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B and IB
in peripheral blood mononuclear cells of trauma patients
* Département de Physiopathologie, Institut Pasteur, 75724 Paris Cedex 15, France, and
Département d’Anesthésie Réanimation, Hôpital du Kremlin Bicêtre, 94275 Le Kremlin Bicêtre Cedex, France
Correspondence: Dr. Pierre Moine, Département d’Anesthésie Réanimation, Hôpital du Kremlin Bicêtre, 78 rue du général Leclerc, 94275 Le Kremlin Bicêtre Cedex, France. E-mail: pierre.moine1{at}fnac.net
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B expression and dimer characteristics were
studied in peripheral blood mononuclear cells (PBMCs) of major-trauma
patients and healthy controls. Analysis of PBMCs on days 1, 3, 5, and
10 after trauma revealed that expression of both p65p50 heterodimers
and p50p50 homodimers was significantly reduced compared with that in
controls. In vitro lipopolysaccharide (LPS) stimulation of PBMCs
induced NF-B translocation. However, throughout the survey, p65p50
activation remained significantly lower in trauma patients than in
controls. After LPS stimulation in vitro, the p65p50/p50p50 ratio was
significantly lower in PBMCs from trauma patients than from healthy
controls. The ex vivo expression of IB
was higher in PBMCs of
controls than of trauma patients. LPS did not induce IB expression
in PBMCs from trauma patients, but strong induction was obtained with
staphylococci, suggesting that this defect is not universal and depends
on the nature of the activating signal. Although no direct correlation
was found between levels of interleukin-10 or transforming growth
factor-ß and NF-B, these immunosuppressive cytokines were
significantly elevated in trauma patients by 10 days after admission.
The long-term low-basal and LPS-induced nuclear translocation of
NF-B recalled long-term immunoparalysis observed in patients with
severe inflammatory stress such as trauma.
Key Words: lipopolysaccharide • inflammation • IL-10 • immunoparalysis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Polytrauma causes a decreased capacity of a patient’s leukocytes to
produce proinflammatory cytokines (TNF-, IL-1ß, IL-6,
interferon-
, and IL-8) in response to endotoxins ex vivo
[14
, 15
]. These phenomena may lead to an
increased susceptibility to sepsis and correlate with increased
mortality in shocked animals subsequently challenged with a septic
insult [5
, 16
]. Several studies indicate
that depression of the patient’s immune function induced by traumatic
injury is etiologically involved in the development of infection or
sepsis [5
, 17
]. Nevertheless, the
mechanisms behind the maintenance of the sustained suppression of the
immune function remain incompletely understood.
The Rel/NF-B family of transcription factors is involved in the
regulation of immune and acute-phase responses at the transcriptional
level [18
]. NF-B regulation may be critical for
cytokine gene expression in trauma, because of its presence in the
enhancer and promoter regions of proinflammatory cytokine (e.g.,
TNF-, IL-1ß, IL-6, and IL-8) genes and its inducibility by several
extracellular signals known to be present in trauma patients, such as
reactive oxygen species, cytokines, endotoxin, and complement fragments
[19
, 20
]. Rel proteins can be divided into
two groups based on their structures, functions, and modes of
synthesis. The first group of Rel proteins consists of p65 (RelA),
c-Rel, and RelB, each of which contains one or more
transcriptional-activation domains necessary for gene induction
[19
]. The second group consists of p105 and p100, which,
upon proteolytic processing, give rise to p50 (NF-B1) and p52
(NF-B2), respectively [18
]. Members of both groups of
Rel proteins can form homo- or heterodimers. Many concordant results
have shown that the transactivator form of NF-B is the p65 unit,
whereas the p50 unit has shown no or minimal activation capacity
[21
22
23
24
].
