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Vol. 55, Issue 4, 658-667, April 1999
B Degradation and
Reduces Microvascular Injury Induced by Lipopolysaccharide in
Multiple Organs
Department of Pharmacology, The University of Illinois, College of Medicine, Chicago, Illinois
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Summary |
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 Top
 Summary
 Introduction
Materials and Methods
Results
Discussion
References
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Lipopolysaccharide (LPS) is a key mediator of multiple organ injury
observed in septic shock. The mechanisms responsible for LPS-induced
multiple organ injury remain obscure. In the present study, we tested
the hypothesis that the LPS-induced injury occurs through activation of
the transcription factor, nuclear factor-
B (NF-B). We examined
the effects of inhibiting NF-B activation in vivo in the rat on
LPS-induced: 1) gene and protein expression of the cytokine-inducible
neutrophil chemoattractant (CINC) and intercellular adhesion molecule-1
(ICAM-1); b) neutrophil influx into lungs, heart, and liver; and c)
increase in microvascular permeability induced by LPS in these organs.
LPS (8 mg/kg, i.v.) challenge of rats activated NF-B and induced
CINC and ICAM-1 mRNA and protein expression. Pretreatment of rats with
pyrrolidine dithiocarbamate (50, 100, and 200 mg/kg, i.p.), an
inhibitor of NF-B activation, prevented LPS-induced I-B
degradation and the resultant NF-B activation and inhibited, in a
dose-related manner, the LPS-induced CINC and ICAM-1 mRNA and protein
expression. Pyrrolidine dithiocarbamate also markedly reduced the
LPS-induced tissue myeloperoxidase activity (an indicator of tissue
neutrophil retention) and the LPS-induced increase in microvascular
permeability in these organs. These results demonstrate that NF-B
activation is an important in vivo mechanism mediating LPS-induced CINC
and ICAM-1 expression, as well as neutrophil recruitment, and the subsequent organ injury. Thus, inhibition of NF-B activation may be
an important strategy for the treatment of sepsis-induced multiple
organ injury.
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Introduction |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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Multiple
organ injury, a frequent complication of sepsis, is characterized by
microvascular injury and increased vascular endothelial permeability
(Brigham et al., 1979
; Fowler et al., 1985; Meyrick et al., 1986). One
crucial event that leads to microvascular endothelial damage and
multiple organ injury is endothelial "activation" and neutrophil
infiltration and accumulation in various organs (Brigham et al., 1979;
Meyrick et al., 1986; Bone, 1991; Parrillo, 1995; Ward, 1996).
Bacterial endotoxin (lipopolysaccharide, LPS) initiates this
pathophysiological process through multiple but cooperative pathways.
LPS induces the expression of adhesion molecules [E-selectin,
P-selectin, and intercellular adhesion molecule-1 (ICAM-1)] on
endothelial cells, which mediate neutrophil margination, rolling, and
firm adhesion (Carlos and Harlan, 1994). LPS also activates and
up-regulates integrins (CD11b/CD18) on the neutrophil cell surface
(Carlos and Harlan, 1994). Interactions between ICAM-1 and CD11b/CD18
lead to neutrophil adhesion to the endothelium (Carlos and Harlan,
1994), followed by neutrophil activation, change in neutrophil shape,
and migration through the endothelial cell junctions into surrounding
tissue (Carlos and Harlan, 1994). Activated neutrophils release oxidant
free radicals and proteinases, resulting in endothelial damage (Tate
and Repine, 1983; Weiss, 1989). LPS also causes the expression of
chemokines including interleukin (IL)-8, macrophage inflammatory
proteins (MIPs), and cytokine-induced neutrophil chemoattractant (CINC)
in several constitutive cells and resident macrophages (Ben-Baruch et
al., 1995; Frevert et al., 1995; Koh et al., 1995; Blackwell et al., 1996; Ward, 1996). Release of these chemokines as well as leukotriene B4 and complement C5 within the interstitium sets up a chemoattractant gradient for neutrophil migration into tissue (Ward, 1996; Mulligan et
al., 1996). LPS also releases other proinflammatory cytokines, which
can amplify the inflammatory response and mediate tissue damage (Bone,
1991; Parrillo, 1995). The sequential events and cooperative
interactions of adhesion molecules and chemokines mediating LPS-induced
inflammation suggest that common molecular mechanisms such as
activation of the transcription factor nuclear factor-B (NF-B)
may be important in orchestrating the response.
