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Departments of Surgery and Medicine, University of Toronto, and the Toronto Hospital, Toronto, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA levels are unaffected. This finding is not unique to the lung,
because a similar effect on PMN influx was recapitulated in a rodent
model of chemical peritonitis. Further, in vitro studies demonstrated
that pretreatment of HUVEC monolayers with DEM prevented both ICAM-1
up-regulation and PMN transendothelial migration. These data indicate
the presence of a thiol-sensitive mechanism for modulating ICAM-1 gene
expression and suggest a potential novel therapeutic strategy for
diseases characterized by PMN-mediated tissue injury. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lung neutrophil sequestration in ARDS is thought to occur as a
result of a cascade of cellular events initiated by either infectious
or noninfectious inflammatory stimuli. Local activation of inflammatory
cells in the lung interstitium and alveolus leads to elaboration of
several proinflammatory cytokines and chemokines including TNF-,
IL-1, and IL-8 (7, 8, 9, 10, 11). Through both a direct effect of the
inflammatory stimulus and secondary effects mediated by products of
activated local inflammatory cells, the pulmonary capillary endothelium
becomes activated leading to expression of adhesion molecules on the
endothelial cell. This event, in concert with the local increase in
chemokines results in neutrophil activation and sequestration. Because
cell activation is necessary for lung leukosequestration and subsequent
injury, it follows that strategies aimed at preventing cell activation
may attenuate lung injury.
Recent studies have suggested that manipulation of the intracellular
redox state of the cell can have profound effects on cellular function.
This regulation has been shown to occur at any one of several levels of
cellular activation including pretranscriptional, transcriptional, or
post-transcriptional events (12, 13, 14, 15). In the whole animal model, one
recent report demonstrated that the antioxidant
N-acetyl-cysteine was able to prevent lung injury, in part
through an inhibitory effect on induction of the gene for the rodent
chemokine, cytokine-induced neutrophil chemoattractant (CINC) (16). We
have recently reported that glutathione (GSH) depletion following
administration of the GSH-depleting agent diethylmaleate (DEM)
prevented lung injury caused by intratracheal challenge with LPS (17).
In these initial studies, DEM appeared to exert its effects by
preventing the accumulation of BALF neutrophils. This effect occurred
despite normal up-regulation of PMN ß2 integrin
expression and normal PMN chemotaxis. Further, the local cytokine
response as measured by BALF fluid levels of TNF- was unaffected.
Considered together, these data suggested the possibility that DEM
might be exerting its effect by modulating endothelial cell activation
during inflammation with a resultant reduction in PMN sequestration. In
the present studies, we demonstrate that GSH depletion prevents the
up-regulation of endothelial cell ICAM-1 expression in vivo and, in so
doing, mitigates PMN influx into the lung. In vitro studies demonstrate
that this is a direct effect on the endothelium and is sufficient to
prevent PMN migration across an endothelial cell monolayer. Finally,
the effect is not specific to the pulmonary capillary bed, since
neutrophil migration into the peritoneal cavity following induction of
chemical peritonitis is prevented by DEM pretreatment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Powdered Brewer’s thioglycollate and phenol-extracted
Escherichia coli O111:B4 LPS were both from Difco
Laboratories (Detroit, MI). Thioglycollate was dissolved in
H2O, autoclaved, and stored in the dark at room temperature
until uniformly green and clear. Murine TNF- was purchased from
Genzyme (Cambridge, MA). DEM, PGE1, human serum albumin
(HSA), o-phenyldiamine hydrochloride (OPD), FMLP, and
glutathione were all obtained from Sigma Chemical Company (St. Louis,
MO). Hexadecyltrimethylammonium bromide (HTAB) and phorone
(diisopropylidene acetone) were purchased from Fluka (Switzerland).
N,N-dimethylformamide was obtained from J.T.
Baker Chemical Co. (Phillipsburg, NJ). 5,5'-dithiobis(2-nitrobenzoic
acid) (DTNB) and 5-sulfosalicylic acid (SSA) were both purchased from
Aldrich Chemical Co. (Milwaukee, WI). 125I-labeled albumin
was obtained from Merck Frosst (Montreal, Quebec) and
Na251CrO4 from Amersham (Oakville,
ON). RPMI 1640 and calcium- and magnesium-free HBSS were obtained from
Life Technologies (Grand Island, NY). Rabbit anti-mouse
biotinylated IgG was used as a secondary Ab for immunohistochemistry
and was purchased from Dimension Laboratories (Mississauga,
ON).
