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Nuclear factor-
B (NF-B) and glucocorticoid receptor-
(GR-)
have diametrically opposed functions in regulating inflammation. We investigated whether unresolving acute respiratory distress syndrome (ARDS) is associated with systemic inflammation-
induced glucocorticoid resistance and whether prolonged methylprednisolone administration accelerates the suppression of systemic inflammatory indices and normalizes the sensitivity of the
immune system to glucocorticoids. Patients enrolled into a randomized trial evaluating prolonged methylprednisolone administration in unresolving ARDS had serial plasma samples collected
before and after randomization. In the plasma, we measured the
concentrations of tumor necrosis factor- (TNF-), interleukins (IL)
IL-1
and IL-6, adrenocorticotropic hormone (ACTH), and cortisol.
The ability of patient plasma to influence the NF-B and GR-signal
transduction systems of normal peripheral blood leukocytes (PBL)
was examined. Patients treated with methylprednisolone had progressive and sustained reductions of TNF-, IL-1, IL-6, ACTH, and
cortisol concentrations over time. Normal PBL exposed to plasma
samples collected during methylprednisolone exhibited significant
progressive increases in all aspects of GR-mediated activity and
significant reductions in NF-B DNA-binding and transcription of
TNF- and IL-1. These findings provide support for the presence
of endogenous glucocorticoid inadequacy in the control of inflammation and systemic inflammation-induced peripheral glucocorticoid resistance in ARDS. Prolonged methylprednisolone administration accelerated the resolution of both systemic inflammation and peripheral acquired glucocorticoid resistance in ARDS.
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Keywords: acute respiratory distress syndrome; glucocorticoid receptors; IB; methylprednisolone; nuclear factor-B
Excessive systemic inflammation is the pathophysiologic hallmark of pulmonary and extrapulmonary organ dysfunction in
patients with acute respiratory distress syndrome (ARDS) (1),
a frequent cause of hypoxemic respiratory failure associated
with a 40% to 60% mortality (2). It is now appreciated that
systemic inflammation in ARDS is sustained over time and
that the final outcome is affected by its degree and duration
(1). Two cellular signaling pathways are central to the host inflammatory response, the stimulatory nuclear factor-B (NF-B)
and the inhibitory glucocorticoid receptor- (GR-)-mediated
signal transduction cascades (3).
NF-B is a heterodimeric protein composed of the DNA-binding proteins p65 and p50 constitutively present in the cytoplasm in an inactive form stabilized by the binding to the inhibitory protein IB (4). Cellular activation by a multitude of
adverse stimuli leads to phosphorylation and proteolytic degradation of IB (4). The liberated NF-B then translocates
into the nucleus and binds to promoter regions of target genes
to initiate the transcription of multiple cytokines including tumor necrosis factor- (TNF-); the interleukins (IL) IL-1,
IL-2, IL-6; and chemokines such as IL-8, cell adhesion molecules, and inflammation-associated enzymes (4). Products of
the genes that are stimulated by NF-B activate this transcription factor. Thus, TNF- and IL-1 both activate and are activated by NF-B by forming a positive regulatory loop that
amplifies and perpetuates inflammation (5).
Glucocorticoid hormones (GC), produced by the adrenal
cortices, are the most important physiologic inhibitors of inflammation. GC exert most of their effects by activating ubiquitously distributed cytoplasmic heat shock protein-complexed
glucocorticoid receptors (GR) with formation of GC
GR-
complexes (6). It is now appreciated that the GCGR- complexes modulate transcription in a hormone-dependent manner by binding to glucocorticoid response elements (GRE) in the promoters of glucocorticoid responsive genes and by interfering with the activity of other transcription factors such as
NF-B on genes regulated by these factors (7). GR-mediated
transcriptional interference is achieved by five important
mechanisms: (1) by physically interacting with the p65 subunit
and formation of an inactive (GR-NF-B) complex (6); (2)
by inducing the transcription of the inhibitory protein IB
gene (6, 8, 9); (3) by blocking degradation of IB via enhanced
synthesis of IL-10 (10); (4) by impairing TNF-
induced degradation of IB (11); and (5) by competing for limited
amounts of GR coactivators such as CREB-binding protein
and steroid receptor coactivator-1 (12).