Deletion mutagenesis of the promoters and reporter gene analysis in
cultured cells, primarily in cell lines, has shown that NF-B plays
an essential role in the transcriptional induction of cell adhesion
molecules ICAM-1, E-selectin, and vascular adhesion molecule-1) and
chemokines (IL-8, CINC, MIP-1, and MIP-2) (Baeuerle and Henkel, 1994;
Carlos and Harlan, 1994; Siebenlist et al., 1994; Collins et al.,
1995). NF-B is also involved in LPS-induced expression of
proinflammatory cytokines and enzymes that contribute to endothelial
damage and development of multiorgan injury (Baeuerle and Henkel,
1994; Siebenlist et al., 1994). Because NF-B activation may be
crucial in the LPS-induced multiple organ injury, in the present study,
we studied the effects of inhibiting NF-B activation using
pyrrolidine dithiocarbamate (PDTC) on LPS-induced: 1) CINC and ICAM-1
mRNA and protein expression; 2) neutrophil infiltration; and 3)
increase in microvascular permeability in multiple organs. Our data
indicate that NF-B activation is an important in vivo signal
orchestrating the LPS-initiated adhesion and chemokine expression,
neutrophil sequestration, and the resultant multiple organ injury.
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Materials and Methods |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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Animal Protocols. Male Sprague-Dawley rats were purchased from Charles River, divided into experimental groups in a randomized manner, and used for experiments when their body weights were in the 300- to 350-g range. We studied six groups of animals: control, saline (1 ml/kg, i.v.); LPS, Salmonella enteritidis LPS (8 mg/kg in saline, i.v.); LPS plus PDTC groups, 50, 100, or 200 mg/kg of PDTC (i.p.) 1 h before LPS challenge; and PDTC alone, PDTC (200 mg/kg, i.p.) for an equivalent period of time. All procedures were approved by the Institutional Animal Care Committee.
Animals were sacrificed by exsanguination, and lung, heart, and liver were collected either at 1 h [for electrophoretic mobility shift assay (EMSA)] or at 4 h post-LPS challenge [for Northern, Western blot, and enzyme-linked immunosorbance assay (ELISA)]. Animals in the PDTC alone group were sacrificed at 2 h (for EMSA) or at 5 h (for Northern, Western blot, and ELISA), and tissues were collected. Tissues were snap-frozen in liquid nitrogen and kept at
70oC for later use.
Nuclear Protein Extract and EMSA.
Lungs and hearts were
minced on ice in 0.5 ml ice-cold buffer A composed of: 10 mM HEPES (pH
7.9), 1.5 mM KCl, 10 mM MgCl2, 0.5 mM
dithiothreitol (DTT), 0.1% IGEPAL CA-630, and 0.5 mM
phenylmethylsulfonyl fluoride (PMSF) (all from Sigma Chemical Co., St.
Louis, MO). The minced tissue was homogenized using Dounce homogenizer,
followed by centrifuging at 5000g at 4°C for 10 min. The
crude nuclear pellet was suspended in 200 µl of buffer B (20 mM
HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, and 4 µM leupeptidin)
and incubated on ice for 30 min. The suspension was centrifuged at
16,000g at 4°C for 30 min. The supernatant (nuclear
proteins) was collected and kept at 70°C for use. Protein
concentration was determined using bicinchoninic acid assay kit with
BSA as standard (Pierce, Rockford, IL).
B consensus oligonucleotide probe
(5'-AGTTGAGGGGACTTTCCCAGGC-3) was end-labeled with
[
-32P] ATP (Amersham Life Science, Arlington
Heights, IL). Nuclear protein (10 or 20 µg for lung or heart) was
incubated with 100,000 cpm 32P-labeled NF-B
consensus oligonucleotide for 30 min in a total volume of 15 µl in a
binding buffer that consisted of 10 mM Tris-Cl, pH 7.5, 1 mM
MgCl2, 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 4%
glycerol, and 2 µg of poly-(deoxyinosinic·deoxycytidylic acid)
(Pharmacia Biotech, Piscataway, NJ). The specificity of the DNA/protein
binding was determined by competition reactions in which 50-fold molar excess of unlabeled NF-B oligonucleotide was added to the binding reaction 10 min before the addition of radiolabeled probe. Reaction was
stopped by adding 1 µl of gel loading buffer and subjected to
nondenaturing 4% polyacrylamide gel electrophoresis in 0.25× TBE buffer (Tris-borate-EDTA). Gel was vacuum-dried and exposed to X-ray film (Fuji hyperfilm).