Induction of LPS-induced acute lung injury and systemic GSH depletion
All animal studies were performed in accordance with guidelines
set forth by the Toronto Hospital Animal Care Committee and the
Canadian Council on Animal Care. Lung injury induced by intratracheal
LPS challenge has been well characterized. It is dependent on
neutrophil influx mediated by up-regulation of ICAM-1 on the pulmonary
capillary endothelium (6) as well as alveolar macrophage induction of
TNF-, IL-1, and CINC (18, 19, 20). Male Sprague Dawley rats weighing 250
to 275 g were obtained from Charles River Laboratories (Constante,
Quebec). Animals were housed in standard wire bottom cages, fed
standard rat chow and water ad libitum, and were allowed to acclimatize
before use. To accomplish systemic GSH depletion, animals were
administered various doses of DEM or phorone by i.p. injection
following an overnight fast. One hour following the administration of
the GSH-depleting agent, animals were anesthetized with sodium
pentobarbital (50 mg/kg i.p.)., and a tracheostomy was performed. LPS
(500 µg) in 0.5 ml of saline was administered by intratracheal
instillation, followed by 20 mechanically ventilated breaths using a
rodent ventilator. Control animals received 0.5 ml of saline alone.
Animals were maintained at 37°C with the use of warming blankets
until recovery from anaesthesia.
Assessment of lung injury
Pulmonary transcapillary albumin transit was assessed by
injection of 1 µCi of 125I-labeled albumin into the
inferior vena cava 30 min before kill. At the end of the experimental
protocol, rats were ventilated, heparin (100 U) was injected into the
right ventricle, and 1 ml of blood was withdrawn by cardiac puncture.
Following exsanguination, lungs were perfused blood-free by cannulating
the pulmonary artery and infusing 10 ml of a low-potassium dextran
solution containing 0.5 µg/L of PGE1. The left ventricle,
left atria, and mitral valve were opened widely to allow free drainage
of effluent. The right lung was immediately frozen in liquid nitrogen
for assay of myeloperoxidase activity, nonprotein thiols, and
evaluation of ICAM-1 and TNF- mRNA expression. The left
lung was used to calculate a permeability index (PI) as follows:
PI = (lung cpm / g tissue) / (blood cpm / ml).
Thioglycollate-induced peritonitis
Male Sprague Dawley rats (250–275 g) were administered DEM as above, followed 1 h later by i.p. injection of thioglycollate (10 ml). Four hours later, animals were killed by carbon dioxide inhalation. Peritoneal exudate cells were obtained following lavage with 60 ml of HBSS. Cell counts were determined using a Coulter counter (Hialeah, FL).
Myeloperoxidase assay
Lung samples were thawed and approximately 0.2 g of tissue was homogenized in 25 ml of potassium phosphate (10 mM, pH 7.4) for 1 min using a Brinkman Polytron (model PT10/35, Brinkman Instruments, Inc., Westburg NY). The homogenate was centrifuged at 12 000 x g for 20 min at 4°C. The supernatant was discarded and the pellet resuspended and homogenized in 25 ml potassium phosphate (50 mM, pH 6.0) containing 0.5% HTAB. The homogenate was frozen overnight at -70°C, rehomogenized for 1 min, and sonicated (model VC 50T, Sonics and Materials Inc., CT) at 40 W for 1 min. After centrifugation as above, the supernatant was collected and used for both MPO and protein assay.
MPO activity was assessed at pH 5.4 and 37°C. The change in absorbance at 655 nm during the first 3 min was measured using the Cobas FARA II Chemistry System (Roche Diagnostic Systems, Montclair, NJ). Each cuvette contained 25 µl of sample, 25 µl of 16 3,3',5,5'-tetramethylbenzidine dissolved in N,N-dimethylformamide and 175 µl of 220 mM potassium phosphate buffer containing 110 mM NaCl. The reaction was initiated by addition of 25 µl of 3.0 mM H2O2, and the change in absorbance during the first 3 min was measured. The absorbance change per minute was used as a measure of MPO activity. Results are expressed as MPO activity per microgram of protein. Protein concentrations were determined using the Pierce BCA protein assay (Pierce, Rockford, IL).