The activity of Rel/NF-B complexes is regulated by their
interactions with members of the IB family of inhibitors
[18
, 19
]. IB and IBß retain
Rel/NF-B dimers in the cytoplasm through masking of their nuclear
localization sequences and inhibit the DNA-binding activity of NF-B
but not that of the p50 homodimer [25
]. For IB,
induction leads to rapid phosphorylation and degradation of the
IB molecule, allowing translocation of Rel/NF-B dimers to the
nucleus [18
].
To understand the intracellular mechanisms involved in the
immunodepression of circulating leukocytes from major-trauma patients,
we studied NF-B activation in their peripheral blood mononuclear
cells (PBMCs) ex vivo and after lipopolysaccharide (LPS) challenge.
Furthermore, the expression of NF-B p65 and p50 subunits and that of
IB were studied ex vivo and after LPS or staphylococci (SAC)
stimulation to determine whether this hyporeactivity is a generalized
phenomenon or depends on the nature of the activating agent.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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25, were studied [26
].
Exclusion criteria were age < 17 years, pregnancy, preexisting
autoimmune or immune deficiency disease (such as diabetes, lupus
erythematosus, multiple sclerosis, rheumatoid arthritis, or AIDS), or
use of steroids or immune cell-ablative chemotherapy within the
previous 30 days. Blood samples were collected on day 1—the day each
patient arrived in the intensive care unit (ICU)—and, when possible,
on days 3, 5, and 10. The mechanisms of traumatic injury were motor
vehicle accidents, automobile-pedestrian accidents, and falls. Fifteen
male and two female trauma patients with severe injuries {ISS,
37 ± 9 (standard deviation SD); range, 25–54;
simplified acute physiology score II, 40 ± 12, range 22–60)}
were enrolled. The mean age was 28 ± 2 years (age range, 17–47
years). On admission, 10 patients had hemorrhagic shock, and
catecholaminergic support was necessary. In the posttraumatic course,
acute-respiratory-distress syndrome occurred in four patients, and
eight patients developed infections. Three patients died during their
stay in the ICU. The mean age of these nonsurvivors was 33 ± 12
years (range 22–46 years), with a mean ISS of 45 ± 4 (range,
43–50) and a mean simplified acute physiology score II of 47 ± 6
(range, 44–54). Their deaths were attributable to severe progressive
brain injury. The clinical characteristics of the trauma patients are
shown in Table 1
.
|
PBMC isolation and extract preparation.
PBMCs were isolated from blood freshly collected on sodium
citrate by centrifugation with Ficoll-Hypaque (MSL; Eurobio, les Ulis,
France). Before addition of Ficoll, a fraction of the blood was
centrifuged at 600 g for 5 min, and 1 mL of plasma was
collected and immediately frozen at -20°C for further cytokine
measurements. After isolation, cells were used immediately for
preparation of nuclear and cytoplasmic extracts (ex vivo) or were
cultured for 1 h at 37°C in a 5% CO2 incubator in
RPMI-1640 medium (Glutamax; Gibco-Life Technologies, Paisley, United
Kingdom) in the presence of Escherichia coli 0111:B4 LPS at
1 µg/106 cells. Cellular extracts were prepared as
previously described [27
]. Briefly, freshly collected
and cultured PBMCs were washed once with phosphate-buffered saline
(PBS) before extraction. Adherent cells cultured with LPS were
harvested with a cell scraper, added to corresponding nonadherent
cells, and suspended in buffer A [10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol (DTT), 0.1% Nonidet P-40] supplemented with protease
inhibitors. The protease inhibitors consisted of 0.5 mM
phenylmethylsulfonyl fluoride, 25 µg/mL of aprotinin, 10 µg/mL of
chymostatin, 2 µg/mL of antipain, 8 µg/mL of pepstatin, 10 µg/mL
of leupeptin, 0.1 mg/mL of -1 antitrypsin, and 0.5 mM
3,4-dichloroisocoumarin (all from Sigma, St Louis, MO). Cells were
incubated at 4°C for 10 min and then centrifuged at 8,900
g for 2 min. The supernatant corresponding to the
cytoplasmic extract was frozen at -80°C. The pellet was suspended in
buffer C (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2,
0.2 mM ethylenediaminetetraacetate, 25% glycerol, 0.5 mM DTT, and
protease inhibitors) and incubated for 20 min at 4°C. Cells were then
centrifuged at 17,500 g for 10 min. The supernatant
corresponding to the nuclear extract was harvested and kept at
-80°C.
