Received 8 September 1999/Returned for modification 9 November
1999/Accepted 10 January 2000
The relationship between fluctuating cytokine concentrations in
plasma and the outcome of sepsis is complex. We postulated that early
measurement of the activation of nuclear factor 
B (NF-B), a
transcriptional regulatory protein involved in proinflammatory cytokine
expression, may help to predict the outcome of sepsis. We determined
NF-B activation in peripheral blood mononuclear cells of 34 patients
with severe sepsis (23 survivors and 11 nonsurvivors) and serial
concentrations of inflammatory cytokines (interleukin-6, interleukin-1,
and tumor necrosis factor) and various endogenous antagonists in
plasma. NF-B activity was significantly higher in nonsurvivors and
correlated strongly with the severity of illness (APACHE II score),
although neither was related to the cytokine levels. Apart from NF-B
activity, the interleukin-1 receptor antagonist was the only cytokine
tested whose level in plasma was of value in predicting mortality by
logistic regression analysis. These results underscore the prognostic
value of early measurement of NF-B activity in patients with severe sepsis.
 |
INTRODUCTION |
Many reports have focused on aspects
of the proinflammatory cytokine network, which is believed to be
central to the pathophysiology of the sepsis syndrome (5,
8). However, the cytokine responses in patients with sepsis
appears to vary so much between individuals (10) that the
prognostic usefulness of circulating cytokine concentrations is often
less than that of clinical variables, such as the acute physiology and
chronic health evaluation (APACHE) II or III (9). Other
studies indicate that the problem in overwhelming sepsis is not that
inflammatory cytokines are expressed but, rather, that their expression
is not properly modulated by anti-inflammatory mediators (16,
17). Recent investigations by others (3) and ourselves
(1) searching for new clinically reliable markers in
patients with sepsis have shown that circulating leptin levels, whose
secretion is closely linked to the activation of the cytokine cascade
(1), may help to predict mortality in sepsis and septic shock.
Among several transcriptional regulatory factors involved in
immunoregulatory genes expression, nuclear factor kappa B (NF-B) acts at a critical step for directing the transcription of many proinflammatory genes in animal models of inflammatory diseases (6, 7). Investigations regarding the role of NF-B in
human inflammatory diseases are scarce (2, 15). So far, no
study has aimed to examine in patients with sepsis the relationship between the concentrations of some components of the proinflammatory and anti-inflammatory cytokine response in plasma, NF-B expression in peripheral blood mononuclear cells, and clinical outcome. We hypothesized that severe, fatal sepsis could be distinguished from less
severe sepsis by demonstrating greater NF-B activation and decreased
anti-inflammatory response. Thus, this study compared the prognostic
value of combining measurements of NF-B activity in circulating
blood cells and the cytokine profile in plasma in patients with severe sepsis.
| |
MATERIALS AND METHODS |
Patients.
The study population was recruited from a series
of 100 patients with a clinical diagnosis of sepsis, consecutively
admitted to the Department of Internal Medicine or the Intensive Care
Unit over a 4-month period. This study was approved by the local Ethics Committee. The patients were screened daily for severe sepsis criteria
according to the recommendation of the American College of Chest
Physicians/Society of Critical Care Medicine Consensus Conference
Committee (13). A total of 34 patients with
bacteriologically documented infections (24 with positive blood
cultures and 10 with positive bronchial fluid or urine cultures) were
included in the study when they met the severe-sepsis criteria for the first time. The time between admission and inclusion in the study varied from 1 to 12 days (median, 5 days). No patient died during the
first 3 days of entry into the study. Written informed consent was
obtained from all subjects. The following exclusion criteria were
imposed: malignancy and chronic inflammatory diseases, treatment with
steroids or immunosuppresive drugs during the last month, hepatic
failure (serum aspartate aminotransferase and/or alanine aminotransferase level, >100 IU/liter; prothrombin time, <60%; total
bilirubin level, >60 µmol/liter), renal insufficiency (creatinine level in plasma, 200 µmol/liter), AIDS, and gestation. The control group consisted of 20 healthy individuals before elective surgery, who
had normal concentrations of orosomucoid in serum (reference value,
<1.1 g/liter) at the time of blood sampling, to exclude an ongoing
acute-phase reaction.