Northern Blot Analysis.
Rat cDNA probes for CINC (207 bp)
and ICAM-1 (384 bp) were amplified using standard reverse
transcription-polymerase chain reaction procedure. RNA (1 µg) from
LPS-treated rat lungs was reverse transcribed into cDNA. The cDNA probe
fragments were amplified from reverse transcriptase-generated cDNAs
using designed primers corresponding to the published rat CINC (Huang
et al., 1992) and rat ICAM-1 (Kita et al., 1992) cDNA sequences,
purified by gel electrophoresis, and eluted from the gels using a
Jetsorb DNA extraction kit (Genomed, Inc., Research Triangle Park, NC).
Authenticity of the PCR product was confirmed by dideoxy chain
termination sequencing.
.
Poly-(A)+ mRNA was isolated using a poly-(A) mRNA
isolation kit (Promega Corp., Madison, WI). Approximately 3 µg of
mRNA from each sample were separated on a 1.2% denaturing agarose gel
and transferred onto Hybond-N nylon filter (Amersham Life Science). The
filter was incubated at 42oC for at least 4 h in a prehybridization buffer that consisted of 50% formamide, 5×
Denhardt's solution, 5× SSC, 200 µg/ml of sonicated denatured
salmon sperm DNA, and 0.1% SDS. The filter was then hybridized with
1 × 106 cpm/ml
32P-labeled rat CINC, ICAM-1, and glyceraldehyde
phosphate dehydrogenase (GAPDH; Fort et al., 1985) probes at
42oC for 14-16 h and washed sequentially with
decreasing concentrations of SSC/0.1% SDS and at increasing
temperatures (final wash, 0.1× SSC/0.1% SDS at 60°C). The blots
were exposed to Fuji film in the presence of an intensifying screen for
1 to 3 days. Each hybridization was followed by film exposure and
stripping down of previous probe before the subsequent hybridization to
other probes.
Protein Extraction and Western Blot Analysis. Lungs, heart, and liver were finely minced and homogenized in 10 volumes of ice-cold protein extracting buffer containing 25 mM Tris-HCl, 0.5 mM EDTA, 0.5 mM EGTA, 0.1 mg/ml PMSF, 10 µg/ml of leupeptin, and 1 µM pepstain. The homogenate was centrifuged at 17,500g at 4°C for 15 min, and the resulting supernatant was collected as cytosolic fraction. The pellet was resuspended in extracting buffer containing 0.1% Triton X-100, homogenized, and centrifuged at 17,500g at 4°C again for 30 min. The second supernatant was collected and centrifuged at 90,000g at 4°C for 60 min. The resulting pellet was dissolved in extracting buffer and taken as membrane fraction. Protein concentration was determined using bicinchoninic acid assay kit with BSA as standard (Pierce, Rockford, IL).
Equal amounts of proteins (30 µg/lane) were loaded and separated on 7.5% (for ICAM-1), 12.5% (for I-B), or 15% (for CINC) SDS-polyacrylamide slab gel under denaturing conditions. Broad range
protein molecular weight marker (Bio-Rad, Hercules, CA) was used as
standard. Proteins were electroblotted to nitrocellulose membrane
(Bio-Rad). After incubation in blocking solution [5% dry milk in
TBST (Tris buffered saline with Tween 20)] at room temperature
for 2 h, the membrane was immunoblotted to the following antibodies at room temperature for 1 h in separate experiments: anti-I-B monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz,
CA); anti-CINC polyclonal antibody (kindly provided by Dr. Zagorski at
National Institutes of Health, Bestheda, MD); and anti-rat ICAM-1
monoclonal antibody (PharMingen, San Diego, CA). The secondary
antibodies were horseradish peroxidase-conjugated monkey anti-goat and
goat anti-mouse antibodies. Peroxidase labeling was detected with
enhanced chemiluminescence Western blotting detection system (Amersham
Life Science) according to the manufacturer's recommendations.
Tissue CINC and ICAM-1 Content Quantification.
Tissue CINC
content was quantitated using sandwich ELISA as described previously
(Wittwer et al., 1993). Ten µg of lung or 20 µg of heart cytosolic
protein were added into each microplate well. The coating antibody was
affinity-purified goat anti-CINC polyclonal antibody (kindly provided
by Dr. Zagorski). The detecting antibody was polyclonal rabbit
anti-CINC antibody (Peptide International, Louisville, KY) and
horseradish peroxidase conjugated anti-rabbit IgG (Amersham Life
Science). To determine tissue ICAM-1 level, 1 µg of lung or 5 µg of
liver or heart membrane protein in 50 mM carbonate/bicarbonate buffer
(pH 9.5) were added to microtitration plates (Dynatech, Chantily, VA)
overnight at 4°C. The contents of the wells were removed, and wells
were washed for four times with PBS containing 0.05% Tween 20 (PBST).
