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Jian-Ping
Gong, Chong-An Liu, Chuan-Xin Wu, Sheng-Wei Li, Yu-Jun Shi, Xu-Hong
Li, Department of General Surgery, The Second College of
Clinical Medicine & the Second Affiliated Hospital of Chongqing
University of Medical Science, 74 Linjiang Road, Central District,
Chongqing 400010, China
Supported by the National Natural Science Foundation of China,
No.39970719, 30170919
Correspondence to: Dr. Jian-Ping Gong, Department of General
Surgery, The Second College of Clinical Medicine & the Second
Affiliated Hospital of Chongqing University of Medical Science, 74
Linjiang Road, Central District, Chongqing 400010, China. gongjianping11@hotmail.com
Telephone: +86-23-63766701 Fax: +86-23-63822815
Received 2001-09-14 Accepted 2001-10-22
Abstract
AIM: To
determine the NF-kB activity in peripheral blood mononuclear cells (PBMC)
in patients with acute cholangitis of severe type (ACST) and
correlate the degree of NF-kB activation with severity of biliary
tract infection and clinical outcome.
METHODS:
Twenty
patients with ACST were divided into survivor group (13 cases) and
nonsurvivor group (7 cases). Other ten patients undergoing elective
gastrectomy or inguinal hernia repair were selected as control
group. Peripheral blood samples were taken 24 hours postoperatively.
PBMC were separated by density gradient centrifugation, then nuclear
proteins were isolated from PBMC, and Electrophoretic Mobility Shift
Assay (EMSA) used determined. The results were quantified by
scanning densitometer of a Bio-Image Analysis System and expressed
as relative optical density (ROD). The levels of TNF-α, IL-6,
and IL-10 in the plasma of patients with ACST and healthy control
subjects were determined by using an enzyme-linked immunoassay
(ELISA).
RESULTS:
The
NF-kB activity was 5.02±1.03 in nonsurvivor group, 2.98±0.51 in
survivor group and 1.06±0.34 in control group. There were
statistical differences in three groups (P<0.05). The
levels of TNF-a and IL-6 in plasma were (498±53)ng·L-1
and (587±64)ng·L-1 in nonsurvivor group, (284±32)ng·L-1
and (318±49)ng·L-1 in survivor group and (89±11)ng·L-1
and (102±13)ng·L-1 in control group. All patients with
ACST had increased levels of TNF-αand IL-6, which were
many-fold greater than those of control group, and there was an
evidence of significantly higher levels in those of nonsurvivor
group than that in survivor group (P<0.05). The levels of
IL-10 in plasma were (378±32)ng·L-1 , (384±37)ng·L-1
and (68±11)ng·L-1 in three groups, respectively. All
patients had also increased levels of IL-10 when compared with
control group (P<0.05), but the IL-10 levels were not
significantly higher in nonsurvivors than in survivors (P>0.05).
CONCLUSION:
NF-kB
activity in PBMC in patients with ACST increases markedly and the
degree of NF-kB activation is correlated with severity of biliary
tract infection and clinical outcome.
Gong
JP, Liu CA, Wu CX, Li SW, Shi YJ, Li XH. Nuclear factor kB activity
in patients with acute severe cholangitis.World J Gastroenterol
2002;8(2):346-349
INTRODUCTION
Acute
cholangitis of severe type (ACST) is a common problem facing today's
surgeons[1-3]. Despite a multitude of advances in the
area of surgical infection and surgical or nonsurgical interventions
to treat biliary tract diseases, ACST and biliary sepsis remain a
significant cause of morbidity and mortality[4-9]. Many
reports have focused on aspects of the proinflammatory cytokines
which are believed to be central to the pathophysiology of the
sepsis syndrome[10-11]. Recent investigations have shown
that expression of many cytokines is closely linked to the
activation of transcriptional factors[12-13]. Among
several transcriptional regulatory factors involved in immuno-regulatory
genes expression, nuclear factor kappa B (NF-kB) acts as a critical
step for directing the transcription of many proinflammatory
cytokine genes in animal models of sepsis or endotoxemia[14-16].
Investigations regarding the role of NF-kB in human inflammatory
diseases are scarce[17-20]. So far, no study has aimed to
examine in patients with ACST the relationship among NF-kB activity
in peripheral blood mononuclear cells (PBMC), the concentrations of
the pro-inflammatory cytokines in plasma, and clinical outcome. The
purpose of this study was to determine the NF-kB DNA binding
activity in circulating blood cells and the cytokines tumor necrosis
factor alpha (TNF-α), interleukin (IL)-6, and IL-10 profile in
patients with ACST. Attempts were made also to correlate degree of
NF-kB activation with severity of biliary tract infection and
clinical outcome.