Whole-cell extracts were prepared with PBMCs after a 45-min culture in the presence or absence of LPS (1 µg/mL) or SAC (100 µg/mL). At the end of the culture, the cells were washed with PBS and resuspended in high-salt buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1.5 mM MgCl2, 1 mM ethylenediaminetetraacetate, 1 mM ethyleneglycol-bis(aminoethylether)-N,N'-tetraacetic acid, 10% glycerol, 1 mM DTT, and protease inhibitors). The cells were disrupted by three cycles of freezing and thawing in liquid nitrogen. The lysate was then centrifuged at 17,500 g for 20 min at 4°C, and the supernatant corresponding to the whole-cell extract was harvested and kept at -80°C. Protein concentrations were determined according to the method of Bradford.
Electrophoretic mobility shift assay (EMSA).
Double-stranded oligonucleotides corresponding to the consensus
NF-B or Oct-1 sequences (Promega, Madison, WI) were end-labeled with
T4 kinase in the presence of [-32P]ATP. Nuclear
extracts (2 µg each) were incubated in binding buffer (4% Ficoll, 20
mM HEPES, pH 7, 35 mM NaCl, 60 mM KCl, 0.01% Nonidet P-40, 2 mM DTT,
0.1 mg/mL of bovine serum albumin, and 1.5 µg/µL of salmon sperm
DNA) for 15 min at room temperature. After 15 min, the radiolabeled
nucleotide was added (150,000 cpm), and the mixture was again incubated
for 15 min at room temperature. EMSA was performed in a 5% acrylamide
gel in 50 mM Tris–45 mM boric acid–0.5 mM EDTA, pH 8.4. Gels were
dried and subjected to autoradiography. The NF-B complexes were
quantified using a PhosphorImager and the ImageQuant software (both
from Amersham Pharmacia Biotech, Buckinghamshire, UK). Because not all
the samples could be analyzed on the same gel, we used a positive
control (PBMCs from a healthy donor stimulated with LPS) that was the
same for all gels. All of the gels were exposed to the PhosphorImager
screen for the same amount of time, and various amounts of the same
nuclear extract were analyzed to ascertain the linearity of the signal
measurement. The values obtained for the positive control allowed us to
calibrate the EMSA to compare counts per minute between gels. The
counts per minute obtained for this positive control on one gel were
chosen as a reference, and the values of all other gels were corrected
by a multiplying factor that took into account the values of this
positive control. This calibration was not necessary when the
p65p50/p50p50 was calculated. Specificity of binding was assessed by
competition with an excess of cold oligonucleotide and by supershift
experiments using anti-p50 and anti-p65 specific polyclonal antibodies
(Santa Cruz Biotechnology, Santa Cruz, CA).
Western blot
Four micrograms of protein from cytoplasmic extracts or 20 µg
of whole-cell extracts were subjected to sodium dodecyl sulfate-12%
polyacrylamide gel electrophoresis and transferred onto nitrocellulose
sheets (Hybond C; Amersham Pharmacia). Protein transfer was ascertained
by Ponceau red coloration. Membranes were then washed with PBS and
blocked with PBS containing 0.1% Tween 20 and 5% gelatin (PBS-T-G)
for 1 h at room temperature. After five washes with PBS-T,
membranes were incubated with rabbit polyclonal immunoglobulin-G
anti-I-B (C-21; Santa Cruz) at 1:2,000, with anti-p65 (sc114X;
Santa Cruz) at 1:20,000, or with anti-p50 (sc-114X; Santa Cruz) at
1/20,000 in PBS-T-G for 1 h at room temperature. After five
washes, peroxidase-labeled goat anti-rabbit immunoglobulin polyclonal
antibodies (Silenus, Hawthorn, Australia) were added at 1:2,000 in
PBS-T-G and incubated for 1 h at room temperature. After five
washes, blots were developed using enhanced chemiluminescence (Amersham
Pharmacia). Densitometry analysis was performed on the Western-blot
films using the National Institutes of Health (Bethesda, MD) Image
software. Background intensity ranged from 36 to 63.
