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Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3439-3446
By
From Unité d'Immuno-Allergie, Institut Pasteur, Paris; and the
Department of Anaesthesia and Intensive Care Medicine, Hôpital
Lariboisière, Paris, France.
Ex vivo cytokine production by circulating lymphocytes and monocytes
is reduced in patients with infectious or noninfectious systemic
inflammatory response syndrome. Very few studies have addressed the
reactivity of polymorphonuclear cells (PMN). To analyze further the
relative contribution of systemic inflammatory response syndrome alone
or in combination with infection we studied the interleukin-8 (IL-8)
production by PMN isolated from patients who had undergone cardiac
surgery with cardiopulmonary bypass (CPB) and patients with sepsis.
Cells were activated with either lipopolysaccharide (LPS) or
heat-killed streptococci. Compared with healthy controls, the release
of IL-8 by PMN in both groups of patients was significantly reduced
whether activated by LPS, independently of its concentration and
origin, or by heat-killed streptococci. These observations suggest that
stressful conditions related to inflammation, independently of
infection, rapidly dampened the reactivity of circulating PMN. We
investigated whether the observed diminished reactivity of PMN might
reflect an endotoxin tolerance phenomenon. Our in vitro experiments
with PMN from healthy controls indicated that PMN could not be rendered
tolerant stricto sensu. However, our data suggested that LPS-induced
mediators such as IL-10 may be responsible for the observed anergy in
patients.
POLYMORPHONUCLEAR neutrophils (PMN) are
key cells in inflammatory processes and during sepsis syndrome. Their
activation is associated with the release of many inflammatory
mediators such as eicosanoids, free radicals, and proteolytic enzymes.
Activated neutrophils display an upregulated expression of CD11b and
CD351 and bind to endothelial cells, contributing to
vascular endothelium damage.2,3 They migrate toward tissues
where they maintain inflammation and favor organ
dysfunction,4-6 which may lead to lethality.7
The reactivity of neutrophils is modulated by many cytokines.
Pro-inflammatory cytokines such as interleukin-1 (IL-1), IL-8, and
tumor necrosis factor (TNF) are potent activators of PMN functions. The
fact that IL-8 is one of the main cytokines produced by
PMN8-10 allows these cells to perpetuate their own
activation within an autocrine loop. Anti-inflammatory cytokines play a
major role in dampening PMN functions, including IL-8 production. We
and others have shown that IL-10 and, to a lesser degree, IL-4, IL-13, and transforming growth factor- During the course of inflammation, both pro- and anti-inflammatory
cytokines, rapidly released with excessive production, can be detected
in the blood compartment15 where they modulate the
reactivity of circulating leukocytes. Indeed, we have previously shown
that monocytes isolated from septic patients have a reduced capacity to
produce IL-1 Because IL-8 is the most abundant cytokine produced by neutrophils, we
were interested in analyzing the capacity of PMN from septic patients
to produce this specific chemokine. It was of interest to compare the
observable modifications in septic patients consecutive to the
infectious process itself and those associated with the inflammatory
reaction, independently of any microbial stimulus. Indeed, we had
previously shown that inflammation after surgery was sufficient to
reduce the capacity of monocytes to produce TNF- Patients.
Eleven patients with sepsis syndrome, as defined by Bone et
al,28 were studied upon admission to intensive care units
(ICU) or at initiation of their sepsis syndrome. The study included 9 men and 2 women, the average age being 49 ± 5 years (range, 21 to 69 years) and the mean simplified acute physiology score II (SAPS II)
being 49 ± 7. There were 8 cases of pneumonia, 1 of pelvic abscess,
and 2 of peritonitis, including 5 in septic shock. The outcome was
survival for 4 patients. The patients' characteristics are given in
Table 1.
Isolation of human polymorphonuclear cells.
Blood was drawn onto heparin (20 IU/mL) from healthy volunteers or from
patients. Ten volumes of blood was mixed with 2 vol of glucose dextran
(3% glucose; 3% dextran T250; Pharmacia, Uppsala, Sweden) and the
leukocytes were recovered after a 40-minute sedimentation at room
temperature.29 The leukocytes were then diluted 1:2 in
RPMI-1640 medium and layered on Ficoll-Hypaque (Milieu de
Séparation des Lymphocytes [MSL]; Eurobio, Les Ulis, France).