The coated wells were blocked with 5% dry milk in PBST at room
temperature for 2 h, followed by incubation with anti-rat ICAM-1
monoclonal antibody in 5% dry milk/PBST at room temperature for an
additional 2 h. After washing with PBST four times, horseradish
peroxidase-conjugated anti-mouse IgG in PBST was added. Color was
developed by the addition of TMB peroxidase substrate mixture (Sigma
Chemical Co.) and read in a microplate reader at the wavelength of 450 nm after addition of stopping buffer.
Measurement of Tissue Myeloperoxidase Activity.
We used
tissue myeloperoxidase (MPO) activity as an index of tissue neutrophil
accumulation. To measure tissue MPO activity, frozen lungs, hearts, and
livers were thawed and extracted for MPO, following the homogenization
and sonication procedure as described previously (Krawisz et al.,1984).
MPO activity in supernatant was measured and calculated from the
absorbance (at 460 nm) changes resulting from decomposition of
H2O2 in the presence of
o-dianisidine (Krawisz et al.,1984).
Measurement of Microvascular Permeability.
Microvascular
endothelial permeability was assessed using the dual tracer methods,
which has been demonstrated to accurately estimate the
permeability-surface product for albumin (Graham and Evans, 1991).
Animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.)
and ventilated with rodent ventilator at a tidal volume of 1 ml/100 g
of body weight and frequency of 60 breaths/min. Left carotid artery and
right jugular vein were cannulated for monitoring of blood pressure and
for the injection of LPS and tracers. After a 20-min equilibration
period, LPS (8 mg/kg, i.v.) or PDTC (50, 100, or 200 mg/kg, i.p.) was
injected. This was followed by an injection of
125I-labeled human serum albumin (1 µCi in 0.2 ml saline) via jugular vein cannula. At 5 h after LPS, animals
were heparinized, and 1 µCi of 131I-labeled
human serum albumin was injected (via jugular vein). Five minutes after
[131I]-labeled human serum albumin injection, 1 ml of blood was withdrawn, and lungs, hearts, and livers were collected
after the animal was exsanguinated. Tissues were dissected into
small pieces, blotted free of surface water and blood, and weighed.
Plasma and tissue 125I and
131I activities (cpm) were counted using a gamma
counter. Extravascular albumin accumulation was calculated from the
ratio of tissue to plasma 125I counting, with the
intravascular albumin retention being corrected with the ratio of
tissue to plasma [131I] countings.
Statistical Analysis
CINC, ICAM-1, and GAPDH bands on Northern blot and NF-B
bands on EMSA autoradiograph were quantitated using a laser
densitometry (Howtek, Hudson, NH) linked to a computer analysis system
(PDI, Huntington Station, NY). The relative CINC and ICAM-1 RNA levels were expressed as a percentage of their corresponding GAPDH bands. Data
are presented as means ± S.E.M. Statistical analysis of results was performed using Kruskal-Wallis Rank test, followed by
Mann-Whitney U test for stepward comparison.
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Results |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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PDTC Inhibits LPS-Induced NF- B Activation.
We previously
reported that challenge of rat with LPS caused a time-dependent NF-B
activation and that the NF-B/DNA complex predominately consists of
p50 and p65 subunits of NF-B protein family in the lungs (Liu et
al., 1997). Here, we used PDTC, an inhibitor of NF-B activation in
vitro and in vivo (Schreck et al., 1992; Ziegler-Heitbrock et
al., 1992; Kawai et al., 1995; Liu et al., 1997), to study the role of
NF-B activation in mediating LPS-induced CINC and ICAM-1 expression
and multiple organ injury. Figure 1, A
and C, is an autoradiogram of EMSA showing LPS-induced NF-B
activation and its inhibition by PDTC. The NF-B/DNA complex in the
lung formed a single band (Fig. 1A) composed exclusively of p50 and p65
heterodimers, whereas NF-B/DNA complex in the heart showed
two bands (Fig. 1C). The lower band was composed of p50 and p65
heterodimer, whereas the upper band was composed of p65 homodimer, as
demonstrated in our supershift assay. We quantitated these NF-B
bands using densitometry. Figure 1, B and D, is mean arbitrary units of
these bands from lungs and hearts of four animals. The NF-B/DNA
binding activity was low in nuclear protein from control lungs and
heart but was markedly increased in tissues from LPS-challenged rats.
