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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Jan. 2004, p. 161–167 Vol. 11, No. 1
1071-412X/04/$08.00⫹0 DOI: 10.1128/CDLI.11.1.161–167.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Cytokine Production and Monocyte HLA-DR Expression as Predictors
of Outcome for Patients with Community-Acquired
Severe Infections
A. Lekkou,
1
M. Karakantza,
2
A. Mouzaki,
2
F. Kalfarentzos,
3
and C. A. Gogos
4
*
Department of Internal Medicine, Patras University Hospital,
1
and Laboratory of Hematology and Transfusion Medicine,
2
Department of Surgery,
3
and Section of Infectious Diseases,
4
Patras University Hospital, School of Medicine,
26500 Patras, Greece
Received 23 April 2003/Returned for modification 27 June 2003/Accepted 10 November 2003
This study was performed to evaluate the impact of pro- and anti-inflammatory molecules and human
leukocyte antigen DR (HLA-DR) expression as markers of immune status for the final outcome of septic
patients. The study included 30 patients with severe sepsis due to community-acquired infections. Concentra-
tions of tumor necrosis factor alpha (TNF-␣), interleukin-6 (IL-6), IL-8, IL-10, and transforming growth factor
1 (TGF-1) in serum, as well as monocyte HLA-DR expression, were determined on admission and on days
3, 10, 13, and 17 during hospitalization. Of the 30 patients enrolled, 13 survived, while 17 died during their
hospital stay. All patients had significantly lower HLA-DR expression and higher pro- and anti-inflammatory
cytokine levels than healthy individuals. HLA-DR expression was significantly decreased in nonsurvivors at
almost all time points. In nonsurvivors, higher levels in serum of TNF-␣on days 13 and 17; IL-6 levels on day
3; and IL-10 on days 3, 10, and 13 were found. Baseline levels of TGF-1 were significantly higher in survivors.
Independent risk factors of mortality were IL-10 levels on days 3 and 10, while monocyte HLA-DR expression
on admission was a good predictor for survival. Several pro- and anti-inflammatory cytokines are oversynthe-
sized during severe infections, especially in patients with a poor outcome. Monocyte HLA-DR expression is an
early and constant predictive marker for survival in severe sepsis, while serum IL-10 levels on days 3 and 10
have negative prognostic value for the final outcome.
Sepsis is a leading cause of acute hospital admissions and
often complicates the clinical course of patients hospitalized
for other reasons. Despite the advent of innovative therapeutic
strategies and a vast body of knowledge related to its patho-
physiology, the mortality rate from severe sepsis remains high
at 40% (25) and may exceed 50%, especially among patients
who develop organ failure and septic shock.
The immune system plays a pivotal role in the pathogenesis
of sepsis. Recent studies have shown that sepsis is a bimodal
entity. The first phase is characterized by the systemic release
of proinflammatory cytokines, such as tumor necrosis factor
alpha (TNF-␣), interleukin-1 (IL-1), IL-8, IL-12, gamma inter-
feron (IFN-␥), and, possibly, IL-6 (24). Proinflammatory cyto-
kines are necessary for initiating an effective inflammatory
process against infection, whereas excess production of them
has been associated with multiple-organ-system dysfunction
and mortality (3). In the second phase, anti-inflammatory me-
diators such as transforming growth factor (TGF-), IL-10,
IL-13, IL-4, and prostaglandin E2 may be released in an effort
to counteract ongoing inflammation (4, 22, 31).
The expression of major histocompatibility complex (MHC)
class II on monocytes is a prerequisite for effective antigen
presentation to CD4
⫹
T cells, an important component of the
immune response to infection (13). Human leukocyte antigen
DR (HLA-DR) expression on peripheral blood monocytes has
been found to correlate highly with infection in many clinical
scenarios (6). Several investigators have shown that low levels
of HLA-DR expression in patients with infection and sepsis
are linked to recovery and mortality rates (9, 14, 30, 34).