Endogenous glucocorticoids are not always effective in suppressing life-threatening systemic inflammation, even though the degree of cortisolemia frequently correlates with severity of illness and mortality rate (13-16). Failure to suppress inflammation could be due to inadequacy of, and/or tissue resistance to, the concentrations and durations of endogenous glucocorticoid elevations, which allow the systemic inflammatory response to go awry (17). We have recently reported a significant physiologic and survival benefit when prolonged glucocorticoid treatment at moderate doses was administered late (9 ± 3 d) in the course of ARDS to patients failing to improve (18). We hypothesized that if endogenous glucocorticoid inadequacy and/or peripheral tissue resistance are important pathophysiologic factors in a dysregulated, protracted systemic inflammatory response in ARDS, then prolonged glucocorticoid therapy may be useful, not as an antiinflammatory treatment per se, but as hormonal supplementation necessary to compensate for the host's inability to produce appropriately elevated amounts of cortisol for the degree of peripheral glucocorticoid resistance (19).
In this study, we tested the hypothesis that prolonged methylprednisolone versus placebo administration in patients with
unresolving ARDS suppresses inflammation and/or corrects
the glucocorticoid resistance of the inflammatory response of
these patients. To test this hypothesis, we measured serially
the plasma levels of TNF-, IL-1, IL-6, adrenocorticotropic
hormone (ACTH), and cortisol in patients with unresolving
ARDS treated with methylprednisolone or placebo and exposed a healthy volunteer's peripheral blood leukocytes (PBL)
to plasma samples from the patients. In the exposed cells, we
measured upstream and downstream events associated with the NF-B and glucocorticoid transduction cascades as they
oppose each other's actions.
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Patient Selection
The original study was conducted between October 1994 and November 1996 in the intensive care units of Baptist Memorial Medical Center and East hospitals, the Regional Medical Center, and the University of Tennessee Bowld Medical Center, all in Memphis, TN. The
study protocol was approved by each institutional review board, and
informed consent was obtained before enrollment. An active effort
was made to identify and recruit eligible patients with ARDS. Patients
at least 18 years old were eligible if they met previously described
ARDS consensus criteria (20) and had a lung injury score (LIS) (21)
2.5. The ventilator management followed recently developed guidelines aimed at limiting plateau pressure to less than 35 cm H2O (22).
Positive end-expiratory pressure was increased by 3-5 cm H2O (up to
20 cm H2O) to achieve the best lung compliance and oxygen saturation
and to maintain FIO2 less than or equal to 0.6. The presence or absence
of improvement in lung function (as defined by LIS [21]) by Day 7 of
ARDS was used to categorize patients as improvers or nonimprovers
and ARDS as resolving or unresolving. On mechanical ventilation Day
9 ± 3, 24 nonimprovers were enrolled into a prospective, randomized, double-blind, placebo-controlled trial evaluating prolonged methylprednisolone treatment (18). Randomization was done on a 2:1 basis;
16 patients received methylprednisolone, and 8 received placebo. Data
from 17 of the 24 randomized patients are reported in the present article. Serial blood samples were available for analysis in 17 patients, 6 of
whom received placebo and served as control subjects. In 7 of the 24 randomized patients, blood samples were either not obtained (three
patients) or were inadequate for serial measurements (four patients).
Methylprednisolone or placebo was given daily as intravenous push
every six hours (one-fourth of the daily dose) and changed to a single
oral dose when oral intake was restored. If the patient was able to tolerate oral intake and had no obvious gastrointestinal dysfunction (i.e.,
diarrhea, etc.), we presumed that the gastrointestinal tract was functional. A loading dose of 2 mg/kg was followed by 2 mg/kg/day from
Day 1 to 14, 1 mg/kg/day from Day 15 to 21, 0.5 mg/kg/day from Day
22 to 28, 0.25 mg/kg/day on Days 29 and 30, and 0.125 mg/kg/day on
Days 31 and 32. During the study, components of the LIS (21), and
multiple organ dysfunction syndrome score (23) were collected, and
results are published elsewhere (24).
Table 1 shows clinical characteristics at the onset of ARDS. The 11 patients randomized to methylprednisolone improved LIS by study Day 10 and survived. In the placebo group, two patients improved LIS by study Day 10 and survived, whereas four failed to improve LIS. Two of the four nonimprovers died within seven days of randomization.