This LPS-induced NF-B/DNA binding activity was inhibited by
pretreatment with PDTC in a dose-related manner (Fig. 1).
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PDTC Prevents LPS-Induced I-B Degradation in Vivo.
To
investigate the possible mechanism of PDTC action in vivo, we treated
animals with 50, 100, or 200 mg/kg PDTC for 1 h before challenge
with LPS for 1 h and compared I-B protein abundance in lung
homogenates of these rats to animals treated with saline (control) or
LPS alone, using Western blot analysis. As shown in Fig.
2, LPS reduced the tissue I-B protein
content dramatically, whereas this was prevented by pretreatment with
three doses of PDTC. The inhibition by PDTC of the LPS-induced I-B
degradation appears to be dose dependent (Fig. 2).
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PDTC Inhibits LPS-Induced CINC and ICAM-1 mRNA Expression in
Vivo.
We studied the effects of inhibiting NF-B activation with
PDTC on LPS-induced CINC and ICAM-1 mRNA expression. We treated animals
with 50, 100, or 200 mg/kg PDTC for 1 h before challenge with LPS
for 4 h and compared CINC and ICAM-1 mRNA abundance in tissue
homogenates of these rats to rats treated with saline (control) or LPS
alone. As shown in Fig. 3, the 0.93-kb
CINC mRNA transcript was absent or negligible in the control
homogenates of both the lung and heart tissues (Fig. 3, A and C),
whereas the 2.6-kb ICAM-1 mRNA transcript showed organ-dependent
variation in its basal level (Fig. 4, A
and C). ICAM-1 mRNA was negligible in control hearts (Fig. 4C) but was
expressed in control lungs (Fig. 4A). The mRNA abundance of these two
genes increased markedly after LPS challenge (Figs. 3A, 3C, 4A, and
4C). PDTC reduced the LPS-induced induction of these genes in a
dose-related manner. PDTC alone did not affect their expression (Figs.
3 and 4). We quantified CINC, ICAM-1, and GAPDH band intensity using
densitometry and normalized the CINC and ICAM-1 bands to their
corresponding GAPDH bands. Neither LPS nor PDTC had a significant
effect on the GAPDH mRNA transcription (Figs. 3A, 3C, 4A, and 4C).
However, LPS increased the CINC/GAPDH and ICAM-1/GAPDH ratios markedly
(Figs. 3B, 3D, 4B, and 4D). PDTC at concentrations of 50, 100, and 200 mg/kg, respectively, reduced LPS-induced CINC mRNA level by 63, 74, and 67% in the lung (Fig. 3B) and by 25, 79, and 24% in the heart (Fig.
3D). PDTC reduced LPS-induced ICAM-1 mRNA level by 63, 75, and 60% in
the lung (Fig. 4B) and by 22, 43, and 34% in the heart (Fig. 4D) at
the PDTC concentrations of 50, 100, and 200 mg/kg, respectively.
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PDTC Inhibits LPS-Induced CINC and ICAM-1 Protein Expression.
To address whether inhibition of CINC and ICAM-1 mRNA expression by
PDTC resulted in inhibition of protein expression, we compared CINC and
ICAM-1 protein levels in tissue homogenates of lungs, hearts, and
livers from control rats; rats challenged with LPS for 4 h; rats
pretreated with 50, 100, or 200 mg/kg PDTC for 1 h before
challenge with LPS for 4 h; and rats treated with PDTC alone. We
quantitated CINC and ICAM-1 protein level in tissue homogenates using
Western blot and ELISA. Western blot analysis showed that the
Mr 6,500 CINC and
Mr 70,000 ICAM-1 proteins were detectable
in the control homogenates but were markedly up-regulated by LPS
challenge in all three organs (Figs. 5A,
5C, 5E, 6A, 6C, and 6E). Pretreatment with 50, 100, and 200 mg/kg of
PDTC variably prevented the up-regulation of these two proteins (Figs.
5A, 5C, 5E, 6A, 6C, and 6E). A Mr 95,000 ICAM-1 protein was also detected in heart, which was up-regulated by
LPS. This up-regulation was prevented by PDTC in a similar fashion as
seen on the Mr 70,000 ICAM-1 protein (Fig.