Recent evidence indicates that, during sepsis, enhanced pro-
duction of proinflammatory cytokines is paralleled by in-
creased activity of counterregulatory mechanisms. Indeed, el-
evated levels of several pro- and anti-inflammatory cytokines in
plasma have been described in patients with sepsis. In a recent
study, it was found that mainly immunoparalysis, as detected
through an elevated serum IL-10/TNF-␣ratio, characterized
patients who succumbed to infection (12). So far, most studies
had included only measurements upon the admission of pa-
tients, with no or limited follow-up (16, 18, 32, 33). In the
present study, we sequentially measured the monocyte
HLA-DR expression and the serum TNF-␣, IL-6, IL-8, IL-10,
and TGF-1 concentrations in patients with severe sepsis for
up to 17 days after admission. We also investigated their prog-
nostic value for the final outcome and the interrelationships
between cytokines and the monocyte immunophenotype.
The study protocol was approved by the University Hospital
ethics committee, and all subjects gave written consent for
participation.
MATERIALS AND METHODS
Patients. Our study was performed at the Patras University Hospital, a 600-
bed tertiary referral hospital in western Greece. During a period of 24 months,
we included 40 patients (23 men and 17 women) with severe sepsis, admitted to
the intermediate care unit of the Department of Medicine. Ten patients were
excluded from the study because of prior antibiotic use, incomplete sampling, or
because they were lost to follow-up, leaving 30 patients (15 males and 15 females,
* Corresponding author. Mailing address: Section of Infectious Dis-
eases, Patras University Hospital, School of Medicine, Panorama Ele-
kistras 11, 26500 Patras, Greece. Phone: 30 2610993982. Fax: 30
2610993582. E-mail: cgogos@med.upatras.gr.
161
mean age of 64.6 ⫾5.2 years; range, 23 to 86 years) assessable for response. All
patients had recently acquired infections, with a mean duration of symptoms of
3.8 ⫾1.4 days prior to admittance to the hospital. Subjects with trauma, human
immunodeficiency virus disease, neutropenia, end-stage renal disease, or end-
stage hepatic disease or subjects receiving immunosuppressive agents were ex-
cluded. Another group of 12 healthy individuals, matched for age and sex (7
males, 5 females, mean age of 62.8 ⫾6.7 years), were also included in the study
as controls for determination of monocyte HLA-DR expression and serum
cytokine levels.
The definition of sepsis was based on the presence of at least two of the
following criteria (28): fever or hypothermia (temperature, ⱖ38 or ⱕ36°C),
tachycardia (heart rate, ⱖ90 beats/min), tachypnea (respiratory rate, ⱖ20
breaths/min) and leukocytosis or leukopenia (leukocyte count, ⱖ12,000 or
ⱕ4,000 per lor⬎10% immature forms), concurrently with the presence of
confirmed infection.
Additional requirements for severe sepsis were at least one of the following
(28): (i) hypotension (systolic blood pressure, ⱕ90 mm Hg; sustained drop in
systolic blood pressure, ⱕ40 mm Hg; or mean arterial pressure, ⱕ65 mm Hg
corrected within1hbyfluid resuscitation), (ii) arterial hypoxemia (partial pres-
sure of arterial oxygen, ⱕ75 mm Hg without evidence of primary lung disease),
(iii) metabolic acidosis (pH ⱕ7.3 or base deficit ⱖ5 meq/liter), (iv) oliguria (urine
output, ⱕ30 ml/h for at least 2 h despite adequate fluid replacement), (v) acute
alteration of mental status, or (vi) coagulation abnormalities of recent onset
(prothrombin time or activated partial thromboplastin time of ⱖ1.2 times the
upper normal limit plus d-dimers at ⱖ500 or platelets at ⱕ100,000/l).
Septic shock was defined as severe hypotension that lasts ⬎1 h, despite ade-
quate fluid resuscitation and pharmacologic intervention with vasopressor agents
(1). The severity of the patients’condition was assessed according to the simpli-
fied acute physiology score II (SAPS II) (2), a reliable tool for measurement of
illness in intermediate care units.