The ratio was 2 vol of leukocytes to 1 vol of MSL. After centrifugation
for 25 minutes at 15°C and 500g, the cell pellet was washed
and centrifuged once for 5 minutes at 300g. Contaminating
erythrocytes were lysed after a 5-minute incubation of the cell pellet
at 4°C by resuspension in 5 mL of lysis buffer
(NH4Cl = 8.32 g/L; NaHCO3 = 0.84 g/L; Na4EDTA = 43.2 mg/L). Lysis was stopped by adding a large
excess of RPMI-1640 medium (Glutamax; GIBCO Life Technologies, Paisley, UK) and the cells were washed and centrifuged for 10 minutes at 200g. The viability of polymorphonuclear cells (PMN)
was assessed by counting the cells in 0.1% eosine. A nonspecific
esterase staining was performed to evaluate the monocyte contamination,
which never exceeded 0.5%.
In vitro culture.
PMN cells were cultured in RPMI-1640 medium (Glutamax; GIBCO Life
Technologies) supplemented with antibiotics (penicillin, 100 IU/mL;
streptomycin, 100 µg/mL) and 5% heat-inactivated normal human serum
(a pool of sera from healthy volunteers). 0.5-mL aliquots of PMN
suspension per well were incubated in a 5% CO2 incubator in 24-well multidish plates (Costar, Cambridge, MA) for 24 hours at
37°C. Stimuli in volumes Reagents.
Escherichia coli (0111:B4) lipopolysaccharide was purchased
from Sigma (St Louis, MO), TNF- Two-step experiments.
PMN (2 × 106 cells/mL) were cultured in RPMI-1640
medium (Glutamax; GIBCO Life Technologies) supplemented with
antibiotics and 5% heat-inactivated normal human serum (complete
medium). A 0.5-mL aliquot of PMN suspension per well in 24-well
multidish plates was incubated for 24 hours at 37°C in a 5%
CO2 incubator in the presence of different reagents
("pretraitment period"). At the end of this preculture period,
the supernatants were obtained and 0.25 mL of fresh complete medium was
added to each well to preserve adherent neutrophils. Supernatants were
centrifuged for 10 minutes at 200g and 15°C and kept at
Assessement of cell-associated forms of IL-8 in whole-blood samples.
One milliliter of blood was centrifuged for 10 minutes at 500g.
Plasma was obtained and kept at Assessement of cell-associated forms of IL-8 in PMN.
At the end of the culture period (2 × 106 PMN/mL), the
PMN pellets were lysed by adding 100 µL of TRAx lysis buffer before adding 100 µL of diluent buffer and 300 µL of RPMI medium.
Cytokine enzyme-linked immunosorbent assay (ELISA).
IL-8 ELISA was performed as previously described30 using a
monoclonal anti-human IL-8 antibody obtained by Dr J-C. Mazié (Hybridolab, Institut Pasteur) and a rabbit polyclonal anti-IL-8 antibody graciously provided by Dr N. Vita (Sanofi Recherche, Labège, France). The sensitivity of the ELISA was 3 pg/mL. In collaboration with Dr S. Berthold (DPC Biermann, Bad Nauheim, Germany)
the values obtained with our ELISA were compared with those obtained
for the same samples using the chemiluminescent quantification of IL-8
(IMMULITE; DPC Biermann). A correlation of r = .98 (P = .0001) was achieved. We ensured that the TRAx buffer did
not interfere with the accuracy of the ELISA.
Apoptosis assay.
PMN apoptosis was assessed as the percent of cells with hypodiploid DNA
by using the technique described by Nicoletti et al.31 After 24 or 48 hours of culture of PMN with or without activators, cells were centrifuged at 200g for 10 minutes and washed in
phosphate-buffered saline (PBS). The cell pellets were gently
resuspended in hypotonic fluorochrome solution (50 µg/mL propidium
iodide [PI], 0.1% sodium citrate, 0.1% Triton X-100) and stored at
4°C in the dark overnight before the flow-cytometric analysis using a
FACScan flow cytometer (Becton Dickinson Immunocytometry System, San
Jose, CA). The red fluorescence of PI in individual nuclei and the
forward and side scatter were simultaneously measured. Cell debris were
excluded from acquisition by raising the forward scatter threshold.