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Plasma Collection and Description of the Ex Vivo Model of ARDS
Blood samples were obtained on Days 1, 3, 5, 7, and 10 of ARDS, on the
day of randomization (Day 0), and on Days 3 (+3), 5 (+5), 7 (+7), and
10 (+10) of treatment. All blood samples were obtained from a central
venous line or an antecubital venipuncture, collected in a vacutainer
tube containing ethylenediaminetetraacetic acid, placed immediately on
ice after collection, and transported to the laboratory for immediate processing. A complete blood cell count with differential was determined
using the Coulter ACT diff Hematology Analyzer (Beckman-Coulter,
Miami, FL). Blood samples were centrifuged at 500 × g for 10 minutes
in a refrigerated centrifuge, and plasma was aspirated and aliquoted in
plastic storage tubes. All samples were stored at 80° C until assay.
The ex vivo model of ARDS consisted of exposing PBL obtained from a single healthy volunteer to plasma samples obtained from patients with ARDS. Using this ex vivo model, we attempted to simulate the in vivo milieu, but we realize that there are limitations to the inferences that can be made by using this approach.
Laboratory Methodology
The laboratory methodology section is available in the online data
supplement and includes the following: isolation of PBL and exposure
to patients' plasma; determination of plasma cytokine, ACTH, and
cortisol concentrations; cellular fractionation and protein determination; electrophoretic mobility gel shift assay (EMSA); detection of
NF-B and GR- binding to their response elements; quantification
of EMSA, determination of NF-B subunits, GR-, and IB by
Western blotting; and quantitative reverse transcription-polymerase chain reaction.
Statistical Analysis
This study has an unbalanced nested factorial design, with groups
(methylprednisolone and placebo) as the main plot effect; patients
were nested within groups; and time was cross-classified with the
groups and patients. We made the assumption that the variances of
continuous dependent variables on the assessment days were equal. If
the variances were not equal or the distributions were positively
skewed, data were transformed using natural logarithms. Comparisons were made both within and across groups. For comparisons within groups, the variance for each dependent variable was estimated by the pooled within-patient variance from data measured repeatedly over time. For comparisons across groups, that variance was
estimated by the weighted average of the residual mean squares and
the between-patient mean squares from repeated measures analysis of
variance (25). Although constituents in plasma serially collected from
patients were moderately and positively correlated, we assume that
results from cell cultures were independent. For each group, partial
correlation coefficients among selected intracellular markers, adjusted for repeated measurements on patients, were estimated. The
assumptions necessary for this analysis are that (1) the markers are
linearly associated over time and (2) the residuals are independently
and identically distributed normally with variance 
2
. For all preplanned or a priori contrasts stipulated in the main hypotheses, a significance level of 0.05 was chosen for statistical significance. All hypothesis tests are two-tailed. Data were analyzed using the SAS
statistical software package (SAS Institute, Inc., Cary, NC).
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Cytokine Concentrations from Plasma of Randomized Patients
A total of 88 blood specimens was available for analysis, 58 from patients in the methylprednisolone group and 30 from
patients in the placebo group. Figure 1 shows the patients'
plasma TNF-, IL-1, and IL-6 levels before and after randomization. On Day 1 of ARDS and up to randomization,
plasma TNF-, IL-1, and IL-6 levels were similar in both
groups. After randomization, plasma TNF-, IL-1, and IL-6
levels declined rapidly in the methylprednisolone group (on
Days 5 to 7, p < 0.0001 for all three cytokines), whereas the
control group had lesser reductions in plasma TNF- and IL-6 and no reduction in plasma IL-1. After randomization, the
methylprednisolone group had significantly (p < 0.0001) lower
plasma IL-1 concentrations than the control group for each
recorded interval.
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ACTH and Cortisol Concentrations from Plasma of Randomized Patients
Figure 2 shows plasma ACTH and cortisol concentrations in patients before and after randomization. On Day 1 of ARDS and up to randomization, plasma ACTH and cortisol levels were similar in both groups. After randomization, the methylprednisolone group had significant and sustained (on Days 5 to 7, p < 0.005 for both measurements) reductions in plasma ACTH and cortisol concentrations, whereas no reductions were observed in the control group.
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NF-B and Its Subunits from Cells of a Healthy Volunteer
Figure 3 shows NF-B, p65 subunit, and p50 subunit binding
to response elements before and after randomization. On Day
1 of ARDS, densities of NF-B, p65 subunit, and p50 subunit
were similar in cells exposed to plasma from both groups.