Glutathione assay
Quantitation of total lung and endothelial nonprotein sulfhydryls (NPSH) was assessed using a DTNB-based assay as described by Jocelyn (21). Lung tissue was thawed and weighed, and approximately 0.25 g was homogenized in 2 ml of 5% 5-sulfosalicylic acid (SSA). The homogenate was sonicated for 30 s and centrifuged for 10 min at 10,000 x g. The resultant acid thiol extract was assayed for nonprotein sulfhydryls by quantitating the reduction of DTNB through its conversion to 5-thio-2-nitrobenzoic acid (TNB) at 412 nm using a spectrophotometer. Sample values were then calculated from a standard curve generated using known amounts of GSH and are expressed as GSH equivalents per gram of lung tissue.
ICAM-1 immunohistochemistry
At the time of kill, lungs were inflated with approximately 10 ml of methanol/acetic acid (95%:5%) and sections obtained for immunohistochemical analysis. Tissues were stained with anti-rat ICAM-1 mAb (clone 1A29), followed by a biotinylated goat anti-mouse IgG (Dimension Laboratories, Mississauga, ON). After rinsing, a streptavidin-horseradish peroxidase conjugate was added. The peroxidase reaction was developed by immersion in a freshly prepared solution of 0.02% 3,3'-diaminobenzidine and 0.005% H202 in 0.05 M Tris buffer, pH 7.6, followed by hematoxylin counterstaining. Appropriate negative controls were performed using secondary Ab alone.
Lung RNA extraction and Northern blot analysis
Total RNA from lungs was obtained using the
guanidium-isothiocyanate method (22). Briefly, lungs were harvested
from treated animals and immediately frozen in liquid nitrogen. Lungs
were then thawed and homogenized in 10 ml of 4 M
guanidine-isothiocyanate containing 25 mM sodium citrate, 0.5%
sarcosyl, and 100 mM ß-mercaptoethanol. RNA was denatured,
electrophoresed through a 1.2% formaldehyde-agarose gel, and
transferred to nylon membrane. Hybridization was conducted using an
[-32P]dCTP-labeled, random-primed murine TNF-,
ICAM-1 cDNA, or 18S ribosomal subunit probe (23) or, in the case of
CINC, an end-labeled 30-bp oligonucleotide probe (16). mRNA expression
was quantitated using a phosphoimager and accompanying ImageQuant
software (Molecular Dynamics, Sunnyvale, CA) and standardized to the
18S rRNA signal to correct for any variability in gel loading.
ICAM-1 expression in vitro
Rat heart endothelial cells (RHEC) were isolated from 5-day-old rats according to the method of Kasten (24) and allowed to grow to confluence in 75-cm2 flasks. HUVEC were isolated as previously described (25). The endothelial cells were harvested using trypsin-EDTA treatment and seeded into 96-well, flat-bottom microtiter plates in minimal essential media with 20% FCS. When the monolayers had reached confluence (approximately 4 days) cells were pretreated with DEM for 30 min followed by activation with LPS. After 6 h, supernatants were collected for measurement of lactate dehydrogenase (LDH) activity. Cells were washed twice with RPMI 1640/5% FCS and incubated with mouse anti-rat (clone 1A29) or anti-human ICAM-1 (clone R11/1) mAb for 1 h, washed, and then incubated with peroxidase-conjugated goat anti-mouse IgG for 1 h. Cells were washed again and color development initiated by the addition of the substrate, o-phenyldiamine hydrochloride (OPD). After 18 min, the reaction was stopped with 3 M sulfuric acid, and the optical density at 492 nM was determined using a microtiter plate reader. Data are expressed as the change in absorption from baseline. Quantitation of supernatant LDH activity as a measure of cytotoxicity was determined using spectrophotometric methods as previously described (26). Previous reports have demonstrated that LDH activity is not affected by DEM or other sulfhydryl-reactive agents (27).