MATERIALS
AND METHODS
Patients
The study population was recruited from a series of 20 patients with
a clinical diagnosis of ACST. Among them, 13 were male, and 7
female, ranging in age from 27 to 78 yr. All patients had
manifestations of fever, chill, jaundice, and right upper quadrant
pain. Other manifestations included two or more of the following
clinical conditions: Blood cultures were positive; Core body
temperature >39℃
or <36℃;
Heart rate >120 beats/min; Hypotension: A systolic blood pressure
of <12.0 kPa or a reduction of >5.33 kPa from baseline in the
absence of other causes of hypotension; White blood cell count
>1.5×109L-1 . These patients were divided
into nonsurvivor group (7 cases) and survivor group (13 cases). Ten
patients undergoing elective gastrectomy or inguinal hernia repair
were selected as control group. Peripheral blood samples were taken
24h postoperatively.
Isolation
of PBMC
PBMC were separated by density gradient centrifugation, as
previously described[18]. In brief, PBMC were isolated
grom blood freshly collected on sodium citrate by centrifugation on
Ficoll-Hypaue. Before Ficoll, a fraction of the blood was
centrifuged 5min at 1500r·min-1 and 1mL of plasma was
collected and put immediately at -20℃
for further cytokine measurements.
Isolation
of nuclear proteins
Nuclear proteins were isolated from PBMC extract by placing the
sample in 0.8mL of ice-cold hypotonic buffer [10mmol·L-1
HEPES (pH7.9), 10mL KCL, 0.1mmol·L-1 EDTA, 0.1mmol·L-1
ethylene glycol tetraacetic acid, 1mmol·L-1 DTT;
Protease inhibitors (aprotinin, pepstatin, and leupeptin, 10mg·L-1
each)]. The homogenates were incubated on ice for 20min, vortexed
for 20s after adding 50μL of 10 per cent Nonidet P-40, and then
centrifuged for 1 minute at 4℃
in an Eppendorf centrifuge. Supernatants were decanted, the nuclear
pellets after a single wash with hypotonic buffer without Nonidet
P-40 were suspended in an ice-cold hypertonic buffer [20mmol·L-1
HEPES (pH7.9), 0.4mol·L-1 NaCL, 1mmol·L-1
EDTA, 1mmol·L-1 DTT; Protease inhibitors], incubated on
ice for 30min at 4℃,
mixed frequently, and centrifuged for 15min at 4℃.
The supernatants were collected as nuclear extracts and stored at
-70℃.
Concentrations of total proteins in the samples were determined
according to the method of Bradford.
Electrophoretic
Mobility Shift Assay (EMSA)
NF-kB binding activity was performed in a 10-uL binding reaction
mixture containing 1×binding buffer [50mg·L-1 of
double-stranded poly(dI-dC), 10mmmol·L-1 Tris-HCl (pH
7.5), 50mmmol·L-1 NaCl, 0.5mmmol·L-1 EDTA,
0.5mmmol·L-1 DTT, 1mmmol·L-1 MgCl2,
and 100mL·L-1 glycerol], 5μg of nuclear protein,
and 35 fmol of double-stranded NF-kB consensus oligonucleotide
(5'-AGT TGA GGG GAC TTT CCC AGG-3') that was endly labeled with
γ-32P(111TBq mmol-1 at 370GBq L-1) using
T4 polynucleotide kinase. The binding reaction mixture was incubated
at room temperature for 20min and analyzed by electrophoresis on 7
per cent nondenaturing polyacrylamide gels. After electrophoresis
the gels were dried by Gel-Drier (Bio-Rad Laboratories, Hercules,
CA) and exposed to Kodak X-ray films at -70℃.
Quantifying
with the Phosphor Imager
The binding bands were quantified by scanning densitometer of a
Bio-Image Analysis System. The results were expressed as relative
optical density (ROD).
Measurement
of cytokines in plasma
TNF-α, IL-6, and IL-10 levels in the plasma of patients with
ACST and healthy control subjects were determined with using an
enzyme-linked immunoassay (ELISA). The detection limits of the
assays were 50ng·L-1 (TNF-α), 49ng·L-1
(IL-6), and 49ng·L-1 (IL-10). All cytokines assays were
performed in duplicate and had intra- and interassay variations
lower than 8% and 11%, respectively.
Statistical
Analysis
Data were analyzed with using Microsoft Excel with Astute
statistical add-in and were expressed as median ± standard error. A
value of P≤0.05
was considered statistically significant.
RESULTS
Methods
The patients of nonsurvivor group died within 35d, all from
complications of SIRS, sepsis or MOF. The patients of survivor group
were well discharged from hospital postoperatively in 28 days. The
median age in nonsurvivor group was 54 yr, which was not
significantly different from that of survivor group (53yr) (P>0.05).
An admission APACHE II score in nonsurvivor group was 25, which was
also not different from that of survivor group (24) (P>0.05).
The NF-kB activity was 5.02±1.03 in nonsurvivor group, 2.98±0.51
in survivor group and 1.06±0.34 in control group. There were
statistical differences in three groups (P<0.05). The NF-kB
activity increased in all patients with ACST, versus the control
group (P<0.05), and the patients of nonsurvivor group had
higher levels of NF-kB activation than those of survivor group (P<0.05,
Figure 1).