Blood cultures (three sets of aerobic and anaerobic broth) were done in all
cases on admission and during follow-up. Additional specimens were obtained
for bacteriologic culture and specific diagnostic procedures (e.g., ultrasound,
computed tomography, or gallium scan) were done to identify the infection site.
All patients were closely monitored during their hospital stay, and critically ill
patients with septic shock were transferred to the intensive care unit.
Sampling and assays. Sera for cytokine determination from patients and
healthy subjects were obtained from whole blood on admission (day 0) and on
days 3, 10, 13, and 17. They were clotted for 30 min at 37°C and stored at ⫺70°C
until analyzed. Determination of cytokine levels was performed by enzyme-
linked immunosorbent assays (ELISAs) as instructed by the manufacturers. For
TGF-1, we used ELISA kits from R&D Systems (Quantikine, Minneapolis,
Minn.); for TNF-␣and IL-10, the ELISA kits were obtained from Endogen
(Woburn, Mass.); and for IL-6 and IL-8, the ELISA kits were obtained from
Diaclone (Besancon, France). The detection limits of the assays for TGF-1,
TNF-␣, IL-10, IL-6, and IL-8 were 7, 1, 3, 2, and 25 pg/ml, respectively.
The monocyte HLA-DR expression was investigated on the same days. A
double-immunofluorescent whole-blood technique was used. The following di-
rectly conjugated monoclonal antibodies (MAbs) were used: a CD14 MAb-
fluorescein isothiocyanate (FITC) conjugate (clone MP9) and an anti-HLA-
DR–phycoerythrin (PE) conjugate (clone L243) from Becton Dickinson. After
completion of the incubation, the erythrocytes were lysed, and the leukocytes
were fixed with the Multi-Q-Prep system (Coulter). The appropriate isotype
controls were used. At least 10,000 cells from each sample were analyzed on the
EPICS XL (Coulter) flow cytometer, and the data were processed with XL2
software. The analysis was performed in a scattergram gate of cells with inter-
mediate side and forward scatter. Results were expressed as percentages of
HLA-DR-positive monocytes and as arbitrary units (mean fluorescent intensity
[MFI]). The percentage of HLA-DR-positive monocytes was calculated by the
coexpression of CD14 and HLA-DR antigens in the total CD14
⫹
population.
The arbitrary units were calculated as follows: the MFI of the isotype control was
subtracted from the MFI of the sample, and the difference was divided by the
MFI of the isotype control.
The serum lactate concentration was measured on admission by an enzymatic
method (Sigma Diagnostics), while C-reactive protein (CRP) was determined
quantitatively on days 0, 3, 10, 13, and 17 by rate nephelometry (Beckman
IMMAGE Immunochemistry Systems).
Statistical analysis. Values are expressed as prevalence rates or as the mean
⫾standard deviation, and in case of a skewed distribution, as the median and
range. Differences in quantitative variables between healthy individuals and
septic patients were analyzed by the unpaired ttest or its nonparametric equiv-
alent, the Mann-Whitney U test. Categorical data were analyzed with the
2
test.
Stepwise multiple regression analysis was utilized to explore relationships be-
tween noncategorical variables. Correlations between variables on septic patients
were tested by Pearson’s correlation coefficient or its nonparametric equivalent,
the Spearman’s rank correlation coefficient value. Logistic regression analysis
was used to detect which variables were most predictive of final outcome in septic
patients. Mortality odds ratio (ORs) were expressed with their 95% confidence
intervals (CI). A Pvalue of ⬍0.05 was considered significant. All statistical
analyses were performed with SPSS, version 9.0, software (SPSS, Chicago, Ill.).
