min in substrate solution, they were centrifuged and 150 µl of supernatant was collected. The colorimetric reaction was stopped by adding 50 µl of 0.2 N H2SO4 and absorbance was read at 405 nm in microplate reader.
Myocyte NF-B activation.
Isolated myocytes were washed with cold PBS, centrifuged, and
homogenized on ice in a Dounce homogenizer in a total volume of 5 ml of
buffer
E+
[0.3% Nonidet P-40 (NP-40), 10 mM Tris (pH 8.0), 5 mM
MgCl2, 5 mM dithiothreitol (DTT),
0.3 M sucrose, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM
PMSF]. The homogenates were centrifuged at 500 g for 5 min, at 4°C. The
supernatant was then removed, the pellets (nuclei) were resuspended in
5 ml of buffer E
(buffer E+
without Nonidet P-40) by several strokes in a Dounce homogenizer and
sucrose concentration in homogenates was adjusted to 1.65 M by adding
2.32 g of a solid sucrose and allowing it to dissolve by gentle
inversion of the tubes at 4°C. Subsequently, the homogenates were
layered on a 3-ml sucrose cushion (2 M sucrose, 2 mM
MgCl2, 10 mM
Tris · HCl, pH 7.5) and centrifuged at 25,000 g for 1 h, at 4°C. The pellet
fractions (nuclei) were resuspended in 30-50 µl of
buffer C (22) (20 mM HEPES, 0.75 mM
spermidine, 0.15 mM spermine, 0.2 mM EDTA, 2 mM EGTA, 2 mM DTT, 20%
glycerol, 1 mM PMSF, 4°C) and the ionic strength was adjusted to
0.4 M with NaCl. Samples were gently mixed on ice for 20 min. Finally,
the samples were centrifuged for 10 min at 10,000 g (4°C) and the supernatants collected and saved as the nuclear protein fraction. The protein concentration was determined by the Bradford assay (4) and samples were
stored at 80°C.
B activation was assessed by measuring NFB in nuclear protein
obtained from isolated myocytes using an electrophoretic mobility shift
assay (EMSA) as previously described (19). The double-stranded
oligonucleotide containing consensus
(5'-AGGGACTTTCCGCTGGGGACTTTCC-3') binding sites for NF-B (provided by Dr. T. Archer) were labeled with
[
-32P]ATP (Amersham Canada,
Oakville, ON), by using T4 polynucleotide kinase (MBI Fermentas,
Flamborough, ON) as previously described (19). One picomole of the
labeled oligonucleotide was incubated with 5 µg of nuclear protein in
the presence or absence of 50× excess of cold oligonucleotide for
30 min and the reaction mixture was then loaded onto native 5% PAG and
electrophoresed at 250 V in 0.5× Tris-borate-EDTA
buffer. The dried gels then were exposed to X-ray films
(Kodak) for 16 h in cassettes with intensifying screens.
Statistical Analysis
All results are presented as means ± SE. Statistical comparisons between the groups were carried out by ANOVA with an adequate post hoc test. P < 0.05 was accepted as statistically significant for all analyses.| |
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Animal Studies
As shown in Table 1 induction of CLP (saline + CLP) had no effect on mean arterial blood pressure or arterial blood gases compared with the sham-operated animals. Induction of CLP did, however, produce neutropenia and systemic acidosis with an increase in lactate compared with the sham animals. These observations are consistent with the development of sepsis. Compared with saline pretreated animals exposed to CLP, LPS-pretreated animals exposed to CLP had a lower systemic lactate concentration and higher neutrophil counts. These latter observations indicate that the LPS-pretreated animals had a lesser degree of CLP-induced systemic manifestations of sepsis.
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As shown in Fig. 1, heart MPO activity was
increased after induction of CLP (saline + CLP) compared with the sham
animals. LPS pretreatment ameliorated the CLP-induced increase in heart MPO activity (saline + CLP vs. LPS + CLP).
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As shown in Table 2, LVDP and its first
derivatives (i.e., +dP/dt and
dP/dt) were decreased 6 h
after induction of CLP (saline + CLP) compared with sham animals. LPS
pretreatment largely prevented the effects of CLP on these variables
(saline + CLP vs. LPS + CLP). Similarly, sham and LPS-pretreated CLP
animals exhibited LVDP-preload and LV volume-preload relationships that
were shifted upward and to the left of the saline pretreated CLP
animals (Fig. 2). These observations
indicate that contractile function of the heart was impaired in
response to CLP and that LPS pretreatment protected the hearts from
this CLP-induced dysfunction.
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Myocyte Studies
Myocyte function.
Ventricular myocytes from sham animals responded by shortening during
electrical field stimulation (Fig. 3).