Laboratory methods.
Body mass index (weight in kilograms
divided by the square of height in meters) and APACHE II score
(12) were calculated on entry into the study. Plasma samples
were simultaneously obtained at the time of systolic hypotension and
every 6 h thereafter for 24 h. Tumor necrosis factor alpha,
interleukin-1
(IL-1), IL-6, and IL-10 levels were determined by
an enzyme-linked immunoassay (Medgenix Diagnostics, Fleurus, Belgium),
and concentrations of soluble TNF receptor type I (sTNF-RI) and IL-1
receptor antagonist (IL-1ra) were measured using a quantitative
sandwich enzyme immunoassay (Quantikine; R&D systems, Minneapolis,
Minn.). The detection limits of the assays were 10 (TNF-
), 21 (IL-1), 8 (IL-6), 12 (IL-10), 90 (sTNF-RI), and 250 (IL-1ra) pg/ml.
All cytokine assays were performed in duplicate and had intra- and
interassay variations lower than 8 and 10%, respectively.
Nuclear protein extraction and electrophoretic mobility shift
assay (EMSA).
Human peripheral blood mononuclear cells (PBMC) were
isolated from freshly drawn heparinized blood by centrifugation on
Lymphoprep (Nycomed Pharma AS, Oslo, Norway) as specified by the
manufacturer. Nuclear protein extracts were individually obtained from
PBMC of the 34 patients and 20 healthy control subjects by the method of Montaner et al. (14). Briefly, cells were lysed in cold
buffer A (20 mM HEPES [pH 8], 1.25% Nonidet P-40, 10 mM KCl, 0.15 mM EGTA, 0.15 mM EDTA, 1 mM dithiothreitol [DTT], 0.2 mM
phenylmethylsulfonyl fluoride). Nuclei were pelled by centrifugation at
400 × g and 4°C for 5 min and washed in cold buffer
B (20 mM HEPES [pH 8], 50 mM NaCl, 25% glycerol, 0.15 mM EGTA, 0.25 mM EDTA, 1.5 mM MgCl2, 1 mM DTT, 0.2 mM
phenylmethylsulfonyl fluoride). After centrifugation, nuclear proteins
were extracted by incubation for 30 min in cold buffer C (buffer B but
with 400 mM NaCl). Then, nuclear extracts from all control subjects
were pooled and used subsequently as the control sample.
Double-stranded oligonucleotide probes of the immunoglobulin gene
containing the NF-B binding site (18) with the following
sequences were synthesized:
5'-TCGACGAGCTCGGGACTTTCCGAGC-3' 3'-GCTCGAGCCCTGAAAGGCTCGAGCT-5'
The DNA binding reaction was carried out by incubation
of 15 µg of nuclear proteins with 32P-labeled
double-stranded oligonucleotides (0.3 ng) in a final volume of 20 µl
of reaction mixture [5 mM HEPES (pH 7.8), 50 mM KCl, 0.5 mM DTT, 5 mM
MgCl2, 10% glycerol, 0.15 µg of poly(dI-dC)-poly(dI-dC) per µl]. The specificity of binding was confirmed by competition with a 400-fold excess of unlabeled B oligonucleotide in the reaction mixture. After incubation, the samples were loaded onto a 5%
polyacrylamide (in 0.5× Tris-borate-EDTA buffer) gel, which was run at
10 V/cm. One control sample was always loaded onto each gel along with
several (two to four) patient samples. After fixation with 10% acetic
acid, the gels were dried and radioactivity was quantified using an
Instant Imager (Packard). The NF-B activity value for each patient
sample was expressed relative to the NF-B binding activity obtained
in the control sample run in the same gel (taken to be 100%). All
samples from septic patients were assayed in two or three different
gels; the final value considered for each sample was the mean of the
individual values obtained, which were quite reproducible.