6C). The 95-kD ICAM-1 protein band was
not seen in lung and liver homogenates (Fig. 6, A and E), suggesting a
differential glycosylation pattern in the myocardium.
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PDTC Reduces LPS-Induced Tissue MPO Activity.
We studied the
functional consequence of inhibiting NF-B activation by PDTC on
neutrophil influx into organs using MPO activity as an index of tissue
neutrophil accumulation. As shown in Fig. 7, control tissues of all three organs
had low MPO activity. The MPO activity increased markedly in all three
organs 4 h after LPS challenge (Fig. 7). PDTC, at the
concentration of 50, 100, and 200 mg/kg, respectively, reduced the
LPS-induced tissue MPO activity by 51, 59, and 49% in the lungs; by
49, 56, and 49% in the heart; and by 41, 47, and 40% in the liver
(Fig. 7). PDTC alone had no significant effect on tissue MPO activity
in heart and liver but increased MPO activity in the lung (Fig. 7).
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PDTC Reduces LPS-Induced Increase in Microvascular
Permeability.
We assessed the functional consequence of inhibiting
NF-B activation by PDTC on LPS-induced organ injury. We compared the microvascular endothelial permeability index in the lungs, heart, and
liver of control, LPS alone, LPS plus various doses of PDTC, and PDTC
alone treated animals. Challenge with LPS caused a 4.4-, 2.6- and
6.2-fold increase in the microvascular endothelial permeability index
in lungs, heart, and liver, respectively (Fig.
8). Pretreatment of the LPS-challenged
animals with 50, 100, and 200 mg/kg of PDTC reduced the LPS-induced
elevation in permeability by 52, 53, and 47% in the lungs; by
41, 47, and 41% in the heart; and by 58, 61, and 56% in the liver
(Fig. 8). The maximal inhibition of LPS-induced increase in
microvascular permeability was observed at the PDTC concentration of
100 mg/kg in all three organs. This was the same concentration at which
PDTC maximally inhibited LPS-induced CINC and ICAM-1 mRNAs (Figs. 3B,
3D, 4B, and 4D), CINC protein expression (Fig. 5, B and D), and tissue
MPO activities (Fig. 7) in these organs.
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Discussion |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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The major focus of the present study was to determine the role of
NF-B activation in mediating LPS-induced multiple organ injury. We
inhibited the activation of NF-B using PDTC and addressed the
effects of this inhibition on LPS-induced CINC and ICAM-1 mRNA, protein
expression, tissue neutrophil sequestration, and the subsequent
LPS-induced increase in microvascular permeability in multiple organs.
We demonstrated that PDTC inhibited LPS-induced I-B degradation
and the resultant NF-B activation in vivo in a dose-related manner.
Pretreatment of rats with PDTC, ranging from 50 to 200 mg/kg body
weight, also inhibited LPS-induced CINC and ICAM-1 mRNA and CINC and
ICAM-1 protein expression. We found that the reduced expression of CINC
and ICAM-1 correlated with important functional consequences because
PDTC also abrogated the LPS-induced tissue neutrophil sequestration and
increase in microvascular endothelial permeability in multiple organs.
Because PDTC is a potent inhibitor of NF-B activation both in vitro
and in vivo (Schreck et al., 1992; Ziegler-Heitbrock et al., 1992; Kawai et al., 1995; Liu et al., 1997), the inhibition by PDTC of
NF-B activation and the suppression of LPS-induced CINC and ICAM-1
expression induced by LPS suggest that NF-B activation mediates in
part the LPS-induced expression of these genes. The results indicate
that NF-B activation is a critical mechanism in LPS-induced
neutrophil sequestration and multiple organ injury.
The role NF-B in LPS- or cytokine-induced CINC and ICAM-1 gene
expression has been defined in cultured cells (Jahnke and Johnson,
1994; Hou et al., 1994; van de Stolpe et al., 1994; Collins et
al., 1995; Ledebur and Parks, 1995; Ohmori et al., 1995; Ohtsuka et
al., 1996). LPS has also been shown to activate NF-B and induce CINC
and ICAM-1 mRNA expression in vivo (Manning et al., 1995; Blackwell et
al., 1996). However, the in vivo function of NF-B in mediating CINC
and ICAM-1 gene and protein expression and its role in the development
of organ injury has not been established. Our studies thus extend these
previous observations by showing that inhibition of NF-B activation
in vivo suppresses LPS-induced CINC and ICAM-1 mRNA and protein
expression, reduces neutrophil accumulation, and prevents the
LPS-induced increase in microvascular endothelial permeability in
multiple organs. Our study also establishes the in vivo linkage between
NF-B activation, adhesion molecule and chemokine expression,
neutrophil sequestration, and the development of tissue injury.