Myocytes obtained from CLP animals had a significantly reduced degree
of shortening (saline + CLP). The contractile function of myocytes
obtained from LPS-pretreated animals exposed to CLP was at normal
levels (sham vs. LPS + CLP).
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Myocyte-PMN adhesion.
When myocytes and PMN obtained from CLP-treated animals were interacted
in an adhesion assay, there was an increase in PMN adhesion to myocytes
(Fig.
4A).
This hyperadhesion response was substantially diminished when PMNs and
myocytes from CLP animals pretreated with LPS were used (saline + CLP
vs. LPS + CLP).
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Myocyte ICAM-1.
Because previous studies indicated that LPS, TNF-, or IL-1 can
induce an increase in ICAM-1 expression on isolated cardiac myocytes
and make them more adhesive for PMNs (24), we assessed whether myocyte
ICAM-1 plays a role in the development of LPS tolerance in our model.
As shown in Fig. 5, surface levels of ICAM-1 were increased on myocytes obtained from CLP animals (saline + CLP) compared with those obtained from sham animals. This increased level of ICAM-1 expression in response to CLP was significantly reduced
when myocytes obtained from CLP animals pretreated with LPS were used.
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Myocyte NF-B.
The nuclear transcription factor, NF-B, appears to be intimately
involved in the inflammatory process by transactivating the gene
encoding ICAM-1 (23). Thus we assessed whether NF-B activation and
translocation to the nucleus was involved in the development of LPS
tolerance in myocytes. As shown in Fig. 6, there was more NF-B in the nuclei of myocytes obtained from CLP animals (saline + CLP) compared with those from sham animals. NF-B
was virtually undetectable in nuclei of myocytes obtained from CLP
animals pretreated with LPS.
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A recent study in mice demonstrated that polymicrobial sepsis induced by CLP increases mRNA levels for a variety of cytokines and chemokines in the lung and liver 6 h after CLP and that prophylactic administration of monophosphoryl lipid A (a nontoxic derivative of LPS) attenuated these responses (21). In the present study we focused on the heart and functional manifestations of CLP-induced sepsis. We show that 6 h after induction of CLP in rats there was evidence of myocardial inflammation (Fig. 1) and contractile dysfunction (Table 2 and Fig. 2) and that LPS pretreatment ameliorates these cardiac manifestations of sepsis. The mechanisms involved in the development of this LPS-induced tolerance are not completely clear. Most studies have focused on the role of inflammatory cells of myeloid origin or endothelial cells in the development of LPS tolerance (15, 33). Although previous studies have shown that LPS tolerance to a subsequent LPS challenge can be induced in a parenchymal cell, i.e., myocytes (with respect to cell shortening) (14), the present study represents the first systematic evaluation of the role of myocytes in the development of LPS tolerance with respect to inflammation.
In the present study myocardial contractile activity was depressed by
CLP-induced sepsis as evidenced by
1) a decrease in the LVDP and its
first derivatives +dP/dt and
dP/dt (Table 2), as well as
2) a shift in the LVDP and LV
volume-preload relationships downward and to the right (Fig. 2). These
indexes were near normal in rats pretreated with LPS before induction
of CLP (Table 2 and Fig. 2). In analogous in vitro experiments,
isolated myocytes from rats subjected to CLP had a reduced ability to
shorten in response to pacing (Fig. 3). The mechanisms responsible for
this shortening defect are unclear. Previous studies indicate that the
LPS-induced decrease in myocyte shortening is not associated with
alterations in intracellular Ca2+
transients, indicating that a decrease in myocyte myofilament response
to Ca2+ may be involved (31). A
similar mechanism may be operative in the present study. Irrespective
of the mechanisms involved in the depressed contractile activity of
myocytes from CLP animals, the contractile activity of myocytes from
CLP animals that had been pretreated with LPS were near normal,
indicating that myocytes are an active participant in the development
of LPS tolerance in vivo. These observations are consistent with a
previous in vitro study showing that LPS-induced myocyte shortening can
be prevented by a previous LPS challenge (14). Thus the major
significance of our observations is that LPS tolerance (with respect to
myocyte shortening) can be induced to polymicrobial sepsis (CLP).
In the present study, when myocytes and PMNs isolated from CLP animals were reacted in an adhesion assay, there was a fivefold increase in PMN adhesion to myocytes (Fig. 4A). This hyperadhesive response was ameliorated when myocytes and PMNs from CLP animals that had been pretreated with LPS were reacted in the adhesion assay. Although these experiments demonstrate that LPS tolerance with respect to PMN-myocyte adhesion was induced, they do not allow for a delineation of the relative roles of myocytes and PMNs in this response. Our subsequent experiments using myocytes from CLP animals and PMNs from sham animals and vice versa, indicate that both cell types contribute to the development of LPS tolerance in this system.