Apoptotic nuclei were easily distinguishable from residual debris by
the high size side scatter value due to the condensation of nuclear chromatin. Ten thousand events were collected and analyzed using the
software Lysis II program. Apoptotic PMN nuclei were distinguished by
their hypodiploid DNA content from the diploid DNA content of normal
PMN nuclei.
Statistical analysis.
Statistical analyses were performed using the nonparametric
Mann-Whitney U-test for comparing data between healthy controls and
patients and the Wilcoxon signed-rank test for the two-step experiments
performed with the PMN from healthy controls.
Circulating and cell-associated IL-8.
Levels of circulating IL-8 were under the detection limit in controls
and in all but two patients undergoing cardiac surgery before
cardiopulmonary bypass (Fig 1), whereas
after cardiopulmonary bypass all patients but two had detectable levels
of plasma IL-8. Similarly, IL-8 was measurable in all but two septic
patients. The highest values were found among septic patients but the
mean levels between post-CPB and sepsis were not significantly
different.
IL-8 production by isolated PMN.
We studied the capacity of isolated circulating PMN to release IL-8 in
response to either 0.1 or 1 µg/mL of LPS of two different bacterial
origins or heat-killed streptococci (Fig
2). A significant reduction in the levels
of IL-8 produced in response to the different concentrations of LPS
from different origins and to heat-killed streptococci was observed in
both groups of patients. Sepsis or infectious systemic inflammatory
response syndrome as well as noninfectious inflammation both led to a
reduced capacity of PMN to release IL-8 upon in vitro stimulation. IL-8
production in patients undergoing cardiac surgery was also tested in a
few patients before the initiation of the cardiopulmonary bypass, and
the levels of released IL-8 were similar to those obtained in healthy
controls (data not shown).
In vitro modulation of IL-8 production by PMN from healthy donors.
The reduced levels of IL-8 release by PMN isolated from septic patients
or patients who underwent cardiopulmonary bypass could be the
consequence of in vivo exposure of PMN to endotoxin and/or to
IL-10. We attempted to mimic this situation by preculturing PMN from
healthy donors in the presence of LPS, IL-10, or both, before further
activation by LPS. Numerous investigators have reported a high percent
of apoptotic PMN after a 24-hour culture period.33,34
However, our PMN preparations (dextran + glucose sedimentation and a
Ficoll-Hypaque [MSL] step) and our culture conditions in the presence
of normal human serum led to a low percentage of apoptotic cells after
a 24-hour culture period. Recovery of live cells after an overnight
culture period was 89% ± 14% with 15% ± 2% and 9% ± 2%
apoptotic cells in the absence or in the presence of LPS, respectively.
After 48 hours the recovery was 46% ± 15% live cells and 35% ± 11% apoptotic cells. Thus, it was possible to perform the
two-step experiments over 48 hours. The mean values of four experiments
are shown in Fig 3: PMN maintained for 24 hours in culture medium alone were fully responsive to LPS during an
additional 24-hour culture period and release significant amounts
of IL-8 in a dose-dependent fashion, illustrating the good viability of
the cells. Such IL-8 production (24 to 48 hours) also occurred when the
cells had been precultured in the presence of increasing amounts of LPS
and then left in cultured medium alone. LPS-pretreated cells responded
in an additional fashion when reexposed to LPS. Similarly,
LPS-pretreated PMN had an enhanced IL-8 production upon a further
challenge by recombinant human TNF (10 ng/mL) compared with cells
maintained for 24 hours in culture medium alone (8,631 ± 3,880
v 3,293 ± 925, P = .04; mean of five
experiments). Thus, in contrast to monocytes which upon preexposure to
LPS are "tolerized" and have a lower capacity to release
cytokines,35 PMN were not rendered tolerant to LPS by prior
encounter with LPS. To see whether this effect was specific for LPS or
could be obtained with other activating signals, PMN were first
cultured in the presence of TNF, and further cultured in the presence
of LPS (Table 3). TNF pretreated PMN
continue to release IL-8 once TNF has been withdrawn. Addition of LPS
acted synergistically and led to an enhanced release of IL-8, while a
second TNF stimulation had an insignificant enhancing effect.