PMN transendothelial migration
Human PMN were isolated from healthy volunteers by collecting blood into heparinized tubes. Neutrophils were isolated by dextran sedimentation and centrifugation through a discontinuous Ficoll gradient (28). PMN purity as assessed by size and granularity on flow cytometry was consistently greater than 95%. PMN were labeled with Na251CrO4 and then resuspended in RPMI 1640 containing 0.5% HSA and 10 mM HEPES (pH 7.4) at a concentration of 10 x 106/ml for 30 min and then washed. Transwell chambers with polycarbonate membranes of 3.0 µM pore size (Costar, Cambridge, MA) were coated for 1 h with fibronectin (50 µg/ml) and then seeded with 1 x 105 HUVEC. Cells were incubated in MEM with 20% FCS until confluence as demonstrated by prevention of 125I-labeled albumin diffusion across the endothelial monolayer. PMN transendothelial migration was assessed as described previously (29). Briefly, after treatment of endothelial cells with LPS ± DEM, the upper and lower surfaces of the HUVEC filter units were washed with medium and then transferred to a new, clean well (lower compartment). To this well, 0.6 ml of RPMI 1640 (10 mM HEPES, pH 7.4, 0.5% HSA) was added containing the chemotactic peptide FMLP (10-7 M). Before immersion of the HUVEC filter unit, 0.1 ml of medium containing 2 x 105 labeled PMN was added above the HUVEC. After a 45-min incubation, migration was stopped by washing the upper compartment twice with 0.1 ml of RPMI 1640 to removed nonadherent PMN. The undersurface of the filter was then vigorously rinsed with 2 ml of ice-cold PBS, 0.2% EDTA solution and collected into the lower compartment. The cells that migrated into the lower compartment or that were detached from the undersurface of the filter were lysed by the addition of 0.5% Triton X-100 and analyzed for 51Cr using a Wallac 1480 Wizard automatic gamma counter (Turku, Finland). The results are expressed as the percentage of the total 51Cr PMN added above the HUVEC monolayer that migrated through the HUVEC filter unit. All the stimulation conditions were performed with triplicate replicates.
Statistical analysis
Results are expressed as mean ± SEM of the indicated number of studies. Statistical significance among the group means was assessed by one-way analysis of variance. Post hoc testing was performed using the Bonferroni modification of the t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intratracheal challenge with LPS caused a significant increase in
the lung permeability index (Control: 0.04 ± 0.02 vs LPS:
0.22 ± 0.03; (p < 0.001)) as assessed by
the transcapillary flux of 125I-labeled albumin (Fig. 1
A). Pretreatment with
a single dose of DEM (6 mmol/kg, i.p.) 1 h before intratracheal
challenge had no effect on lung permeability in control animals, yet
completely prevented the increase in permeability associated with LPS
challenge. This protective effect was not evident at doses less than 6
mmol/kg (Fig. 1B). Two lines of evidence suggest that
the effect of DEM was related to its ability to deplete glutathione.
First, DEM conferred protection at 6 mmol/kg, a dose that was capable
of reducing GSH by more than 80%, but not at lower doses that had
limited ability to reduce GSH levels (Fig. 1B).
Second, we evaluated the effect of phorone, another rapidly acting
GSH-depleting agent. Phorone (250 mg/kg) administered by i.p.
administration 1 h before intratracheal LPS challenge induced a
comparable reduction in lung GSH levels to that seen with DEM (Fig. 2A). As shown in Figure 2B, treatment with phorone recapitulated the effect on lung
permeability demonstrated with DEM, with almost complete prevention of
the increase in lung permeability induced by LPS challenge.