RESULTS
Characteristics of the study population. In the patients’
group, severe sepsis was the consequence of pneumonia (n⫽
12), intra-abdominal infection (n⫽9), urinary tract infection
(n⫽5), severe soft tissue infection (n⫽2), or osteomyelitis (n
⫽2). Microorganisms were isolated in 18 patients (gram-neg-
ative bacteria, n⫽10; gram-positive bacteria, n⫽8). The most
frequently isolated bacteria were Escherichia coli (n⫽4),
Streptococcus pneumoniae (n⫽3), Proteus mirabilis (n⫽2),
methicillin-resistant Staphylococcus aureus (n⫽2), Staphylo-
coccus epidermidis (n⫽2), and Enterococcus spp. (n⫽1).
Other gram-negative bacteria (Pseudomonas aeruginosa,Kleb-
siella pneumoniae,Enterobacter aerogenes, and Acinetobacter
baumannii) were cultured from four patients. Candida was
isolated from one patient.
Of the 30 patients enrolled, 13 survived, while 17 died during
their hospital stay. The main patient characteristics are shown
in Table 1. No difference was detected between survivors and
nonsurvivors concerning age and sex distribution. The mean
SAPS II, lactate concentration, and CRP values were not sig-
nificantly different between these two groups. There was also
no difference in the prevalence of bacteremia and gram-nega-
tive infection between survivors and nonsurvivors. No correla-
tion between the types of organisms and the final patients’
outcome was detected. Twelve (40%) of the patients devel-
oped septic shock within 48 h after admission, and 8 of them
TABLE 1. Characteristics of survivors and nonsurvivors with
severe sepsis
Characteristic
a
Result for:
Survivors
(n⫽13)
Nonsurvivors
(n⫽17)
Age (yr) 62.8 ⫾4.1 65.7 ⫾5.7
Sex (no. of males/females) 6/7 8/9
SAPS II
b
42.7 ⫾14.6 48.6 ⫾14.3
No. (%) with gram-negative
infection
5 (38.5) 5 (29.4)
No. (%) with bacteremia 7 (53.8) 8 (47)
Lactate concn (mg/dl) 23.3 ⫾13.7 28.4 ⫾19
CRP (mg/dl)
c
15.5 ⫾14.6 23.3 ⫾16.1
No. (%) with hypoxemia
d
5 (38.5) 9 (52.9)
No. (%) with renal failure
e
5 (38.5) 8 (47)
No. (%) with DIC
f
3 (23) 6 (35.3)
a
All comparisons (P) were nonsignificant.
b
Score on admission.
c
CRP on admission.
d
Ratio of partial pressure of arterial oxygen to forced inspiratory oxygen
fraction of inspired oxygen: ⬍100.
e
Creatinine concentration of ⬎3.5 mg/dl or hemodialysis or oliguria of ⬍250
ml/24 h.
f
DIC (disseminated intravascular coagulation) represents prothrombin time
or partial thromboplastin time of ⱖ1.2 times the upper normal limit plus d-
dimers ⱖ500 or platelet count of ⱕ100,000/l.
162 LEKKOU ET AL. CLIN.DIAGN.LAB.IMMUNOL.
(66.7%) died, whereas the remaining 18 patients had no he-
modynamic deterioration during their hospital stay.
Cytokine production. Serum cytokine levels in healthy indi-
viduals were undetectable or ⱕ13 pg/ml for TNF-␣,ⱕ2.6 pg/ml
for IL-6, ⱕ2 pg/ml for IL-8, and ⱕ4 pg/ml for IL-10. Mean
levels of serum TGF-1 were 14,500 ⫾3,500 pg/ml (range, 11
⫻10
3
to 8 ⫻10
3
pg/ml).