Additionally, dried gels were subjected to autoradiography to visualize
the NF-B activity and the specificity of the binding.
Statistical analysis.
Mean cytokine levels were tabulated by
adding serial values and dividing by 4, the total number of
determinations. Data are presented as geometric means and extremes.
Comparisons were performed by one-way analysis of variance.
Scheffé's simultaneous confidence intervals were determined to
account for multiple comparisons. Effectiveness in predicting outcome
was compared by calculating the sensitivity and specificity of the best
cutoff value for each variable, and the accuracies of the various
variables were determined by constructing their receiver operating
characteristic (ROC) curves with software from MedClac. Stepwise
logistic regression analysis was used to compare survivors and
nonsurvivors and to determine which variables best predicted mortality.
| |
RESULTS |
Eleven patients died within the first 10 days, and the other 23 patients survived and were discharged from hospital. NF-B binding
activity on study admission and average serial cytokine concentrations
in plasma over the first 24 h are shown in Tables 1 and 2.
Figure 1 shows a representative
experiment of the EMSA for NF-B activity in control subjects and in
two patients with sepsis. Compared with survivors, patients who died
had significantly higher NF-B activity in peripheral blood cells and
APACHE II scores (P < 0.01); moreover, there was a
strong correlation between these two variables (r = 0.616;
P < 0.001). Differences in NF-B activity between patients
with positive blood cultures and those without proven bacteremia (364%
activity [range, 135 to 820%] versus 231% activity [range, 123 to
740%]) did not reach statistical significance. All patients had
increased levels of IL-6, which were manyfold greater than normal
levels, and there was evidence of significantly higher levels in those
who died (Table 1). In contrast, the IL-10 concentrations in plasma
were significantly higher in survivors than in nonsurvivors. TNF-
and IL-1 were detected in only 6 of the nonsurvivors (55%) compared
with 14 of the survivors (61%) (
2 = 2.7;
P = 0.1). sTNF-RI and IL-1ra were detected in plasma
from all patients in concentrations greatly in excess of the TNF- and IL-1 concentrations, respectively. Survivors had significantly higher concentrations of sTNF-RI and IL-1ra and a higher ratio of
IL-1ra to IL-1 than did nonsurvivors (Table 2).
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|
FIG. 1.
Representative EMSA analysis for NF-B binding
activity in PBMC from healthy controls and patients with sepsis.
Nuclear extract (15 µg) from the control sample (lanes 1 and 2), from
a survivor with sepsis (lanes 3 and 4), or from a nonsurvivor (lanes 5 and 6) were incubated with 32P-labeled B oligonucleotide
as described in Materials and Methods and loaded in the same gel. In
lanes 2, 4, and 6, the reaction mixtures also contained excess cold
unlabeled B oligonucleotide, which completely inhibited binding of
the labeled probe.
|
|
Comparisons of ROC curves for the APACHE II score, NF-B activity,
and IL-1ra assay are shown in Fig. 2.
Evaluation of these ROC curves indicated that 21 was the best cutoff
point of the APACHE II score for predicting mortality (sensitivity,
96%; specificity, 92%), while 372%, expressed as the relative
percentage of the value obtained with nuclear extracts pooled from 20 healthy control subjects, was the best cutoff value for NF-B
activity (sensitivity, 97%; specificity, 70%). The area under the
curve for NF-B activity was significantly higher than that for the
APACHE II score and IL-1ra assay.
View larger version (16K):
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|
FIG. 2.