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We had previously shown reduced numbers of PMN in the
bronchoalveolar lavage fluid of animals treated with DEM before LPS
challenge. Histologic evaluation of lung tissue following LPS treatment
in the presence or absence of DEM provided some insight into the
mechanism whereby DEM exerted its effect (Fig. 3). LPS challenge causes thickening of
the interalveolar septa due to interstitial edema and influx of PMN
into the lung interstitium. By contrast, in animals pretreated with
DEM, PMN are absent from both within the alveolar spaces and the
interstitium. Further, PMN do not appear to be trapped within the
pulmonary capillaries. The reduction in PMN sequestration in the lung
is also evident upon evaluation of lung MPO activity. Unlike the
quantitation of bronchoalveolar lavage fluid neutrophils, lung MPO
activity evaluates total lung PMN content and is a quantitative measure
of PMN demonstrated on histologic sections. As shown in Figure 4, LPS induces a 6-fold increase in lung
MPO activity. DEM has no effect on MPO activity in control animals, yet
significantly attenuates the increase in lung MPO following LPS
challenge. We have previously demonstrated that DEM induces a
neutrophilia, thus excluding systemic neutrophil depletion as a
possible mechanism for this effect (17). Taken together, these data
suggest that DEM prevents LPS-induced PMN sequestration into the lung
and the ensuing transmigration into the interstitium and alveolar
spaces.
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Lung PMN sequestration is dependent on interactions between
adhesive ligands on the PMN and the pulmonary capillary endothelium.
Abs directed against ß2 integrins or ICAM-1 have
been reported to prevent lung PMN influx in LPS-induced acute lung
injury (6). Having previously demonstrated that PMN up-regulation of
the ß2 integrin CD11b/CD18 was unaffected by GSH
depletion (17), we assessed whether pretreatment with DEM attenuated
LPS-induced up-regulation of its complementary endothelial ligand,
ICAM-1. Analysis of lungs obtained from sham animals stained with an
anti-ICAM-1 Ab demonstrated low level ICAM-1 expression within the
alveolar septa and the epithelial surface (Fig. 5), a reflection of low level
constitutive ICAM-1 expression on the pulmonary capillary endothelium
and alveolar epithelial cells, respectively. Four hours following
intratracheal challenge with LPS, ICAM-1 protein expression is markedly
up-regulated. By contrast, up-regulation of ICAM-1 expression following
LPS challenge was not evident in animals pretreated with DEM,
suggesting that GSH depletion may be preventing lung leukosequestration
through an inhibitory effect on ICAM-1 expression.
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mRNA
To determine whether the observed effect of DEM on LPS-induced
ICAM-1 up-regulation was due to modulation of ICAM-1 gene expression,
we evaluated lung ICAM-1 mRNA expression using Northern analysis. As
demonstrated in Figure 6A,
constitutive ICAM-1 mRNA expression was detectable in whole lungs
obtained from control animals. In animals receiving LPS there was a
significant increase in ICAM-1 mRNA expression. However, consistent
with its effects on tissue expression of ICAM-1 protein, DEM
pretreatment almost completely prevented up-regulation of ICAM-1 mRNA
expression following challenge with LPS. The effect of DEM on the
expression of two other genes whose products are considered to
contribute to lung injury was next evaluated. The LPS-stimulated
increase in mRNA levels of CINC, a chemoattractant protein clearly
shown to be important in LPS-induced lung neutrophil sequestration
(20), was modestly reduced by DEM pretreatment, although this effect
did not reach statistical significance (Fig. 6B).
Further, DEM pretreatment had no effect on LPS-induced lung TNF-
mRNA expression. Together, these findings suggest that the effect of
DEM on ICAM-1 mRNA expression was not due to a global suppressive
effect on gene induction.
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ICAM-1 is expressed on a number of cells present in the lung
including endothelial cells, macrophages, neutrophils, and alveolar
epithelial cells (30). Since DEM appeared to act by reducing lung
leukosequestration, we hypothesized that an effect on endothelial cell
ICAM-1 expression might be responsible. To test this possibility, we
modeled the system in vitro using RHEC activated by LPS. In preliminary
studies, DEM at 250 µM was sufficient to cause a comparable reduction
(
75%) in intracellular GSH levels (No DEM: 6.74 ± 0.08 vs
DEM: 1.66 ± 0.1 nmol/106 cells,
p < 0.01) to that observed in vivo. At this
concentration, DEM did not affect cell viability as determined by
trypan blue exclusion, nor alter the integrity of the cell monolayer as
assessed by permeability to 125I-labeled albumin. Moreover,
supernatant lactate dehydrogenase levels were unaffected, demonstrating
that DEM was not cytotoxic to the RHEC (Table I). LPS (1 µg/ml) treatment of RHEC for
6 h significantly increased ICAM-1 expression (Fig. 7A). Pretreatment with
DEM or treatment concomitant with cell stimulation prevented
up-regulation of ICAM-1 (Fig. 7, A and B).