On admission, patients with severe sepsis had significantly
higher levels of all measured cytokines (P⬍0.001) than did
controls. We found significantly higher levels of TNF-␣on days
13 and 17 in nonsurvivors compared to survivors (P⬍0.05),
while there was no significant difference on admission and at
days 3 and 10 (Table 2). Nonsurvivors had significantly higher
serum IL-6 concentrations than survivors only on day 3 (P⬍
0.05). Serum IL-8 concentrations were similar in the two
groups at all time points. IL-10 levels were significantly higher
in nonsurvivors than in survivors on days 3 (P⬍0.01) and 10
and 13 (P⬍0.05). In surviving patients, IL-10 levels decreased
steadily from admission to day 17. Baseline levels of TGF-1
were significantly higher (P⬍0.05) in survivors than in non-
survivors (Fig. 1). On day 10, there was a peak serum TGF-1
level in both groups, and then TGF-1 concentrations de-
creased steadily in survivors, while they remained high in non-
survivors. However, we didn’tfind significant differences be-
tween them at any time point after admission. Finally, no
correlation was found between serum cytokine levels and the
focus of infection.
HLA-DR expression on CD14
ⴙ
mononuclear cells. The per-
centage of peripheral blood monocytes expressing the
HLA-DR antigen in healthy donors was ⱖ85%. Likewise, the
mean level of HLA-DR per monocyte, expressed as MFI, was
52.3 ⫾20 in healthy controls. On admission, the percentage of
HLA-DR-bearing monocytes and the density of HLA-DR in
patients with severe sepsis were significantly lower than those
in controls (P⬍0.01). As shown in Table 3, monitoring of
monocyte HLA-DR expression revealed that nonsurvivors
were characterized by significantly lower levels on admission
and at days 3 and 13 (P⬍0.05) and 17 (P⬍0.01) compared
to survivors, while on day 10, this difference did not achieve
statistical significance (P⫽0.057). It is noteworthy that all
patients with monocyte HLA-DR expression of ⬎40% sur-
vived, while those who died had ⬍40% expression at all time
points (Fig. 2). In surviving patients, the percentage of mono-
cytes expressing HLA-DR (as well as MFI) increased progres-
sively from admission to day 17.
Correlation between HLA-DR expression and serum cyto-
kine levels. Stepwise multiple regression analysis was used to
detect the underlying correlations between monocyte
HLA-DR expression and cytokine levels at different time
points. A significant negative effect of day 3 serum IL-10 levels
on day 10 monocyte HLA-DR expression (P⫽0.041) was
demonstrated, while no other cytokine had any impact on
HLA-DR. There were significant negative bivariate correla-
tions between monocyte HLA-DR expression and concentra-
tions of IL-10 (day 3, r⫽⫺0.479 and P⫽0.031; day 13, r⫽
⫺0.537 and P⫽0.026; day 17, r⫽⫺0.822 and P⬍0.0005),
IL-8 (day 10, r⫽⫺0.533 and P⫽0.028), and TGF-1 (day 10,
r⫽⫺0.518 and P⫽0.04; day 17, r⫽⫺0.661 and P⫽0.007)
(Table 4). The IL-10/TNF-␣ratio was inversely correlated with
HLA-DR on days 3 (r⫽⫺0.479 and P⫽0.021) and 17 (r⫽
⫺0.683 and P⫽0.005).
Concerning the severity of disease, the SAPS II in relation to
monocyte HLA-DR expression and serum cytokine levels was
evaluated. A significant inverse correlation of monocyte
HLA-DR expression (r⫽⫺0.533, P⫽0.009) on day 3 with the
respective SAPS II was noted. The IL-10/TNF-␣ratio was
correlated with SAPS II on the same day (r⫽0.536, P⫽
0.008). Regarding standard clinical variables, there was a sig-
nificant inverse relationship between the baseline IL-10/TNF-␣
ratio and the serum lactate concentration (r⫽⫺0.687, P⫽
0.01).