IL-10 and transforming growth factor (TGF) -ß1 measurements
IL-10 and TGF-ß1 were quantified in the plasma of patients
with major trauma and in healthy controls with specific enzyme-linked
immunosorbent assay (ELISA) kits (R&D Systems, Abington, United
Kingdom). For TGF-ß1, platelet-poor plasma samples were subjected to
acidification and subsequent neutralization, according to the
manufacturer’s instructions, before the ELISA test.
Statistical analysis
Nonparametric statistics were used for intra- and
intergroup comparisons. A Mann-Whitney U test was used to
compare day-1 NF-B and day-1 LPS-stimulated NF-B activities with
the NF-B activities of controls and LPS-stimulated controls,
respectively. The LPS-stimulated NF-B activities were compared with
nonstimulated NF-B in both groups, that is, control and trauma
patients at day 1, using a Wilcoxon test. The surveys of IL-10 and
TGF-ß levels and of NF-B activity and LPS-stimulated NF-B
activity were compared at days 1, 3, 5, and 10 using a Friedman test.
P < 0.05 was considered the minimal level of
significance. Data are given as means plus or minus standard errors
(SEs).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B in PBMCs from trauma patientsB was analyzed by EMSA. Two representative
examples of healthy controls and a representative patient with major
trauma are shown in Figure 1A
. Ex vivo, NF-B was constitutively activated within nuclear
extracts of PBMCs from healthy controls, as shown by the retardation of
migration of the labeled B oligonucleotide. As previously found,
activation of NF-B was increased in PBMCs from healthy controls
after LPS stimulation. In contrast, trauma patients had a lower ex vivo
nuclear activation of NF-B at admission, and this depressed level of
activation persisted until day 10. Moreover, NF-B activation
remained low despite LPS stimulation in vitro. Recovery of the nuclear
translocation in response to LPS was, however, observed for some
patients at day 10.
|
B complexes were observed by EMSA, we
characterized them by competition with cold oligonucleotides and p50-
or p65-specific antibodies. As shown in Figure 1B
, the addition of an
excess of cold oligonucleotide, corresponding to the NF-B-binding
site, turned off the signal of the two upper bands, showing that the
lowest band was nonspecific. An excess of an irrelevant oligonucleotide
[corresponding to the activator protein-1 (AP-1) transcription
factor] had no effect. The addition of an anti-p50 antibody caused a
supershift of the two upper bands, whereas the anti-p65 antibody
affected only the uppermost band. Thus, the upper complex corresponded
to the p65p50 heterodimer, and the intermediate band is the p50p50
homodimer. The low NF-B binding was not observed for an unrelated
and ubiquitous transcription factor Oct-1 [28
]. Oct-1
binding was low ex vivo, especially for the patients, but it was
induced by LPS stimulation in the PBMCs of both healthy controls and
trauma patients (data not shown). These data illustrate that the low
NF-B binding activity detected in the patients’ PBMC extracts was
not caused by proteolytic degradation of the samples.