We observed that PDTC had a greater inhibitory effect on LPS-induced
NF-B activation than on the expression of ICAM-1 gene in the heart,
suggesting that additional transcription factors are also involved in
LPS-induced ICAM-1 expression in vivo. This is consistent with results
from cultured Mel Juso (human melanoma cell line) and Hep G2 cells
(human liver heptoma cells), showing that tumor necrosis factor-
(TNF-)-mediated induction of ICAM-1 promoter activity required the
synergistic cooperation between NF-B and C/EBP (nuclear factor for
IL-6) (Jahnke and Johnson, 1994; Hou et al., 1994). Our results differ
from the reported in vitro studies in human umbilical vein endothelial
cells and U937 cells (human monocytic cell line) in which NF-B alone
was found to be sufficient for the induction of ICAM-1 promoter
activity by in response to LPS or TNF- (van de Stolpe et al., 1994;
Ledebur and Parks, 1995).
Numerous in vitro studies have demonstrated PDTC to be a relatively
selective inhibitor of NF-B activation (Schreck et al., 1992;
Ziegler-Heitbrock et al., 1993; Kawai et al., 1995; Munoz et al.,
1996). It appears that PDTC is also an effective in vivo inhibitor of
NF-B activation (Liu et al., 1997). We observed that PDTC inhibited
LPS-induced NF-B activation in vivo but had no effect on the
activation of other transcription factors, cAMP response element
binding protein, Sp-1 (promoter-selective transcription factor), AP-1
(activating protein-1), and AP-2 (Liu et al., unpublished observation).
Thus, PDTC is a useful pharmacological tool for the in vivo analysis of
NF-B activation and NF-B-regulated gene expression.
One limitation to the use of PDTC is that a high dose of PDTC (200 mg/kg) has nonspecific effects. We observed that 200 mg/kg PDTC
markedly augmented LPS-induced AP-1 activation and that this dose of
PDTC alone activated AP-1 (Liu et al., unpublished observation). Because the promoter region of both ICAM-1 and CINC genes contain an
AP-1 binding site (Collins et al., 1995; Ohmori et al., 1995 ) and
activation of AP-1 is an important mechanism of LPS- or cytokine-induced expression of these genes (Collins et al., 1995), the
inhibitory effect of PDTC mediated through NF-B inhibition may be
partially offset by its stimulation of AP-1 activation. This could
explain why PDTC at 200 mg/kg was only partially effective as compared
with 100 mg/kg of PDTC in preventing LPS-induced CINC and ICAM-1 mRNA
and protein expression and LPS-induced MPO activity and the increase in
microvascular permeability.
Nathens et al. (1997) have reported that PDTC failed to inhibit
LPS-induced NF-B activation in rats, in contrast to our in vivo
finding and several in vitro studies (Schreck et al., 1992; Ziegler-Heitbrock et al., 1993; Kawai et al., 1995; Liu et al., 1997).
Differences in cell types and duration of PDTC preincubation may
explain this discrepancy. Nathens used nuclear protein from peritoneal
macrophages of LPS-challenged rats for EMSA, whereas we used nuclear
protein from whole lung and heart tissues; thus, it is possible that
different cells can variably respond to PDTC. Nathens pretreated
animals for 30 min, whereas we pretreated them for 60 min. Cell studies
showed that PDTC exerted greater inhibitory effect on TNF-- or
PMA-induced NF-B activation when the preincubation time was 1 h
or longer compared with cells preincubated with PDTC for <1 h (Schreck
et al., 1992; Ziegler-Heitbrock et al., 1993). We also observed that
PDTC had little inhibitory effect on LPS-induced NF-B activation
when pretreatment time was <20 min, suggesting that a minimum
preincubaion time is required for maximum PDTC inhibitory effect on
NF-B activation. Other factors such as time point at which EMSA was
performed (3 h versus 1 h after LPS) and route of LPS
administration (local versus systemic) could also contribute to this discrepancy.