TABLE 2. Cytokine levels of survivors and nonsurvivors with severe sepsis from admission to day 17
Day and group Cytokine level (pg/ml)
a
n
TNF-␣IL-6 IL-8 IL-10 TGF-
1
0 (Admission)
Survivors 59.4 (33–157.4) 179.3 (40–417.6) 24.1 (0–297.5) 55.6 (36.9–367.2) 30,213 (20,643–64,419)* 13
Nonsurvivors 115.9 (46.8–138.6) 184.2 (6.9–408.8) 4.3 (0–433.4) 66.4 (41.5–143.2) 25,144 (9,462–39,499) 17
3
Survivors 54.3 (32.9–149.9) 57.6 (13–274.9)* 3.7 (0–49) 40.2 (34.2–82.5)†39,357 (12,683–81,901) 13
Nonsurvivors 117.8 (43–126) 200.7 (10.8–407) 0.8 (0–26.9) 62.5 (40.2–102.8) 40,541 (9,983–68,826) 10
10
Survivors 48 (27.9–131) 33.3 (6.8–154.3) 0 (0–26.9) 39.6 (35–62.7)* 56,981 (21,307–94,551) 13
Nonsurvivors 110.3 (40.5–117.2) 83.2 (7.4–266.1) 5.1 (0–17.6) 54.8 (41.8–84.1) 67,428 (39,263–72,047) 4
13
Survivors 49.3 (17.6–119.7)* 12.6 (5.4–66.7) 0 (0–11.2) 35.8 (11.3–54.5)* 44,900 (29,560–90,003) 13
Nonsurvivors 115.93 (56.8–131.2) 74.6 (3.6–291.6) 14.3 (0–35) 54.5 (40.7–114.2) 44,829 (38,457–49,354) 4
17
Survivors 29.5 (11.8–128.5)* 4.9 (1.3–76.6) 0 (0–8.3) 4.6 (0–50.7) 25,671 (12,591–94,267) 13
Nonsurvivors 122.8 (104.6–141) 62.8 (5.7–120) 0 (0–0) 41.3 (40.2–42.3) 51,580 (34,382–68,778) 2
a
Shown are the median and range (in parentheses). *, P⬍0.05; †,P⬍0.01.
VOL. 11, 2004 IMMUNOLOGICAL PROGNOSTIC MARKERS IN SEVERE SEPSIS 163
Clinical significance of the immune parameters. When we
compared the monocyte HLA-DR expression and the serum
cytokine levels as risk factors of mortality, we found that IL-10
levels on days 3 (OR, 0.93; 95% CI, 0.86 to 0.99; P⫽0.035)
and 10 (OR, 0.80; 95% CI, 0.65 to 0.98; P⫽0.037) were
independent predictors of poor outcome. On the contrary, we
found that monocyte HLA-DR expression on admission (OR,
1.04; 95% CI, 1.00 to 1.08; P⫽0.03) was a good predictor for
survival in patients with severe sepsis. A most interesting find-
ing of the present study was that all patients (100%) with
serum TNF-␣levels below the median value (ⱕ89.52 pg/ml)
and monocyte HLA-DR expression of ⱖ30% on admission
survived (P⫽0.037).
DISCUSSION
Mononuclear phagocytes are central elements in host de-
fense against a variety of invading microorganisms and in the
pathogenesis of sepsis. They play a central role in the immune
response by presenting microbial antigens to T lymphocytes
and producing cytokines, thus initiating and regulating both
cellular and humoral immune responses. HLA-DR expression
plays a central role in the processing of antigen by macro-
phages and helper T cells. Several previous studies demon-
strated that low HLA-DR expression on monocytes was found
in patients with sepsis (13, 17), trauma (9), and severe burns
(26). Our study extends those observations and demonstrates
significantly lower monocyte HLA-DR expression in all pa-
tients with severe sepsis compared to healthy controls. We also
revealed that septic patients who survived had significantly
higher levels of HLA-DR expression compared to nonsurvi-
vors during a 17-day follow-up. This was found at almost all
time points (except day 10), which means that HLA-DR ex-
pression may serve as a dynamic prognostic marker not only on
admission but also during the whole septic episode in patients
with severe infections.