DEM did not impair ICAM-1 binding by the primary Ab, as normal levels
of ICAM-1 expression were detected with delayed addition (30–60 min)
of DEM. To determine whether the effect of GSH depletion on ICAM-1
expression was species specific, this parameter was similarly evaluated
in HUVEC. As is evident in Figure 7C, HUVEC pretreated with
DEM, followed by activation with LPS, demonstrated a similar
attenuation in ICAM-1 up-regulation.
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Transendothelial migration in vitro is a functional measure of
PMN-endothelial cell interactions. To determine whether the reduction
in ICAM-1 expression mediated by thiol depletion was sufficient to
impair PMN transmigration, we evaluated the effects of endothelial cell
thiol depletion on PMN transmigration in vitro. HUVEC monolayers atop a
3-µm pore polycarbonate filter in a dual compartment chamber were
pretreated with DEM for 30 min followed by activation with LPS (1
µg/ml) for 6 h. Human peripheral blood neutrophils were then
placed above the monolayer, and the rate of transendothelial migration
into the bottom chamber in response to FMLP (10-7)
was evaluated. As shown in Figure 8,
there is an increase in the number of PMN transmigrating across the
filter following activation of the endothelium by LPS. By contrast, DEM
pretreatment attenuates this increase such that the rate of
transmigration is equivalent to that seen under basal conditions. In
parallel studies, pretreatment with anti-ICAM-1 Ab resulted in
inhibition of transmigration comparable to that observed for DEM (data
not shown), suggesting that the reduction in ICAM-1 expression is
sufficient to account for the observed effect.
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Antibody strategies aimed at preventing PMN sequestration at
various extravascular sites have demonstrated site specificity. To
determine whether the effect of GSH depletion on PMN trafficking was
specific to the pulmonary microvasculature, we evaluated the effect of
DEM on thioglycollate-induced PMN influx into the peritoneal cavity.
Four hours following the administration of thioglycollate by i.p.
injection, the number of peritoneal exudate neutrophils increased from
0.71 ± 0.9 x 107 to 4.1 ± 5.5
x 107 (p < 0.01). This increase
was almost completely attenuated by pretreatment with DEM (Fig. 9). DEM had no effect on the basal cell
number within the peritoneal cavity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and CINC
mRNA was unaffected. Further, DEM exerted a similar effect on rat and
human endothelial cells in vitro without cytotoxicity or an alteration
in barrier function. This finding suggests that these agents might
exert their actions directly on endothelial cells in vivo, although it
does not rule out the possibility that DEM might inhibit other cells in
vivo from releasing cytokines that induce endothelial cell ICAM-1
expression. However, as mentioned above, one candidate mediator,
TNF-, did not differ between untreated and DEM-treated animals at
the protein or mRNA level.
Although the principal effect of DEM in vivo is GSH depletion,
nonspecific effects of this agent have been reported. For example DEM
may inhibit protein synthesis, a phenomenon that is an unlikely
contributor to the current observations because phorone recapitulates
the effect of DEM but has no effect on protein synthesis (31). Further,
BALF levels of TNF- are equivalent in DEM-treated and control
animals. DEM has also been reported to reduce core body temperature by
approximately 3.0°C (31). This hypothermic effect is an unlikely
explanation for these data because core body temperature was maintained
through the use of warming blankets.
Several lines of evidence suggest that the protective effects of DEM are related specifically to the thiol-depleting properties of this agent. First, the effect of DEM correlated with its ability to deplete lung GSH. Specifically, concentrations of DEM able to deplete GSH were protective, while lower concentrations had no effect. Secondly, in a previous report we have demonstrated that the beneficial effects of DEM parallel the kinetics of GSH depletion (17). Finally, phorone, an unrelated thiol-depleting agent, had a similar protective effect, excluding the possibility that this phenomenon was due to a conjugate or metabolite of DEM. Taken together, these data suggest that the salutary effects of DEM are related to the ability of this agent to deplete cellular glutathione.