The PDTC analog, dithiocarbamate, has been shown to inhibit HIV
progression in patients (Reisinger et al., 1990), suggesting clinical
usefulness of PDTC. However, there are limitations to the application
of PDTC as a therapeutic NF-B inhibitor: 1) the effective PDTC
concentration range is narrow. According to our data, the minimal
concentration of PDTC required to produce in vivo inhibition of NF-B
activation lies between 25 and 50 mg/kg, whereas at the PDTC
concentration of 200 mg/kg animals showed toxic effects manifested as
hypersalivation, excitability, and neuromuscular irritability; 2) PDTC
has to be administered before the challenge, because PDTC was
ineffective in inhibiting NF-B activation when given concurrently
with LPS (data not shown). A similar observation was reported in cell
culture studies (Schreck et al., 1992; Ziegler-Heitbrock et al., 1993;
Kawai et al., 1995). This probably reflects the time required for the
accumulation of effective PDTC concentration in cytoplasm, where PDTC
exerts its inhibitory action on NF-B activation. The present results suggest the potential usefulness of a structurally modified PDTC with
less toxic effects and greater efficacy.
The mechanisms of the action of PDTC remain obscure. PDTC is a
well-known antioxidant. There is evidence supporting that PDTC suppresses NF-B activation through its antioxidant property (Schreck et al., 1992; Satriano and Schlondorff, 1994), but contrary evidence also exists (Ziegler-Heitbrock et al., 1993; Satriano and Schlondorff, 1994; Brennan and O'Neill, 1995). We showed that PDTC prevented the
LPS-induced I-B degradation, suggesting that PDTC acts upstream of I-B degradation in the signaling cascade (perhaps at the level of
I-B phosphorylation or I-B kinase activation) to prevent NF-B
activation in vivo.
We observed that the dose-response data in PDTC-mediated inhibition of ICAM-1 protein expression obtained by Western blot was not correlated with data obtained using ELISA (Fig. 6: A, C, and E versus B, D, and E). This inconsistency might be the result of an intrinsic variation. ICAM-1 protein, as determined in Western blot, is fully denatured by SDS treatment and boiling, whereas ICAM-1 protein, as determined in ELISA, may not be similarly denatured. The anti-rat ICAM-1 antibody used in these experiments was a blocking antibody. Because blocking antibody is designed to best recognize ICAM-1 protein in its natural conformation, it may have differential affinity to the determined ICAM-1 proteins. Thus, changes in ICAM-1 protein expression as determined by Western blot may not fully parallel measurement of ICAM-1 protein by ELISA.
In summary, we have shown that challenge of rats with LPS activated
NF-B and increased microvascular endothelial permeability in lungs,
heart, and liver. Pretreatment of rats with PDTC inhibited the
LPS-induced I-B degradation and resultant NF-B activation in a
dose-related manner and suppressed the LPS-induced CINC and ICAM-1 mRNA
and protein expression in multiple organs. PDTC also reduced neutrophil
accumulation in lungs, heart, and liver and attenuated the increase in
microvascular endothelial permeability induced by LPS in these organs.
These results suggest that NF-B activation is a critical in vivo
mechanism mediating LPS-induced multiple organ injury. Thus, inhibition
of NF-B activation may represent a novel therapeutic strategy for
the treatment of sepsis-induced multiple organ injury.
| |
Acknowledgment |
|---|
We appreciate the technical support of Eun Mee Cheon.
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Footnotes |
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Received August 12, 1998; Accepted December 15, 1998
1 Current address: Division of Pulmonary & Critical Care Medicine, Albert Einstein College of Medicine, New Hyde Park, New York.
This work was supported by National Heart, Lung, and Blood Institute Grants HL46350 (to A.B.M.) and the American Heart Association Grant-in-Aid 9650733N (to S.F.L.).
Send reprint requests to: Dr. A. B. Malik, Department of Pharmacology (M/C 868), University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, IL 60612. E-mail: abmalik{at}uic.edu
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Abbreviations |
|---|
EMSA, electrophoretic mobility shift assay;
GAPDH, glyceraldehyde phosphate dehydrogenase;
PDTC, pyrrolidine
dithiocarbamate;
NF-B, nuclear factor-B;
AP, activating protein;
ICAM-1, intercellular adhesion molecule-1;
CINC, cytokine-inducible
neutrophil chemoattractant;
MPO, myeloperoxidase;
MIP, macrophage
inflammatory protein;
TNF-, tumor necrosis factor-;
IL, interleukin;
DTT, dithiothreitol;
PMSF, phenylmethylsulfonyl
fluoride.
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References |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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Annu Rev Cell Biol
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J Biol Chem
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6185-6192B mobilization and TNF production in human monocytes.
J Immunol
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