1 Departement de Pneumologie Pediatrique-Institut National de la Santé et de la Recherche Médicale E213, Hopital Armand Trousseau, 75012 Paris; and 2 Unite Postulante Cytokines and Inflammation, Institut Pasteur, 75015 Paris, France
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ABSTRACT |
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Inflammation plays a critical role
in lung disease progression in cystic fibrosis (CF). This inflammatory
process is dominated by a neutrophil influx in the airways. To
determine whether the accumulation of neutrophils in the airways of CF
patients is associated with an altered function, we analyzed the
capacity of neutrophils isolated from the lung compartment and the
blood to release the major neutrophil pro- and anti-inflammatory
cytokines IL-8 and IL-1-receptor antagonist (ra) spontaneously and in
the presence of LPS. Comparison of cytokine production by blood
neutrophils from CF patients and from control subjects showed
significantly increased IL-8 and decreased IL-1ra release by CF
neutrophils. Comparison of cytokine production by airway and blood
neutrophils from CF patients also documented distinct profiles: the
spontaneous release of IL-8 and IL-1ra by airway neutrophils was
significantly higher than that from blood neutrophils. Culture in the
presence of LPS failed to further enhance cytokine production. Analysis of the effect of dexamethasone confirmed the difference in the responsiveness of lung and blood neutrophils in CF. Used at a concentration effective in reducing IL-8 production by blood
neutrophils, dexamethasone (106 M) was unable to repress
secretion of IL-8 by airway neutrophils. In addition, comparison of
cytokine production by airway neutrophils from children with CF and
children with dyskinetic cilia syndrome also documented distinct
profiles of secretion. These results are consistent with a dysregulated
cytokine production by lung and blood neutrophils in CF. They provide
support to the hypothesis that not only the CF genotype but also the
local environment may modify the functional properties of the neutrophils.
inflammation; cytokines
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INTRODUCTION |
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CYSTIC FIBROSIS (CF) is one of the most frequent lethal autosomal hereditary disorders in Caucasian populations (10). It is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective cAMP-dependent chloride ion conductance. In patients with CF, lung disease is the major cause of morbidity and mortality (9, 16, 24). The progressive decline of pulmonary function is due to a vicious cycle of airway infection and inflammation. Indeed, there is now evidence that inflammation plays a pivotal role and may be present very early in life, even before the onset of respiratory manifestations (2, 8, 11).
The inflammatory process in the CF lung is dominated by a
polymorphonuclear neutrophil influx (7). Accumulation of
neutrophils in the airways is associated with high concentrations of
neutrophil-derived mediators, in particular proinflammatory cytokines
such as IL-8 and TNF- (5). Neutrophils also release
numerous toxic agents, e.g., proteases and reactive oxygen species,
which contribute to the damage of lung tissue (4, 20, 32).
If the consequences of the neutrophil-dominated inflammation in CF with
an altered repair of the respiratory structures can be explained by an
overwhelming neutrophil toxicity, the mechanisms leading to neutrophil
accumulation and activation in the CF airways are poorly understood
(34). Several possibilities can be discussed. The
proinflammatory and anti-inflammatory imbalance with excessive
concentrations of the neutrophil chemotactic cytokine IL-8 certainly
plays an important role in the influx of neutrophils in the inflamed
airways (14, 19). Locally, bacterial toxins and
inflammatory mediators can directly activate the neutrophils to carry
out their cytotoxic activities. In addition, in CF patients, impaired
neutrophil functions may contribute to an abnormal release of
inflammatory mediators. Recently, Witko- Sarsat et al.
(33) provided data suggesting that
myeloperoxidase-dependent oxygenation activities are altered in blood
neutrophils from CF heterozygotes and homozygotes.
Therefore, in the airways of children with CF, it is unclear whether the excessive presence of neutrophils is solely a consequence of an increased influx of these cells or whether it is associated with a cellular dysfunction. To address this question we examined in the present study the capacity of neutrophils to release the major neutrophil pro- and anti-inflammatory cytokines, respectively, IL-8 and IL-1-receptor antagonist (ra) (23). We compared the production of these molecules by neutrophils isolated from the sputum and from the blood of children with CF. The capacity of neutrophils from CF children to release IL-8 and IL-1ra was also compared with cytokine production by blood neutrophils obtained from control subjects and by airway neutrophils obtained from children with chronic pulmonary disease related to dyskinetic cilia syndrome. In addition, we analyzed the response of airways and blood neutrophils to the anti-inflammatory action of dexamethasone.
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MATERIALS AND METHODS |
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Study populations and samples.