On the other hand, during inflammatory syndromes, a num-
ber of proinflammatory molecules are released and serve as
modulators of the immune response. Several studies have al-
ready been published that show increased concentrations of
various cytokines in the serum of patients with sepsis, empha-
sizing their possible contribution to predicting the final out-
FIG. 1. TGF-1 levels in survivors (gray bars) and nonsurvivors (black bars) with severe sepsis on admission and at days 3, 10, 13, and 17. Data
are presented as box plots with median lines, 25- and 75-percentile boxes, and 10- and 90-percentile error bars. A circle represents the outliers.
TABLE 3. Expression of HLA-DR on CD14
⫹
monocytes in
patients with severe sepsis from admission to day 17
Day
HLA-DR expression in
a
:
Survivors Nonsurvivors
% MFI % MFI
0 (admission) 43.1 ⫾25.4 3.9 ⫾3.3 23.4 ⫾20.7* 1.4 ⫾1.5*
3 45.6 ⫾29.3 8.1 ⫾9 25.7 ⫾15.4* 2.2 ⫾2.1*
10 58.4 ⫾19.3 9.4 ⫾7.7 33.4 ⫾12.7 5.2 ⫾3.7
13 60.3 ⫾23.2 14.7 ⫾13.8 32.4 ⫾10.3* 4.5 ⫾4.6*
17 71.2 ⫾16.8 15.8 ⫾9.1 31.6 ⫾4.1†5⫾0.3†
a
*, P⬍0.05; †,P⬍0.01.
164 LEKKOU ET AL. CLIN.DIAGN.LAB.IMMUNOL.
come in these patients (5, 8). Elevated levels of TNF-␣and
IL-6 have been associated with increased mortality from sepsis
(16, 29), and some investigators have indicated that IL-6 levels
are a good prognostic parameter in the early phase of sepsis.
However, sepsis is associated not only with the exacerbation of
the production of proinflammatory cytokines but also with the
release of many anti-inflammatory cytokines, such as IL-10 and
TGF-1. Previous investigations have indicated that TGF-1
represses the production of inflammatory cytokines by acti-
vated macrophages and induces the release of soluble TNF
receptor and IL-1 receptor ␣. It has been shown that mean
plasma TGF-1 levels were increased in patients with sepsis
syndrome compared to healthy donors (19). Except for TGF-
1, IL-10 is one of the immunosuppressive mediators postu-
lated to play an important role in down-regulation of the im-
mune response. Elevated levels of IL-10 have been associated
with increased mortality from septic shock (27, 32).
Our data indicated that all cytokine levels measured were
significantly higher in septic patients than healthy individuals,
as expected. Monitoring the serum cytokine levels was a good
marker of the immune status and outcome, but at different
time intervals, because TNF-␣levels were significantly higher
in nonsurvivors on days 13 and 17, IL-6 was higher only on day
3, and IL-10 was higher on days 3, 10, and 13. These findings
are in accordance with previous studies of ours (12) and other
investigators (33), who noted that TNF-␣and IL-10 had a
significant prognostic value for the final outcome.
On the contrary, HLA-DR expression on monocytes was
FIG. 2. Percentage of CD14
⫹
monocytes expressing HLA-DR in survivors (gray bars) and nonsurvivors (black bars) with severe sepsis on
admission and at days 3, 10, 13, and 17 during hospitalization. Data are expressed as means and 95% CIs.