From Day 1 of ARDS to randomization, densities of all three
proteins increased significantly and similarly in cells exposed
to plasma from both groups. After randomization, cells
treated with plasma from the methylprednisolone group exhibited significant progressive reductions in NF-B and its
subunits (on Day 3, p < 0.02 for all three measurements), whereas no changes were observed in those exposed to plasma
from the control group. After randomization, significant differences (p < 0.01) between the two groups were observed for
NF-B for each recorded interval.
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GR--Mediated Activity
Figure 4 shows cytoplasmic GR- bound and unbound to NF-B
before and after randomization. On Day 1 of ARDS, cells exposed to plasma of both groups had similar densities of GR-.
After exposure to plasma obtained from Day 1 of ARDS up
to randomization, the amounts of GR- bound to NF-B did
not change, whereas the amounts of GR- unbound decreased
significantly. After randomization, exposure to plasma from
the methylprednisolone group was associated with significant
increases (on Day 3, p < 0.0001 for both measurements) in
GR- bound and unbound, whereas lesser increases were observed in cells exposed to plasma from the control group. After randomization, a significant difference (p < 0.0001) between the two groups was observed for cytoplasmic GR-
bound and unbound to NF-B for each recorded interval.
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Figure 5 shows cytoplasmic IB and GR- binding to response elements. On Day 1 of ARDS and up to randomization, cells exposed to plasma from both groups had similar
densities of cytoplasmic IB and GR- bound to the response elements. After randomization, exposure to plasma
from the methylprednisolone group was associated with significant increases (on Day 3, p < 0.0001 for both measurements) in cytoplasmic IB and GR- bound to the response
elements. In contrast, cells exposed to plasma from the control
group had lesser increases in GR bound to the response elements and significant reductions in cytoplasmic IB densities. After randomization, a significant difference (p < 0.0001)
between the two groups was observed for cytoplasmic IB
and GR- binding to response elements for each recorded interval.
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Cytokine Transcription
Figure 6 shows TNF-, IL-1, and IL-10 mRNA levels measured in media of cells exposed to plasma obtained before and
after randomization. On Day 1 of ARDS and up to randomization, TNF-, IL-1, and IL-10 mRNA levels were similar in
media of cells exposed to plasma from both groups. TNF-
and IL-1 mRNA were significantly lower after exposure to
plasma from the methylprednisolone group after randomization (p < 0.0001 for both types of mRNA on each recording),
whereas cells exposed to plasma from the control group after
randomization had no reductions in TNF- and IL-1 mRNA levels. Cells exposed to plasma from the methylprednisolone
group after randomization had significant (p < 0.0001) and
progressive increases in IL-10 mRNA levels, whereas no
changes were observed in cells exposed to plasma from the
placebo group after randomization until Day 10. After randomization, significant differences (p < 0.01) between the two
groups were observed for IL-1 and IL-10 mRNA levels for
each recorded interval.
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Relations among Selected Variables after Randomization
Figure 7 depicts the relations on natural logarithmic scales between mean levels of nuclear NF-B and nuclear GR- (top)
and between mean levels of nuclear NF-B and cytoplasmic
GR- bound to NF-B (bottom), a factor affecting the translocation of activated NF-B to the nucleus. Untransformed
means of these transcription factors were depicted separately
in Figures 3 and 4. After natural logarithmic transformation
and adjustment for repeated measurements, partial correlations among responses to plasma from the methylprednisolone group were 0.92 (p < 0.0001) both for nuclear NF-B and
nuclear GR- and for nuclear NF-B and cytoplasmic GR-
bound to NF-B. For responses to plasma from the placebo
group, no significant relationship was found between nuclear
NF-B and nuclear GR- (r = 0.11; p = 0.70) or between NF-B
and cytoplasmic GR- bound to NF-B (r = 0.33; p = 0.23).