The CF population included 15 children, seven boys and eight girls
(mean age: 12.6 ± 0.4 yr). In all patients, the diagnosis of CF
was confirmed by sweat chloride concentration >60 meq/l (21,
25). Over a 3-mo period, all CF children who visited the
outpatient department were invited to participate in the study. The
criteria for eligibility were the ability to produce an adequate volume
of sputum and the absence of pulmonary exacerbation at the time of the
study. Results of physical examination, chest radiographs, and
pulmonary function tests with determination of forced vital capacity
(FVC), forced expiratory volume in 1 s (FEV1), oxygen
saturation, and sputum quantitative bacterial cultures were recorded at
the time of the study. The main characteristics of the CF patients are
summarized in Table 1 with the results of
genetic analysis, the clinical score determined by the
Schwachman-Kulkuczycki score (60 ± 9), the results of the
FVC and the FEV1 (57 ± 16% and 43 ± 18%,
respectively), as well as the presence of Pseudomonas aeruginosa.
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Isolation of neutrophils in blood samples. Blood was drawn onto heparin (20 IU/ml) in both groups (12). Ten volumes of blood was mixed with two volumes of glucose dextran (3% glucose; 3% dextran T250; Sigma, Saint Quentin Fallavier, France). After sedimentation, the leukocytes were recovered and diluted 1:2 in RPMI 1640 medium and then layered on Ficoll-Paque (Amersham Pharmacia Biotech, Orsay, France). The ratio was two volumes of leukocytes to one volume of Ficoll-Paque. After centrifugation for 25 min at 15°C and 500 g, the cell pellet was washed and centrifuged once for 5 min at 300 g. Contaminating erythrocytes were lysed after a 5-min incubation of the resuspended cells at 4°C in 5 ml of lysis buffer (8.32 g/l NH4Cl, 0.84 g/l NaHCO3, 43.2 mg/l Na4EDTA). Lysis was stopped by the addition of a large excess of RPMI 1640 medium (Life Technology, Saint Cergy Pontoise, France), and the cells were washed and centrifuged for 10 min at 200 g. The neutrophils were then purified after incubation with pan anti-human human leukocyte antigen (HLA) class II-coated magnetic beads (Dynabeads M450; Dynal, Oslo, Norway) for 20 min at 4°C with gentle rotation to deplete monocytes, B cells, and activated T cells (17). Cells were counted, and viability was assessed by the trypan blue dye exclusion test. The purity of the neutrophil suspension was >99%, as assessed by staining with May-Grünwald-Giemsa.
Isolation of neutrophils in sputum samples. Each child with CF or with dyskinetic cilia syndrome was asked to rinse his or her mouth, swallow the water, and blow his or her nose to minimize contamination with saliva and postnasal drip. The sputum was then collected in sterile cups and processed immediately.
The sputum was transferred in a petri dish and weighed. As described by Pang et al. (15), an aliquot of 10 ml of trypsin-EDTA containing 0.05% trypsin and 0.50 mM EDTA (Sigma) was added to each gram of sputum. The mixture was shaken vigorously and then incubated at 37°C on a rotator for 30 min. The procedure was repeated after the addition of an equal volume of fresh trypsin-EDTA. The cell suspension was filtered through a 40-µm nylon gauze (Falcon) to remove debris and then centrifuged for 5 min at 300 g and 4°C. The cells were incubated 5 min with trypsin inhibitor (volume equal to the volume of trypsin-EDTA) and then washed three times with cold phosphate-buffered saline (PBS). As described for blood samples, the neutrophils were then purified after incubation with pan anti-human HLA class II-coated magnetic beads. Cells were counted, and viability was assessed by the trypan blue dye exclusion test. The purity of the neutrophil suspension was >99%, as assessed by staining with May-Grünwald-Giemsa. In the present work, blood and airway neutrophils were isolated by a procedure based on the method described by Reglier et al. (17). This procedure, which includes an immunomagnetic depletion of cells other than neutrophils in sputum and blood samples, allows the collection of highly purified populations of neutrophils, with <1% of contaminating cells. The influence of this technique of isolation on the capacity of neutrophils to produce IL-8 was evaluated. Neutrophils were isolated from samples obtained from CF patients and control subjects by the dextran-Ficoll technique alone or dextran-Ficoll technique followed by immunomagnetic depletion. They were then cultured in medium without or with LPS, and the production of IL-8 was measured. In all samples tested, the use of immunomagnetic depletion was associated with a lower concentration of IL-8, confirming the elimination of contaminating cells. In the presence of LPS, the magnitude of increase of IL-8 secretion was similar in the two protocols, indicating that the functional response of neutrophils was not modified by the use of immunomagnetic beads (data not shown). Also, trypsin-EDTA had no effect on IL-8 production by blood neutrophils (data not shown).Neutrophil cultures.