ROC curves for NF-B activity in PBMC, APACHE II
score, and IL-1ra levels in plasma. The areas under the curve (AUC) for
these variables were compared. Overall, NF-B activity was the best
predictor of mortality.
|
|
Significant linear correlations were found between NF-B activity and
the IL-1ra concentration in plasma (r = 0.556; P < 0.001) or the IL-1/IL-1ra ratio (r = 0.37; P < 0.05). A stepwise logistic regression analysis revealed that only
NF-B activity ( = 1.90; standard error = 0.69; odds
ratio = 6.7; 95% confidence interval = 1.7 to 26.3) and
IL-1ra ( = 1.35; odds ratio = 3.2; 95% confidence interval = 0.6 to 9.7) were found to predict mortality
(P < 0.001). No other variable was of sufficient
predictive value to be included in the model.
| |
DISCUSSION |
Mortality in patients with sepsis reflects a multifactorial
pathology, and neither cytokine concentrations in plasma nor even the
APACHE II score can be expected to accurately predict patient outcomes.
Therefore, we chose to focus on NF-B as an important transcriptional
regulatory factor that regulates the expression of multiple cytokine
genes in animal models of sepsis (6, 7). Previous
observations showed a significantly higher NF-B activity in the
blood monocytes of 5 patients with sepsis who died than in 10 patients
who survived, but the predictive value of NF-B was not compared with
that of other sepsis mediators (2). In this study, by
comparing the predictive value of measuring NF-B activity in PBMC
and the concentrations of some pro- and anti-inflammatory mediators in
plasma in patients with severe sepsis, we found that the NF-B
activity measured in a single blood sample on admission into the study
was a better overall predictor of mortality than the balance and time
course of pro- and anti-inflammatory cytokines released in plasma. The
APACHE II score correlated strongly with NF-B activity on study
admission, but neither was related to cytokine levels in plasma. The
analysis of the ROC curves showed that NF-B activity had a better
predictive value for mortality than did the APACHE II score and IL-1ra
assay. Apart from NF-B activity in mononuclear cells, the IL-1ra
level in plasma was the only cytokine concentration determined that had
an independent correlation with prognosis in the logistic regression analysis.
The predictive value of the levels of circulating proinflammatory
cytokines and their inhibitors in human sepsis is controversial, since
patients with sepsis do not represent a homogeneous population (4,
5). In addition, to be most useful for prognosis, daily determinations should be made because the peak value is the most informative. In this study, we measured various mediator concentrations at particular time points, i.e., every 6 h over the first day after the onset of systolic hypotension, when the concentrations in
plasma probably better reflect their paracrine activity. Although significant differences in the concentrations of the cytokines tested
were observed between survivors and nonsurvivors in the group as a
whole, the degree of overlap limited the predictive value of these
concentrations. Interestingly, we noted a profound imbalance between
the levels of cytokines and their antagonists. Surviving patients had
levels of IL-1ra in plasma in great excess, approximately a mean value
of 75-fold higher than those of IL-1, whereas nonsurvivors had
significantly less overproduction of IL-1ra. A high IL-1ra level or
IL-1ra/IL-1 ratio thus appears to modulate inflammatory responses by
inhibiting IL-1 activity and is associated with recovery. This finding
is consistent with data obtained for experimental endotoxemia,
suggesting that high circulating IL-1ra levels could have a modulating
effect as a defense mechanism (11).
In summary, unlike measurements of pro- and anti-inflammatory cytokine
levels in plasma at particular time points, which only indirectly
reflect tissue secretion, our data showed that NF-B activity in PBMC
assessed once on study admission better reflects the complex pattern of
immunologic events in patients with sepsis and may accurately predict
the outcome of infection. An overproduction of IL-1ra appears to play a
protective role. Thus, both parameters used together provided excellent
prognostic information.
This work was supported by a grant (expediente 082/0010/1997)
from the Direccion General de Investigación, Comunidad
Autónoma de Madrid, Madrid, Spain.
We are grateful to David Cabestrano and Manuel Cidoncha (Intensive Care
Unit) for helping with data collection. We thank the patient care staff
and our patients for their willing involvement in the study.
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B Activity
and Plasma Cytokine Levels in Patients with Sepsis
Top
Abstract
Introduction