The mechanism whereby thiol depletion might prevent ICAM-1
up-regulation and thus exert its beneficial effect in vivo requires
further study. Northern analysis of total lung mRNA demonstrates that
thiol depletion mediates its effect through prevention of the
LPS-induced increase in ICAM-1 mRNA expression. ICAM-1 mRNA expression
is regulated at the transcriptional level and by modulating the rate of
mRNA degradation (32, 33). In other cell systems, both of these
processes have been shown to be modulated by changes in intracellular
thiol concentrations. The promoter region of ICAM-1 contains a
consensus binding motif for NF-
B. Although thiol oxidation has been
reported to increase nuclear translocation of NF-B, recent evidence
suggests that the overall effect may be more complex due to
redox-sensitive events at the level of DNA binding (34). In the
nucleus, thiol oxidation has been shown to prevent the binding of
NF-B to its DNA binding site by forming mixed disulfides at a
redox-reactive cysteine in the DNA binding region of NF-B,
potentially leading to a reduction in NF-B-dependent gene
expression. Relevant to this proposed mechanism, alveolar macrophage
expression of TNF- and CINC is also dependent on NF-B activation
and is responsive to redox manipulation (16, 35). The lack of any
significant effect on these macrophage products suggests either a
greater resistance of macrophages to glutathione depletion or the
presence of a greater potential for redox buffering in these cells.
Alternatively, the systemically administered DEM may achieve greater or
more sustained levels in endothelium as compared with macrophages.
Alternatively, thiol depletion may play a role in modulating ICAM-1 mRNA stability. The 3' untranslated region of ICAM-1 mRNA has several reiterations of the pentamer adenosine-uridine-uridine-uridine-adenosine (32). This adenosine-uridine-rich element (ARE) is a relatively well-conserved motif in several labile transcripts. Recently several trans-acting mRNA binding proteins that interact with the ARE have been identified. The binding activity of these proteins may either proportionally or inversely correlate with transcript stability (36). Further, critical thiol groups at the mRNA binding site may modulate binding to the ARE and thus alter mRNA stability (37), suggesting that a reduction in ICAM-1 mRNA stability may account for the reduction in steady state ICAM-1 mRNA observed following thiol depletion.
ICAM-1 is an important ligand for the ß2 integrin family of leukocyte membrane glycoproteins. Interactions between these complementary surface molecules mediate firm adhesion of the PMN to the endothelial cell and are a prerequisite for PMN transmigration across endothelial cell monolayers in vitro and into the interstitium in vivo (38). The observed effects of GSH depletion on PMN influx into the lung and peritoneal cavity are thus consistent with the importance of ICAM-1 in these processes. However, the profound reduction in lung neutrophil sequestration in animals pretreated with DEM suggests that this agent may have additional effects on PMN-endothelial interactions apart from the effect on ICAM-1 expression demonstrated in the present studies. We previously reported that DEM administration in vivo had no effect on the levels of CD11b/CD18 on the surface of circulating neutrophils. Recent studies have demonstrated that CD11a/CD18 in addition to CD11b/CD18 may play a significant role in PMN sequestration during inflammation (6). Further, these studies did not specifically address alterations in the adhesive interactions of these cells. Alternatively, DEM may have additional effects on other endothelial cell adhesion molecules involved in lung leukosequestration including E-selectin and VCAM. These possibilities require further study.
A limited number of other studies have demonstrated beneficial effects associated with GSH depletion, although the mechanisms for these effects were not evaluated. Both buthionine sulfoximine (BSO), an agent that prevents de novo GSH synthesis, and DEM reduced mortality following cerebral ischemia-reperfusion injury (39). Thiol depletion has also been reported to be protective in a murine model of endotoxin/galactosamine-induced hepatic necrosis (40). The common pathogenetic link between these reported observations and the current studies is the central role of PMN-endothelial cell interactions leading to PMN sequestration and target organ injury. The present studies provide sound evidence that the salutary effect observed in each model may have been related to impaired up-regulation of endothelial cell adhesion molecules. Importantly, this information suggests that treatment with thiol-depleting agents may be generally effective in pathologic processes mediated by endothelial cell-PMN interactions involving ICAM-1, a phenomenon demonstrated by the abrogation of thioglycollate-elicited PMN influx into the peritoneal cavity in animals pretreated with DEM in the current study.