Figure
1(PDF)The
activity of NF-kB in three groups. aP<0.05, vs
other groups, cP<0.05, vs control.
The
levels of TNF-a and IL-6 in plasma were (498±53)ng·L-1
and (587±64)ng·L-1 in nonsurvivor group, (284±32)ng·L-1
and (318±49)ng·L-1 in survivor group and (89±11)ng·L-1
and (102±13)ng·L-1 in control group. All patients with
ACST had increased levels of TNF-α and IL-6, which were
many-fold greater than those of control group, and there was an
evidence of significantly higher levels in those of nonsurvivor
group than that in survivor group (P<0.05). The levels of
IL-10 in plasma were (378±32)ng·L-1 , (384±37)ng·L-1
and (68±11)ng·L-1 in three groups, respectively. All
patients had also increased levels of IL-10 when compared with
control group (P<0.05), but the IL-10 levels were not
significantly higher in nonsurvivors than in survivors (P>0.05).
(Figure 2).
Figure
2(PDF)Changes of cytokines in plasma. aP<0.05, vs
other groups, cP<0.05, vs control
DISCUSSION
The
overwhelming inflammatory response in patients with ACST is a major
cause which induces systemic inflammatory response syndrome (SIRS)
and MOF[1-4]. Mortality in patients with ACST reflects a
multifactorial pathology, and neither cytokine concentrations in
plasma nor even the APACHE II score can be expected to accurately
predict patients' outcomes[5-9]. NF-kB is a protein found
in inflammatory cells such as lymphocytes, monocytes, and
macrophages. NF-kB activation is stimulated by LPS, TNF-α, and
IL-1, the very early mediators or factors in the inflammatory
cascade[21,22]. Once stimulated NF-kB activates various
parts of the inflammatory resposes: TNF-α, IL-6 and IL-10, and
adhesion molecules such as selectins and integrin[23-25].
These mediators and factors then promote further activity of the
inflammatory cascade and “off” goes the SIRS and MOF[2,8,9,24].
Therefore, we chose to focus on NF-kB as an important regulatory
factor to regulate the expression of multiple cytokine genes. NF-kB
is a ubiquitous transcription factor involved in the signal
transduction pathway of many inducers of the inflammatory response
and is therefore a potentially attractive target for
immunomodulatiom to reduce sepsis and organ dysfunction[26],
but we are not yet clear about changes of NF-kB and relation between
NF-kB and cytokines in patients with ACST. Foulds et al[20]
reported that levels of nuclear-bound NF-kB (activated NF-kB) were
greater in patients who developed organ dysfunction after surgery,
and patients with lower levels of nuclear NF-kB who recovered from
surgery without organ dysfunction. Bohrer et al investigated
activity of NF-kB in nuclear extracts from PBMC of 15 patients with
sepsis, of whom 10 survived. NF-kB activity was measured on days 1,
2, 3, 4, 5, 6, 8, 10, and 14 after admission where available. All
patients with NF-kB binding activty exceeding 200% of day 1 died.
This small study concluded that NF-kB activation might be an
important event in clinical sepsis. But, Adib-Conquy et al [27]
found the expression of NF-kB was signifcantly reduced for all
patients with sepsis and trauma as compared with control subjects.
In our study, by comparing the predictive value of measuring NF-kB
activity in PBMC and the concentrations of some pro- and
anti-inflammatory mediators in plasma in patients with ACST, we
found that the NF-kB activity measured in the PBMC was a better
overall predictor of mortality than the balance and time course of
pro- and anti-inflammatory cytokines released in plasma. On the
basis of these results NF-kB would seem to be a more sensitive
molecular marker assay when compared with cytokines used as an
indicator of sepsis.The expressions of many genes involved in the
inflammatory and immune processes are regulated by NF-kB, TNF-α,
IL-6, and IL-10, and they possess NF-kB binding sites in the
promoter region, enabling messenger ribonucleic acid to express in
response to extracellular stimuli[28-31]. TNF-α,IL-6,
and IL-10 have all been implicated in the pathogenesis of SIRS and
MOF that results from trauma, injury, infection, and sepsis[32-43].
In the present study, we found that TNF-α and IL-6 were
elevated in the patients with ACST and there were higher levels of
TNF-α and IL-6 in patients who survived than in patients who
died, which is in agreement with previous reports. These previous
studies also showed that levels were higher in patients with sepsis
when compared with trauma patients. We found significant
relationship between NF-kB activation and circulating concentrations
of TNF-α and IL-6. IL-10, as an anti-inflammtory mediator, was
elevated in the patients with ACST but reduced in patients who died.
There might be other transcription factor as AP-1 involved in the
signal transduction pathway of the inflammatory response[16,21,23,44-48]
and its mechanism is going on.
In conclusion, we have shown NF-kB activation in PBMCs in
patients with ACST increased markedly before death and was related
to plasma TNF-αand IL-6 concentrations. These findings have
important implications for the development of future therapeutic
interventions in the critically ill and support the need for further
study of the role of NF-kB activation in mortality from ACST and MOF.
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