PMN obtained from CLP-treated animals adhered to naive myocytes (obtained from sham animals). This observation is consistent with the observation that LPS-stimulated PMN are more adhesive to endothelium (30). In the present study, this hyperadherence response was no longer present if the PMN were obtained from CLP animals previously pretreated with LPS (Fig. 4B). This latter observation indicates that PMN contribute to the development of LPS tolerance in vivo. This is not entirely surprising considering the numerous observations that cells of myeloid origin can be rendered tolerant to LPS with respect to cytokine production (33) and that PMN obtained from LPS- tolerant rats are less adherent to nylon fibers (3). Nonetheless, our findings are the first to demonstrate that LPS tolerance can be induced in PMN with respect to adhesivity for myocytes.
Of further relevance to the present study is the role of myocytes in the development of LPS tolerance. Myocytes from CLP animals were more adhesive to naive neutrophils, whereas myocytes from CLP animals pretreated with LPS developed LPS tolerance with respect to PMN adhesion (Fig. 4C). These findings are the first to show that a parenchymal cell, i.e., cardiac myocyte, can develop LPS tolerance with respect to PMN adhesion.
Previous studies indicate that LPS, TNF-, and IL-1 can induce
canine myocytes to be more adhesive for PMNs (6, 24). The adhesion of
PMN to cardiac myocytes results in an intracellular oxidative stress
within the myocytes and myocyte dysfunction, i.e., contracture (6).
This cytokine-induced 1) PMN
adhesion and 2) PMN-induced injury
to the myocytes is a result of an increased surface level of ICAM-1 on
the myocytes (24), because both of these events can be prevented by
monoclonal antibodies directed to ICAM-1 (6). In the present study
myocytes from CLP animals had an increased surface level of ICAM-1, a
finding consistent with the aforementioned studies. More importantly,
the surface levels of ICAM-1 were not altered in CLP animals pretreated
with LPS. This latter observation indicates that the development of LPS
tolerance in myocytes (with respect to PMN adhesion) is associated with
a diminished ability of myocytes to increase their surface levels of
ICAM-1 in response to CLP (Fig. 5).
Increases in surface levels of ICAM-1 are dependent on protein
synthesis apparently resulting from the transactivation of the ICAM-1
gene. Transactivation of the ICAM-1 gene is initiated by nuclear
transcription factors, such as NFB (19). In the present study, there
were increased levels of NFB in the nucleus of myocytes from CLP
animals, indicating that NFB had been activated and translocated to
the nucleus. This is consistent with previous reports showing that
exposing isolated neonatal cardiac myocytes to the cytokine, IL-1
(16), results in translocation of NFB to the nucleus. The novel
observation of the present study is that there was no detectible NFB
in the nucleus of myocytes obtained from CLP animals pretreated with
LPS (Fig. 6). These latter observations indicate that the development
of LPS tolerance in myocytes is associated with a lack of NFB
translocation to the nucleus in response to CLP. These observations are
also consistent with a role for NFB in the development of LPS
tolerance in monocytes and macrophages where LPS- tolerant cells are
unable to mobilize this nuclear transcription factor in response to
further LPS stimulation (11, 12, 26).
In summary, the results of the present study indicate that parenchymal
cells may contribute to the LPS-induced tolerance to polymicrobial
sepsis in vivo. Specifically, our results support a role for cardiac
myocytes in the development of LPS-induced tolerance to CLP-induced
peritonitis. In a rat model 6 h after induction of CLP:
1) myocyte shortening is impaired,
2) myocyte NFB is activated and
translocates to the nucleus, 3)
myocyte surface levels of ICAM-1 are increased and
4) myocytes are adhesive for naive
PMN. All of these events are prevented if the rats are pretreated with
LPS before induction of CLP. A similar scenario may also occur in the
parenchyma of other organs and contribute to the reduced PMN
sequestration and infiltration in LPS-tolerant animals or humans
subsequently exposed to sepsis. Thus development of therapeutic
modalities for the amelioration of sepsis-induced multiple organ
failure should also take into account the contribution of parenchymal
cells to the development of LPS tolerance.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from Medical Research Council MT-13940.
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FOOTNOTES |
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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 and other correspondence: P. R. Kvietys, Vascular Biology Program, London Health Sciences Centre-Research, 375 South St., Rm. C210, London, ON, Canada N6A 4G5 (E-mail: pkvietys{at}julian.uwo.ca).
Received 30 November 1998; accepted in final form 14 April 1999.
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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P < 0.05 compared with saline + CLP.
, Sham; 
, saline + CLP; 
, LPS + CLP. See Fig. 