Neutrophils were cultured in RPMI 1640 medium supplemented with
L-glutamine and antibiotics (100 IU/ml penicillin, 100 µg/ml streptomycin; Life Technology) and 5% heat-inactivated normal human serum (a pool of sera from healthy volunteers) (12).
We incubated 0.5-ml aliquots (5 × 105 cells) of
neutrophil suspension per well in a 5% CO2 incubator in
24-well multidish plates (Nunc; ATGC Biotechnology, Marne La Vallée, France) for 18 h at 37°C. We added 1 µg/ml LPS (Escherichia coli 0111:B4 LPS; Sigma) and/or
106 M dexamethasone (Sigma) in a volume not exceeding 10 µl at the beginning of the culture. At the end of the culture, the
supernatants were harvested, centrifuged 10 min at 300 g and
15°C, and kept at
20°C before cytokine measurements.
Measurement of cytokine concentrations. IL-8 and IL-1ra measurements were made using enzyme-linked immunosorbent assays (ELISA). We performed IL-8 ELISA as previously described using a monoclonal anti-human IL-8 antibody obtained by Dr. J. C. Mazie (Institut Pasteur, Paris, France) and a rabbit polyclonal anti-IL-8 antibody graciously provided by N. Vita (Sanofi Recherche, Labege, France) (13). The sensitivity of the ELISA was 3 pg/ml.
We set up an ELISA specific for IL-1ra using a monoclonal antibody (no. 84.1, prepared by Dr. J. C. Mazie) against a recombinant human IL-1ra (Synergen, Boulder, CO) and a rabbit polyclonal anti-human IL-1ra antiserum. Immunization of rabbits with recombinant human IL-1ra was performed with IL-1ra in complete Freund's adjuvant, and two boosters at 3-wk intervals were performed in incomplete Freund's adjuvant. Ammonium sulfate precipitation was performed on sera, and immunoglobulins were stored in 50% glycerol (20 mg/ml). Microtitration plates (96 wells, Maxisorp; Nunclon, Rockville, Denmark) were coated with 100 µl/well of mouse monoclonal anti-human IL-1ra (5 µg/ml in carbonate buffer, 0.05 M, pH 9.6) overnight at 4°C. After five cycles of washing with PBS-Tween 0.1% buffer, we carried out a protein blocking step using 100 µl/well of bovine serum albumin (BSA, 2% in carbonate buffer) for 60 min at 37°C. After additional washings, standards of recombinant human IL-1ra, diluted in PBS-0.1% Tween-1% BSA containing 10% RPMI 1640 medium were added in triplicate (100 µl/well). Cell supernatants of activated cell cultures in RPMI 1640 medium, tested 1:10 in PBS-Tween-BSA, were added in duplicate (100 µl/well). Incubation was performed for 2 h at 37°C. After being washed, 100 µl/well of rabbit polyclonal anti-human IL-1ra (diluted 1/375 in PBS-0.1% Tween-1% BSA) were added for 90 min at 37°C. After five cycles of washing, 100 µl/well of a peroxidase-labeled goat anti-rabbit immunoglobulin (Silenus-AMRAD Biotech, Victoria, Australia; 1/2,500 in PBS-0.1% Tween-1% BSA) were added. After 1 h of incubation at 37°C, plates were washed five times, and enzymatic activity was revealed by the addition of 100 µl/well ortho-phenylene-diamine substrate (1 mg/ml; Sigma) extemporaneously prepared in 0.05 M citrate buffer, pH 5, containing 0.06% hydrogen peroxide. Plates were kept in the dark, and the reaction was stopped by the additional of 50 µl/well 3 N HCl. Optical density at 490/630 nm was measured on microplate reader spectrophotometer (Dynex Technologies, Compiegne, France). The lower limit of sensitivity of the assay (blank ± 2 SD) was 40 pg. The coefficients of variation of intra-assay and interassay were 5.6 ± 3% and 13 ± 8.4%, respectively, for samples ranging from 40 pg/ml to 3 ng/ml. The ELISA was specific for IL-1ra and was negative for other cytokines such as IL-1Statistical analysis. Results are expressed as means ± SE. Statistical comparison was evaluated by analysis of variance followed by the Wilcoxon test. A P value <0.05 was considered significant.
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RESULTS |
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IL-8 secretion by blood neutrophils.
We first compared IL-8 production of blood neutrophils from control
subjects and from CF patients. Results are shown in Fig. 1. In control subjects (Fig.