There is substantial evidence to suggest that alterations in the redox state of the cell may modulate cellular and gene activation. The data presented herein demonstrate that cellular redox manipulation achieved by depletion of intracellular thiol levels may prove useful in modulating the broad range of disease processes characterized by neutrophilic infiltration. While lowering of the predominant endogenous cellular antioxidant may seem an unlikely therapy for disease processes characterized by neutrophil-mediated tissue injury, this strategy appears to be effective by virtue of its ability to prevent neutrophil sequestration at the inflammatory site. This suggests that redox manipulation may be most effective if directed toward the modulation of cell activation rather than post hoc attempts at minimizing PMN-mediated injury to target organs, a strategy that has achieved only modest success in man (41).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ori D. Rotstein, Eaton North 9-232, The Toronto Hospital, 200 Elizabeth St., Toronto, Canada, M5G 2C4.
3 Abbreviations used in this paper: ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; CINC, cytokine-induced neutrophil chemoattractant; DEM, diethylmaleate; GSH, glutathione; MPO, myeloperoxidase; RHEC, rat heart endothelial cells; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); LDH, lactate dehydrogenase; ARE, adenosine-uridine-rich element; HSA, human serum albumin; PMN, polymorphonuclear neutrophil.
Received for publication June 4, 1997. Accepted for publication November 18, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B site and p65 homodimers. J. Biol. Chem. 270:933.B mobilization and TNF production in human monocytes. J. Immunol. 151:6986.[Abstract]
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 G. Zhang, R. D. Nichols, M. Taniguchi, T. Nakayama, and M. J. Parmely Gamma Interferon Production by Hepatic NK T Cells during Escherichia coli Infection Is Resistant to the Inhibitory Effects of Oxidative Stress Infect. Immun., May 1, 2003; 71(5): 2468 - 2477. [Abstract] [Full Text] [PDF]
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 T. H. Wyman, A. J. Bjornsen, D. J. Elzi, C. W. Smith, K. M. England, M. Kelher, and C. C. Silliman A two-insult in vitro model of PMN-mediated pulmonary endothelial damage: requirements for adherence and chemokine release Am J Physiol Cell Physiol, December 1, 2002; 283(6): C1592 - C1603. [Abstract] [Full Text] [PDF]
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 E.-B. Haddad, K. McCluskie, M. A. Birrell, D. Dabrowski, M. Pecoraro, S. Underwood, B. Chen, G. T. De Sanctis, S. E. Webber, M. L. Foster, et al. Differential Effects of Ebselen on Neutrophil Recruitment, Chemokine, and Inflammatory Mediator Expression in a Rat Model of Lipopolysaccharide-Induced Pulmonary Inflammation J. Immunol., July 15, 2002; 169(2): 974 - 982. [Abstract] [Full Text] [PDF]
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M. J. Parmely, F. Wang, and D. Wright Gamma Interferon Prevents the Inhibitory Effects of Oxidative Stress on Host Responses to Escherichia coli Infection Infect. Immun., April 1, 2001; 69(4): 2621 - 2629. [Abstract] [Full Text] [PDF]
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 J. Nilsson, O. Soderberg, K. Nilsson, and A. Rosen Thioredoxin prolongs survival of B-type chronic lymphocytic leukemia cells Blood, February 15, 2000; 95(4): 1420 - 1426. [Abstract] [Full Text] [PDF]
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F. Wang, L. Y. Wang, D. Wright, and M. J. Parmely Redox Imbalance Differentially Inhibits Lipopolysaccharide-Induced Macrophage Activation in the Mouse Liver Infect. Immun., October 1, 1999; 67(10): 5409 - 5416. [Abstract] [Full Text] [PDF]
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p < 0.001 vs LPS, no DEM. B,
Effect of increasing the administered dose of DEM on lung injury and
lung thiol levels. Data are expressed as mean ± SEM of four
animals per group. Bars, permeability index; solid line, lung thiol
levels. *, p < 0.001 vs LPS, no DEM (0 mmol/kg). 