After EMSA, p50p50 and p65p50 complexes were quantified using a
PhosphorImager. The results corresponding to NF-B activation on days
1, 3, 5, and 10 for trauma patients (n = 13) are shown
in Figure 2
. These data were compared with those of 13 healthy controls. We
found similar results for survivors and nonsurvivors, and because the
nonsurvivor group was small (three patients) despite the high ISS
scores of these patients, the values for all trauma patients are shown
together. In addition, no difference was found between patients who
developed an infection during their ICU hospitalization and those who
did not. As shown in Figure 2A
, the ex vivo nuclear activation of the
p65p50 heterodimer of NF-B on the first day of trauma was
significantly lower in comparison with that of healthy controls
(P = 0.04), and it remained low even on day 10
posttrauma. After LPS in vitro stimulation, an enhanced nuclear
translocation of p65p50 was noticed. However, this expression remained
significantly lower than that of controls throughout the survey. The ex
vivo expression of p50p50, the inactive homodimeric form of NF-B,
was also very low (with statistical significance) in nuclear extracts
taken from trauma patients on days 1 through 10 (Fig. 2B)
. After LPS in
vitro stimulation, the expression of p50p50 was lower than in the
controls, but the difference was not found to be significant.
|
B:
the absolute number of NF-B complexes in the nucleus and the ratio
of active p65p50 needed to inactive p50p50 complexes. The ratios of
p65p50 to p50p50 are shown in Figure 3
. The ex vivo ratios were lower for trauma patients in comparison
with controls, but the differences did not reach statistical
significance. After LPS stimulation, the ratios were significantly
lower than those of controls on day 1 and remained lower than those of
healthy controls until day 10. These results showed that an
inflammatory insult without infection is sufficient to block NF-B
nuclear translocation in response to LPS and that this effect persists
until day 10 posttrauma, independently of the occurrence of an
infectious process.
|
B and p65 by Western blotB, to determine whether its
up-regulation could explain the low NF-B activation. Indeed,
IB belongs to the family of cytoplasmic inhibitors of NF-B,
and by binding to p65p50, it prevents its nuclear translocation. We
also analyzed the cytoplasmic expression of p65 by Western blot
analysis. Representative examples obtained with ex vivo cytoplasmic
extracts of PBMCs from healthy controls and trauma patients are shown
in Figure 4A
. As expected, we saw IB and p65 in all the extracts from
healthy controls, with some individual variability of expression. In
contrast, their ex vivo expression was quite low for patients with
major trauma and remained low for the majority of them until the end of
the survey on day 10. No difference was seen between survivors and
nonsurvivors. Densitrometric analysis of Western blots is shown in
Figure 4B
. It can be seen that IB expression was significantly
lower in patients’ PBMCs on day 1 and remained low throughout the
survey. Upon LPS stimulation, no further reexpression of IB was
observed in patients’ PBMCs (data not shown). The expression of p65
was also found to be significantly lower in patients’ PBMCs on day 1
in comparison with that in healthy controls. No significant difference
was seen in the values obtained for trauma patients on day 1 versus
days 3, 5, and 10 (Friedman test).
|
B, p65, and p50 in whole-cell extracts by
Western blotB is degraded and then rapidly
resynthesized under the control of NF-B (within 1 h). It can
then enter the nucleus, bind p65p50, and stop NF-B-induced
activation [29
]. To ensure that the low expression of
IB in the cytoplasm was not caused by its reinforced presence in the
nucleus, we prepared whole-cell extracts from PBMCs of four consecutive
trauma patients (patients 14–17) on day 3 and compared them with PBMCs
from four healthy controls. As shown in Figure 5A
, in whole-cell extracts without any stimulation, the expression
of IB, p65, and p50 was lower for PBMCs derived from patients in
comparison with those from controls. Densitometric analysis showed that
the difference was significant only for p65 (Fig. 5B)
. LPS stimulation
induced an up-regulation of IB in PBMCs of healthy controls but
not in those of major-trauma patients. In contrast, patients’ PBMCs
were responsive to SAC, and an up-regulation of IB and p50 was
similar to that of healthy controls. For healthy controls, the p65
expression was not modified by cell activation nor was that of p50 with
LPS, whereas an up-regulation of p65 was observed for trauma patients
after SAC stimulation.
|
|
Despite the increased plasma levels of these immunosuppressive
cytokines during the survey of the patients, no direct correlation was
found with the NF-B expression in mononuclear cells.