1A), the release of IL-8 by blood neutrophils was
significantly increased in the presence of 1 µg/ml of LPS
(P < 0.001). We then tested the inhibitory effect of
dexamethasone. Spontaneous and LPS-induced IL-8 production levels were
significantly decreased in the presence of 106 M
dexamethasone (P < 0.001).
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IL-8 secretion by airway neutrophils.
Analysis of IL-8 production by neutrophils present in the airways of
children with CF revealed a profile different from that documented in
blood neutrophils (Fig. 2A).
First, the amount of spontaneously released IL-8 was significantly
higher than that from blood neutrophils from the same patients (+70%,
P < 0.01). Second, addition of LPS failed to further
enhance the secretion of IL-8. Third, dexamethasone, when used at
106 M, was unable to repress the production of IL-8 in
the absence or presence of LPS. Notably, the levels of IL-8 production
were similar in CF patients without or with P. aeruginosa
colonization.
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IL-1ra secretion by blood neutrophils.
Results of IL-1ra secretion by blood neutrophils are shown in Fig.
5. In control subjects (Fig.
5A), the spontaneous IL-1ra release by blood neutrophils was
1,550 ± 250 pg/ml and could be enhanced by the addition of LPS
(P < 0.05). Interestingly, dexamethasone failed to
reduce both the spontaneous and the LPS-induced production of IL-1ra.
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DISCUSSION |
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An excessive inflammatory response is likely to play an important role in the pathogenesis of lung disease in CF (18, 29, 30). This is supported by several studies that have documented increased levels of proinflammatory mediators in the CF airways and decreased production of anti-inflammatory molecules such as IL-10 (14, 17). Furthermore, the observation of a dramatic accumulation of neutrophils in the CF lung strongly suggests a role of these cells in the perpetuation of an abnormal airway inflammation (34).
The present work is the first report comparing airway and blood neutrophils from children with CF in terms of pro- and anti-inflammatory cytokine production and their respective responsiveness to glucocorticoids. Comparison of airway and blood neutrophils from the same CF patients showed distinct profiles of cytokine production spontaneously and in the presence of LPS, as well as differences in the response to dexamethasone, supporting the view that the local environment may modify the functional properties of the cells. In addition, comparisons of cytokine production by circulating neutrophils from children with CF and controls and by airway neutrophils from children with CF or dyskinetic cilia syndrome revealed significant differences, suggesting that genetic components may also participate in the altered neutrophil function in CF.
Results reported herein indicate that in CF patients the responsiveness
of neutrophils was different in the lung and in the systemic
circulation. Indeed, we show that the amount of spontaneous release of
IL-8 by airway neutrophils was significantly higher than that by blood
neutrophils from the same patients and that addition of LPS failed to
further enhance the secretion of IL-8. Increased production of IL-8 by
airway neutrophils corroborated the elevated levels of IL-8 we have
previously reported in the sputum of children with CF
(14). For IL-1ra, similar results were observed: the level
of IL-1ra produced spontaneously by lung neutrophils was significantly
increased compared with the level secreted by circulating cells and was
not modified by LPS. There is now cumulative evidence that neutrophils
can display distinct functional capacities depending on their local
environment. Pang et al. (15) analyzed the activity of
neutrophils isolated from sputum and blood in bronchial sepsis. They
documented higher production of IL-8, IL-1, and TNF- by sputum
neutrophils. They also showed that cytokine production by these cells
is constitutive, with little increase in the presence of LPS. In
addition, in contrast to blood neutrophils, anti-TNF-
antibodies did
not inhibit IL-8 production by sputum cells. Together these data
support the view that specific regulatory mechanisms of neutrophil
activation may be present in the airways that differ from those
involved in the systemic circulation. These mechanisms may vary,
depending on the underlying disease (3). In CF,
differences in blood and airway neutrophil behavior can be linked to
the intense inflammation and infection of the respiratory tract. They
can also be related to ionic composition of the airway surface fluid.
Tager et al. (26) demonstrated that exposure of
neutrophils to elevated chloride concentrations increases IL-8
synthesis and accelerates cell apoptosis and lysis.
Analysis of the effect of dexamethasone on IL-8 production confirmed
the difference in the responsiveness of lung and blood neutrophils in
CF patients. When used at a concentration effective in reducing IL-8
production by blood neutrophils (106 M), dexamethasone
was unable to repress the secretion of IL-8 by airway neutrophils
either in the absence or presence of LPS. An inhibitory effect of
dexamethasone on these lung cells could be observed only at a very high
concentration (10
3 M). These results share similarities
with the data reported by Pang et al. (15). In their
study, culture of sputum neutrophils from patients with chronic
bronchial sepsis with various concentrations of the anti-inflammatory
cytokine IL-10 showed no significant effect on IL-8 production, whereas
a major reduction of IL-8 secretion was observed with blood neutrophils
under the same experimental conditions. The resistance of IL-8
secretion by airway neutrophils provides additional evidence that the
mechanisms, which regulate neutrophil cytokine production within the
inflamed lung, differ from those controlling blood neutrophil response.