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Figure 8 depicts the relations on natural logarithmic scales
between mean levels of IB and factors affecting its formation (nuclear GR-) and degradation (IL-10 mRNA and TNF-
mRNA). Untransformed means of these factors were depicted
separately in Figures 5 and 6. After natural logarithmic transformation and adjustment for repeated measurements, partial
correlations were +0.97 (p < 0.0001) between IB and nuclear GR-, +0.98 (p < 0.0001) between IB and IL-10
mRNA, and 0.95 (p < 0.0001) between IB and TNF- mRNA. In contrast, for responses to plasma from the placebo
group, the partial correlation coefficients were 0.73 (p = 0.003) between IB and nuclear GR-, 0.85 (p < 0.0001)
between IB and IL-10 mRNA, and +0.27 (p = 0.33) between IB and TNF- mRNA. Figure E1 in the web repository shows the EMSA of nuclear extract of NF-B over time
from one patient randomized to methylprednisolone and from
one patient randomized to placebo.
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Patients treated with methylprednisolone had rapid, progressive, and sustained reductions in plasma TNF-, IL-1, IL-6,
ACTH, and cortisol concentrations over time. These were associated with parallel improvements in pulmonary and extrapulmonary organ dysfunction scores (previously reported in
reference [18]). Normal PBL exposed to plasma samples collected during methylprednisolone versus placebo treatment
also exhibited rapid, progressive significant increases in GR--mediated activities (GR- binding to NF-B, GR- binding to GRE DNA, stimulation of inhibitory protein IB, and
stimulation of IL-10 transcription) and significant reductions
in NF-B binding and transcription of TNF- and IL-1.
These findings provide strong mechanistic evidence for the
efficacy of prolonged methylprednisolone treatment in unresolving ARDS.
In the observation period before randomization, the biologic and physiologic characteristics of the methylprednisolone and placebo groups were similar. Patients had persistent
elevations in plasma concentrations of inflammatory (TNF-,
IL-1, and IL-6) cytokines and hypothalamic-pituitary-adrenal (HPA)-axis (ACTH and cortisol) hormones and similar severity of organ dysfunction scores. We hypothesized that inadequate secretion of cortisol and/or immune tissue resistance to
endogenous glucocorticoids might explain the observed failure to suppress inflammation in the presence of persistently
elevated ACTH and cortisol concentrations. Because the GR
ultimately controls GC-mediated activity, anything that affects its binding affinity, concentration, transport to the nucleus, interactions with GRE, or other relevant transcription factors and coregulators can ultimately affect the response of cells to glucocorticoids (6). GR-mediated resistance was originally described as a primary inherited familial syndrome (26,
27) and was recently recognized as an acquired condition.
Among others, acquired immune tissue-specific GR resistance
has been described in patients with asthma (28-31), acquired
immunodeficiency syndrome (32), and severe sepsis (33).
Recent in vitro studies have shown that cytokines may induce resistance to glucocorticoids by reducing GR- binding
affinity to cortisol and/or GRE (34-36). Such abnormalities of
GR- function were demonstrated in T cells incubated with a
combination of IL-2 and IL-4 (35), IL-1, IL-6, and interferon-
(34), or IL-13 (36). Glucocorticoid resistance was induced in a
cytokine concentration-dependent fashion and was reversed
by the removal of cytokines (35). GR-mediated resistance in
the presence of systemic inflammation was also studied in experimental models of sepsis and sepsis-induced ARDS (33, 37,
38). In a sheep model of sepsis-induced ARDS, maximal binding capacity of GR decreased continuously after endotoxin infusion, whereas there was a marked elevation of cortisol
concentrations (37). The reduced GR binding correlated negatively (r = 0.87; p < 0.01) with phospholipase A2 activity, a
gene that is stimulated by NF-B. In a rat model of septic
shock, GR blockade by mifepristone (RU 486) exacerbated
the physiologic and pathologic changes induced by endotoxemia (38). Phospholipase A2 activity in rats with 80% GR
blockade was more marked than in those with 50% GR blockade (38). Monocytes of patients with sepsis developed near-
total glucocorticoid resistance in vitro when cytokines, especially IL-2, were added (33).
Several inflammatory cytokines, including TNF-, IL-1,
and IL-6, activate NF-B (39). It has been proposed that when
cytokine-activated NF-B forms protein-protein complexes
with activated GR-, the availability and activity of effective
GR- molecules are reduced (6, 31). This functional reduction
in GR- availability is associated with decreased GR--GRE
DNA binding and GC-mediated antiinflammatory activity (6,
31), and our findings support this hypothesis. The intracellular
changes observed by exposing leukocytes of healthy volunteers
to plasma samples collected before randomization included
escalating increases in NF-B-mediated activities (NF-B
DNA-binding, p50 and p65 DNA-binding, and transcription of TNF- and IL-1) and modest changes in GR--mediated
activities. The reduction in cytoplasmic IB levels observed
before randomization indicates that NF-B-mediated IB
degradation predominated over GR--mediated IB formation (40).