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate whether this immunodepression reflects intracellular
changes, we analyzed the nuclear expression of the transcription factor
NF-B in PBMCs from patients with major trauma and the cytoplasmic
expression of its specific inhibitor IB. Our results showed that an
inflammatory insult without infection, at least on day 1, is sufficient
to block NF-B translocation, and this phenomenon persists until day
10 posttrauma. The basal nuclear expression of NF-B was low in
trauma patients’ PBMCs. Although LPS was able to induce its nuclear
translocation, NF-B activation remained significantly lower than
that of controls. NF-B expression was affected at two levels: first,
the ratio of the active (p65p50) to the inactive (p50p50) complexes was
considerably diminished in the PBMCs from trauma patients.
Second and most important, we observed a general and persistent
diminution of expression of both forms of NF-B (p65p50 and p50p50)
in comparison with that in healthy controls. The long-term low
expression of NF-B parallels the long-term immunoparalysis reported
in trauma patients [33
]. This observation is reminiscent
of several studies on endotoxin tolerance, which revealed modifications
in the expression NF-B and which associated tolerance with a
depletion of both forms of NF-B [34
35
36
]. It is
interesting that endotoxin tolerance is very similar to other
phenomena, known as deactivation, desensitization, anergy, or
refractoriness, which are events that occur in many inflammatory
stresses [37
]. The mechanism of tolerance in PBMCs after
trauma seems different from that demonstrated by Ziegler-Heitbrock et
al. [38
]. They showed that the mobilization of NF-B
could occur in a tolerized monocytic cell line or monocytes, but it was
associated with a predominance of the p50p50 homodimer. Similarly, the
inability of macrophages derived from p50-deficient mice to develop
endotoxin tolerance [39
] favors a central role played by
the p50 subunit of NF-B in conferring endotoxin tolerance. Our study
showed that NF-B modulation occurs in vivo in patients with SIRS,
independently of the presence of an infectious insult. This occurrence
could be the result of cytokine production during trauma. Indeed,
various cytokines, such as IL-10, TGF-ß, and TNF in combination with
IL-1, can mimic the effects of endotoxins in vivo and in vitro. They
induce tolerance [37
] similar to the immunoparalysis
seen in trauma patients.
Because it has been reported that the immunosuppressive and
anti-inflammatory cytokine IL-10 can alter NF-B expression and
translocation [40
] and contribute to cell
desensitization [18
, 41
], we investigated
whether the absence of nuclear translocation of NF-B found for
major-trauma patients was linked to plasma IL-10 levels. This
immunosuppressive cytokine was detected in the plasma of patients on
day 1, and its level was significantly higher than in healthy controls,
although its presence was less frequent during the following days. This
observation is in agreement with a previous report [13
]
and suggests that IL-10 may be an early actor of cell desensitization
and an alteration of the NF-B cascade in patients with major trauma.
We also measured TGF-ß1 levels in the plasma of trauma patients,
because this cytokine has also been shown to contribute to
immunosuppression [42
]. The effect of TGF-ß1 on
NF-B is not clearly defined. In some experimental models, TGF-ß1
has been shown to inhibit NF-B [43
], whereas in
others, no change or activation of this transcription factor has been
reported [44
, 45
]. In agreement with a
recent report [46
], we found increased levels of
TGF-ß1 in the plasma of major-trauma patients, and these levels
persisted very long after the injury. IL-10 and TGF-ß may contribute
to the long-term leukocyte hyporeactivity, but we did not find any
correlation between their levels and our observation of NF-B
expression. Certainly, other mediators, such as PGE2,
catecholamines, neuropeptides, or glucocorticoids, also contribute to
the hyporeactivity of circulating cells [46
,
47
]. Although increased apoptosis of immune cells has
been reported in trauma [48
, 49
], it is
unlikely that apoptosis is responsible for the observed NF-B
down-regulation, because most apoptosis-inducing agents also activate
NF-B. Furthermore, we found that trauma patients’ PBMCs were
responsive to SAC, suggesting that they were not dead or apoptotic.