Circulating and cell-associated IL-8.
Systemic inflammatory response syndrome of infectious (eg, sepsis
syndrome) or noninfectious (eg, cardiopulmonary bypass surgery) origin
can both be associated with the detection of circulating cytokines.
Although the plasma levels of IL-8 could not discriminate between the
two types of inflammation, significantly higher levels of
cell-associated IL-8 were measured in septic patients (Fig 1). An
enhanced cell-associated IL-8 was found in patients after cardiac
surgery involving CPB as compared to pre-CPB in agreement with a
previous report.36 We have previously shown that a high proportion of IL-8 in the blood compartment was associated with erythrocytes and mainly with mononuclear and polymorphonuclear cells.32 A major proportion of the cell-associated IL-8
found with leukocytes was the result of internalization of surrounding IL-8. Because post-CPB and septic patients had similar levels of
circulating PMN (data not shown), the relative number of circulating cells may have had little influence on the lower level of
cell-associated IL-8 observed in the CPB group. These results may
rather reflect lower IL-8 receptor expression and IL-8 internalization
process in this group of patients. The analysis of cell-associated IL-8 after in vitro activation which led to a diminished level of
PMN-associated IL-8 among CPB patients as compared with healthy
controls (Table 2) further argues for a lower IL-8 receptor expression.
Indeed, C5a generated during extracorporal circulation and IL-8 itself known to modulate IL-8 receptor expression37,38 may be the causative agents which led to low IL-8 receptor expression. Further experiments will test it.
Sepsis and reduced cytokine production/PMN hyporeactivity.
Whole-blood assays have been widely used these last years to
investigate the capacity of circulating leukocytes to produce cytokines
within their local environment. A reduced capacity was observed when
IL-1, TNF- Inflammatory stress and ex vivo cytokine production.
To see whether the reduced IL-8 production by PMN we observed in septic
patients was specific to an infectious process or related to the
systemic inflammatory response, we included another group of patients
who underwent cardiac surgery associated with cardiopulmonary bypass.
Inflammatory stress that occurs during CPB is linked to the surgical
procedure itself as well as to the interaction of circulating
leukocytes with biomaterials during extracorporal
circulation.27 To our knowledge, very few investigators have tested the ex vivo capacity of leukocytes of CPB patients to
release cytokine upon activation. Naldini et al49 have
reported that phytohemagglutinin-induced production of IFN- In vitro tolerization of PMN from healthy donors.
To determine whether an LPS encounter in vivo in patients could alter
PMN reactivity to a later in vitro LPS stimulation, we performed
two-step experiments with PMN prepared from healthy controls. PMN were
first stimulated with LPS overnight and resuspended in fresh medium in
the presence of activating LPS for another 24-hour culture. IL-8
production was then assessed in the cell supernatants. Although a high
percentage of PMN has been reported to become rapidly apoptotic in
cultures,33,34 our culture conditions had a low level of
apoptotic cell after 24 hours and allowed us to perform these two-step
analyses. Although a similar protocol led to tolerized
monocytes,35,62 neutrophils that were first exposed to LPS
before a secondary stimulation with LPS had an enhanced cytokine
production.
Submitted October 1, 1997;
accepted December 22, 1997.
We thank the Cardiopulmonary Unit of the Hôpital
Lariboisière for its support and help, and Dr Dorris Grossmann
for linguistic advice.
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|
Abstract
Introduction
Abstract
(TGF-
, IL-1
(IFN-
10 µL were added at the beginning of the
culture. At the end of the culture, the supernatants were obtained,
centrifuged for 10 minutes at 300g and 15°C, and kept at
20°C before cytokine assessments.