If acquired glucocorticoid receptor resistance played a role in
the pathogenesis of unresolving ARDS, adequate hormonal supplementation should restore glucocorticoid antiinflammatory
function, by decreasing the production of inflammatory cytokines, cytokine-driven HPA-axis activity, and cytokine-driven
organ dysfunction. Indeed, after randomization, the biologic
and physiologic characteristics of the two groups (methylprednisolone versus placebo) rapidly diverged. The responses observed during methylprednisolone administration support the
concept of inflammation-dependent acquired glucocorticoid resistance in patients with ARDS. We found that the methylprednisolone-treated group had significant, progressive reductions in plasma concentrations of TNF-, IL-1, IL-6, ACTH,
and cortisol and a parallel improvement in severity of organ
dysfunction scores (18). The biologic response observed in this
study was similar to that of a prior uncontrolled report (41). In
that study, methylprednisolone treatment was associated with
significant and parallel reductions in plasma and bronchoalveolar lavage (BAL) TNF-, IL-1, IL-6, and IL-8 concentrations; LIS; and BAL indices of pulmonary vascular permeability (BAL albumin and percentage of polymorphonuclear
cells) (41).
Our ex vivo findings reflect the effects of a mixture of inflammatory cytokines and other factors and the variable methylprednisolone and cortisol concentrations in the plasma of
our patients on normal nonactivated circulating blood leukocytes. The concentration of methylprednisolone in the plasma
samples was unknown and may have varied during the study
period. A prior study has shown similar changes in NF-B activation in the peripheral monocytes of patients with septic
shock (42) or with critical illness (43). These data suggest that
the glucocorticoid resistance of the immune system of ARDS
patients simply reflects the inflammatory state of the individual.
The extent to which methylprednisolone in the plasma has
biased the magnitude of responses is unknown. We conducted
an experiment to quantify this bias (data not reported). The
amount of IL-8 released from fresh PBL incubated for four
hours with plasma from a patient who received methylprednisolone was four times greater when the methylprednisolone
had been removed by dialysis compared with undialyzed
plasma. For TNF- release, the effect of removing methylprednisolone was an increase of 1.6 times the amount compared with undialyzed plasma. Thus, the degree of bias probably
varies greatly, depending on what intracellular or extracellular
variable is selected. We point out that in the current report the
results from PBL of a healthy volunteer are consistent with
the outcomes of patients randomized in the clinical trial (18).
However, we explicitly acknowledge this known source of bias
on the magnitude, significance, and possibly the direction of
the differences presented in this report.
Our current understanding of the physiologic antagonism
between the NF-B and GR- cascades explains our findings
on the exposure of normal leukocytes to plasma samples from
patients receiving methylprednisolone or placebo. With methylprednisolone administration, the intracellular relations between the NF-B and GR- signaling pathways changed from
an initial NF-B-driven and GR--resistant state to a GR--
driven and GR--sensitive one. In contrast, exposure to serial
plasma samples collected during placebo administration demonstrated a significantbut lower (in comparison with the
methylprednisolone group, p < 0.0001)increase in GR--
mediated activity over time, and persistently elevated NF-B activity.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. G. Umberto Meduri, University of Tennessee Health Science Center, Division of Pulmonary and Critical Care Medicine, 956 Court Avenue, Room H316, Memphis, TN 38163. E-mail: umeduri{at}utmem.edu
(Received in original form June 6, 2001 and accepted in revised form January 22, 2002).
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.orgAcknowledgments: The authors recognize James Dalton, Pharm.D., and Pierluigi Carratu, M.D., for the critical review of this manuscript. They are indebted to their research nurses, Stephanie Carson, Mary Gibson, and Reba Umberger; to David Patterson for laboratory assistance; and to their collaborating clinical investigators, Drs. Emmel Golden and Stacey A. Headley. They acknowledge Vivian Gomez for the creation of the figures, and Gail Spake and David Armbruster for editing the manuscript.
This study was supported by the Assisi Foundation of Memphis and the Baptist Memorial Health Care Foundation.
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