This conclusion is of importance for the development of
anti-inflammatory therapies in CF (29, 35). Clearly,
evaluation of the efficacy of drugs aimed at reducing lung inflammation
should be performed on cells isolated from the respiratory compartment.
Alternatively, the resistance of airway neutrophils to the effects of
dexamethasone could be due to their precondition status as a reflection
of their activation by either microbial products or inflammatory
agents, independent of their compartmentalization. Indeed, the
LPS-induced IL-8 production by circulating neutrophils from healthy
controls was essentially inhibited by dexamethasone when added
simultaneously with LPS. In contrast, the capacity of dexamethasone to
decrease IL-8 secretion was dramatically reduced when dexamethasone was added after the activation of the cells with LPS. Accordingly, one can
suggest that the altered inhibitory effect of dexamethasone on IL-8
release by airway neutrophils may be due to its inability to act on
cells that are already activated.
The present study also showed that neutrophils from CF patients constitutively secrete higher amounts of IL-8 than those from control subjects. Although this difference in neutrophil behavior could be explained by a sustained in vivo exposure of CF cells to various inflammatory mediators, one cannot exclude a genetic component to altered cytokine production by neutrophils in CF (6, 16). In this view, Russell et al. (22) reported that neutrophils from CF patients displayed a decreased responsiveness with regard to L-selectin shedding. They also showed that this reduction in L-selectin responsiveness was not observed in non-CF bronchiectasis patients, supporting the hypothesis that the response of CF neutrophils differs from that of neutrophils from patients without a defective CFTR gene. The results on neutrophil myeloperoxidase-dependent oxygenation activities reported by Witko-Sarsat et al. (31) and on neutrophil elastase release by Taggart et al. (27) also suggest a relationship between altered neutrophil functions and CFTR mutations. Data reported herein showing significant differences in cytokine production by airway neutrophils from children with two distinct causes of chronic obstructive lung disease, CF and dyskinetic cilia syndrome, provide additional support for a role of genetic component in the altered neutrophil function in CF.
Our results on IL-1ra provide additional evidence for a dysregulation
of the inflammatory response in CF. IL-1ra is a major anti-inflammatory
cytokine that functions as a specific inhibitor of the two other
functional members of the IL-1 family, IL-1 and IL-1
(1). In the present work, the spontaneous IL-1ra secretion
by blood neutrophils from CF patients was significantly lower than that
by neutrophils from control subjects. The striking point is that the
same blood CF neutrophils produced significantly higher concentrations
of the proinflammatory IL-8 than control cells, supporting the concept
of an altered regulation of neutrophil cytokine production in CF
patients. In the airways, CF neutrophils secreted higher amounts of
IL-1ra than blood neutrophils. However, from a consideration of the
intensity of CF lung inflammation and the altered production of IL-10
reported in several studies, it is likely that the local production of
IL-1ra is largely insufficient to control the inflammatory activities.
Indeed, the current understanding of the control of the inflammatory
response is that the anti-inflammatory agents should be present in far
greater concentrations than those of proinflammatory cytokines to
inhibit their actions.
To conclude, data reported in the present work provide additional evidence that the persistent and excessive inflammation in the lungs of CF patients involves a failure of the mechanisms that control the inflammatory response. An altered regulation of cytokine production by neutrophils is certainly an important factor that promotes continued inflammation and injury. Development of therapeutic interventions with specific cytokine inhibitors, anti-inflammatory cytokines, as well as anti-inflammatory drugs, which could target airway neutrophils, appears essential to control CF inflammation.
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ACKNOWLEDGEMENTS |
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The authors thank Marie-Claude Miesch for technical assistance.
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FOOTNOTES |
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This work was supported by grants from Association Vaincre la Mucoviscidose, Assistance Publique Hopitaux de Paris - Association Claude Bernard, Chancellerie des Universités (Legs Poix).
Address for reprint requests and other correspondence: A. Clement, Departement de Pneumologie Pediatrique-INSERM E213, Hopital Armand Trousseau, 26, Ave Dr Netter, 75012 Paris, France (E-mail: annick.clement{at}trs.ap-hop-paris.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 24, 2003;10.1152/ajplung.00156.2002
Received 20 May 2002; accepted in final form 13 January 2003.
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