This result also shows that leukocyte hyporeactivity is not generalized
and depends on the nature of the activating signal. In contrast, one
can suggest that the low expression of NF-B could be responsible for
the reported apoptosis, because numerous studies have clearly
demonstrated an anti-apoptotic role for this nuclear factor
[50
, 51
].
Eight trauma patients developed an infection during their stay in the
ICU. However, the results obtained on NF-B activation in their PBMCs
were different from those observed during sepsis. Indeed, a higher ex
vivo nuclear expression of NF-B during sepsis and an even higher
expression in the nonsurvivor group have been reported
[52
]. However, the latter study reported data on only
the ex vivo expression of this nuclear factor and did not measure the
capacity of cells to translocate NF-B in response to LPS.
Furthermore, it did not include a comparison with healthy controls, nor
did it quantify p65p50 and p50p50 expression. Nevertheless, these two
forms of NF-B were not equivalent because p65p50 is a potent gene
transactivator, whereas p50p50 is not [21
,
23
]. The predominance of p50 homodimers over p65p50 is
another inhibitory mechanism of gene activation by NF-B. We recently
performed a similar study on septic patients and found that the active
form of NF-B (p65p50) is diminished in their PBMCs in comparison
with that in healthy controls [53
].
The results concerning IB seem paradoxical. Indeed, because NF-B
nuclear expression was very low, we could expect to find an
overexpression of its inhibitor, which has been described
for many experiments with endotoxin tolerance in vitro
[54
, 55
]. However, the up-regulation of
IB was not always found. In another study, after the first
exposure to LPS and before the second challenge, cells did not express
cytoplasmic IB [56
], as we found ex vivo with the
PBMCs of the trauma patients. Thus, the absence of nuclear NF-B was
not the consequence of its cytoplasmic sequestration by IB but
rather seems to have been caused by a general down-regulation of this
transcription factor. Indeed, total p65, p50, and IB expression
was found to be lower in the PBMCs of trauma patients ex vivo or after
LPS stimulation. This defect was reversible and could be overcome by
SAC stimulation. In contrast to our observation of mononuclear cells,
IB expression in neutrophils of trauma patients was shown to be
preserved [57
]. In contrast to IB, p50 was not found
to be up-regulated by LPS in the PBMCs of healthy controls after 45
min. This result can be explained by differences in the kinetics of
their induction by LPS. Indeed, p50 levels increase after LPS because
of the induction of its precursor (p105) mRNA and because of
proteolytic processing of p105. However, these events happen about
2 h after LPS stimulation [58
].
It is interesting that our observation of the expression of NF-B in
circulating leukocytes of SIRS patients contrasted with the results of
the NF-B analysis performed with cells derived from other
compartments and tissues. An increased activation of NF-B was
demonstrated by Moine et al. [59
] in alveolar
macrophages of patients with acute respiratory distress syndrome.
Similarly, NF-B activation was reported in lung mononuclear cells
and lung neutrophils after hemorrhage in mice [20
,
60
] and in the lung and liver tissue of mice with
peritonitis [61
, 62
]. Although NF-B
activation was reported in lung neutrophils after hemorrhage or LPS
stimulation, this result was not found for blood neutrophils
[60
]. This observation is in agreement with our study,
which suggests that the consequence of SIRS on NF-B expression may
differ in the blood compartment and other tissues.
|
ACKNOWLEDGEMENTS |
|---|
Received September 13, 2000; revised January 18, 2001; accepted January 19, 2001.
|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.001
versus controls is indicated for day 1 (Mann-Whitney U
test). No significant difference was seen between the values obtained
for trauma patients on day 1 versus days 3, 5, and 10 (Friedman
test).