TABLE 4. Bivariate correlations (Spearman rank coefficient) between various cytokines, SAPS II, CRP, and monocyte HLA-DR expression
in patients with severe sepsis
Data set Correlation coefficient (P) for monocyte HLA-DR expression at day
a
:
0
b
3 101317
TNF-␣⫺0.035 (0.862) ⫺0.122 (0.590) ⫺0.037 (0.899) ⫺0.270 (0.295) ⫺0.700†(0.004)
IL-6 ⫺0.023 (0.911) ⫺0.361 (0.091) ⫺0.233 (0.368) ⫺0.396 (0.116) ⫺0.882†(0.000)
IL-8 ⫺0.334 (0.071) ⫺0.149 (0.499) ⫺0.533* (0.028) ⫺0.341 (0.180) ⫺0.355 (0.194)
IL-10 ⫺0.104 (0.598) ⫺0.471* (0.031) ⫺0.417 (0.108) ⫺0.537* (0.026) ⫺0.822†(0.000)
IL-10/TNF-␣⫺0.007 (0.973) ⫺0.479* (0.021) ⫺0.230 (0.374) ⫺0.256 (0.321) ⫺0.683†(0.005)
TGF-
1
⫺0.118 (0.558) ⫺0.156 (0.477) ⫺0.518* (0.040) ⫺0.323 (0.207) ⫺0.661†(0.007)
SAPS II ⫺0.087 (0.648) ⫺0.533†(0.009) ⫺0.402 (0.110) ⫺0.514 (0.087) ⫺0.522 (0.288)
CRP ⫺0.163 (0.390) ⫺0.197 (0.368) ⫺0.370 (0.144) ⫺573 (0.051) ⫺0.100 (0.873)
a
*, P⬍0.05; †,P⬍0.01.
b
Admission.
VOL. 11, 2004 IMMUNOLOGICAL PROGNOSTIC MARKERS IN SEVERE SEPSIS 165
significantly higher in survivors both at admission and at almost
all time intervals. Therefore, according to our findings, mono-
cyte HLA-DR expression seems to be an early and constant
predictive marker for the prognosis of outcome in severe sep-
sis, while the main pro- and anti-inflammatory cytokine levels
were significantly higher in a rather later stage. There seems to
be a relationship between HLA-DR expression and cytokine
synthesis, because we detected an inverse correlation between
monocyte HLA-DR expression and IL-10 and IL-8 concentra-
tions, as well as with the IL-10/TNF-␣ratio. Interestingly, we
detected that serum IL-10 levels on day 3 inversely affected
monocyte HLA-DR expression on day 10 (P⬍0.05). Similar
findings have been reported by other investigators, who dem-
onstrated that IL-10 induces in vitro the downregulation of
HLA-DR surface expression of “normal”monocytes and of
immature dendritic cells obtained from peripheral mononu-
clear cells, with accumulation of MHC II molecules in intra-
cellular compartments (11, 15, 20).
Concerning TGF-1, we found significantly higher levels of
TGF-1 in survivors with severe sepsis compared to nonsurvi-
vors on admission. These results might be interpreted in terms
of the beneficial effect of TGF-1 on the hemodynamic com-
promise of septic shock in a previous murine model (23). In
that study, it was shown that TGF-1 inhibited inducible nitric
oxide synthetase mRNA and NO production in vivo in vascular
muscle cells, after its induction by cytokines critical in the
sepsis cascade. TGF-1 was shown in vitro to inhibit IFN-␥-
induced HLA-DR surface expression of various cell types
through a transcriptional effect (7, 21). TGF-1 has also been
described as downmodulating the monocyte surface expression
of HLA-DR in association with IL-10 (10). In the present
study, we found a significant inverse correlation between
monocyte HLA-DR expression and TGF-1 concentrations on
days 10 and 17, which is in accordance with the previous ob-
servations.
On a clinical basis, it is noteworthy that there was no differ-
ence in the prevalence of bacteremia and gram-negative infec-
tion between survivors and nonsurvivors, while the frequencies
of organ dysfunction were similar in both groups.
In conclusion, analysis of our data indicates that dynamic
monitoring of the immune status of septic patients may con-
tribute to the estimation of the death risk in relation to other
clinical or laboratory markers. Pro- versus anti-inflammatory
response may be sequentially identified at different time points
by measuring certain serum cytokine levels in correlation with
peripheral blood monocyte HLA-DR expression. The pres-
ence of immunoparalysis seems to be the main risk factor of
poor outcome in patients with severe sepsis, as indicated by the
diminished HLA-DR expression and the increased serum
IL-10 levels. Monocyte HLA-DR expression seems to be an
early prognostic marker for survival, while serum IL-10 levels
may be a good marker for identification of patients with a
guarded prognosis in a rather later stage.
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