Institut National de la Santé et de la Recherche Médicale Unité 408 and 479, Faculté Xavier Bichat, Département d'Anesthésie-Réanimation, Laboratoire de Biochimie A et Service de Pneumologie, Hôpital Bichat, Assistance Publique-Hôpitaux de Paris, 75877 Paris, France
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ABSTRACT |
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We
tested the novel hypothesis that neutrophils in the lung or the
airspaces may produce hepatocyte growth factor (HGF) in ventilated
patients with acute respiratory failure. Neutrophils were purified from
blood and bronchoalveolar lavage (BAL) fluid samples from 16 mechanically ventilated patients who underwent BAL for a diagnostic
workup of ventilator-acquired pneumonia. Most of the patients had
pneumonia (n = 11). Ten nonventilated patients served
as controls. Both blood and BAL neutrophils released HGF in vitro.
Basal HGF secretion by blood neutrophils from controls was 823 (666)
pg · ml1 · 10
7 neutrophils
(median, 25th-75th percentile) and doubled to 1,730 (1,684-2,316)
pg · ml
1 · 10
7 neutrophils
(P = 0.001) with lipopolysaccharide (LPS) stimulation. Basal HGF secretion by blood neutrophils from patients was similar [956 (655-2,140)
pg · ml
1 · 10
7 neutrophils,
P = 0.4] and doubled with LPS stimulation [2,767 (2,165-3,688)
pg · ml
1 · 10
7 neutrophils,
P < 0.0001 vs. controls]. Alveolar neutrophils
released HGF in vitro [653 (397-1,209)
pg · ml
1 · 10
7
neutrophils]. LPS stimulation did not significantly increase the HGF
release from alveolar neutrophils [762 (434-1,305)
pg · ml
1 · 10
7
neutrophils]. BAL HGF positively correlated with the BAL neutrophil count (P = 0.01, R = 0.58). We conclude
that blood and alveolar neutrophils from patients with acute
respiratory failure can produce HGF, a mitogenic factor that may
enhance the alveolar repair process.
alveolar epithelium; alveolar repair; acute respiratory distress syndrome; acute lung injury; growth factors
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INTRODUCTION |
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POLYMORPHONUCLEAR NEUTROPHILS are an essential component of the early response to infection or injury and accumulate in the alveoli in many pathological conditions. Neutrophils are thought to contribute to alveolar injury through the release of reactive oxygen species, potent proteases, and bioactive lipids. However, recent data suggest that neutrophils could also positively influence the tissue repair after injury (10, 11).
Functional restoration of the alveolar epithelium after an injury requires the proliferation and migration of type 2 alveolar epithelial cells (AEC2) and their differentiation into AEC1, a tightly regulated phenomenon. Hepatocyte growth factor (HGF), a heparin-binding growth factor, induces AEC2 proliferation in vitro and in vivo in rodents and promotes alveolar repair in different models of alveolar injury (16, 21, 22, 31). HGF has been detected in the lung after injury where it may be produced through different pathways (30). Active HGF can be synthesized through the cleavage of its circulating inactive precursor, pro-HGF, by an HGF-converting enzyme that is locally activated after lung injury (18). Local synthesis, secretion, and activation of HGF may also take place in the lung (25, 33). Fibroblasts, endothelial cells, and perhaps hyperplastic alveolar epithelial cells themselves are all a potential source of HGF in the human lung (25). Production of HGF by human alveolar macrophages remains a matter of debate (16, 25).
We recently observed that bronchoalveolar lavage (BAL) HGF levels were positively correlated with the total BAL neutrophil count in patients with acute alveolar injury (27). Moreover, preliminary data suggested that tissue neutrophils contain immunoreactive HGF (24). Therefore, we hypothesized that neutrophils could secrete HGF in blood or in the alveolar space and therefore might enhance the alveolar repair process. To test this hypothesis, blood and alveolar neutrophils were purified from critically ill ventilated patients so that we could measure their capacity to secrete HGF ex vivo under basal conditions and after lipopolysaccharide (LPS) stimulation.
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PATIENTS AND METHODS |
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Patients with acute alveolar injury. The study was carried out in an intensive care unit (ICU), where BAL is the standard procedure for the diagnosis of ventilator-acquired pneumonia. All consecutive mechanically ventilated patients who underwent BAL for the diagnosis of new pulmonary infiltrates, with fever or purulent aspirates, were included in the study. The exclusion criteria were HIV infection, end-stage cancer, age <18 yr, arterial PO2 (PaO2)/fractional rate of inspired O2 (FIO2) <70 mmHg, current pregnancy, septicemia, or inclusion in another protocol. The local ethical committee of Paris-Bichat University Hospital approved the study protocol and waived the need for informed consent. This committee did not approve the performance of repeated sequential BAL without a diagnostic rationale.
During the inclusion period, we studied 55 BAL and blood samples from 36 patients. Because of the small volume of BAL recovered from many patients, alveolar neutrophils could be purified from only 20 BAL from 16 patients. There were 13 males and 3 females, age 69 yr (median, 25th-75th percentiles). The patients were hospitalized in a surgical ICU because of cardiac surgery (n = 9), digestive surgery (n = 3), thoracic or vertebral surgery (n = 2), or major trauma (n = 2). None of those patients suffered from extrapulmonary infection at the time of the procedure. Four patients underwent a second BAL procedure 5, 7, 11, and 13 days after the first BAL, and the results were included in the study to increase the total number of samples. The physician in charge of the patients assessed the cause of the lung infiltrates (summarized in Table 1) after reviewing the available clinical and biological data and the results of the bacteriological analysis of BAL. Two patients fulfilled the American-European consensus conference criteria for the acute respiratory distress syndrome (ARDS) at the time of the BAL procedure, and one patient fulfilled the criteria for acute lung injury. One BAL only was studied for each of these patients. The BAL was performed 3.5 (2-6) days after the appearance of the infiltrates.
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Controls. Venous blood samples (5 ml) from 10 healthy volunteers were collected on EDTA to measure plasma HGF concentrations and HGF secretion by blood neutrophils.
We collected BAL fluid from 10 nonventilated patients who underwent a fiberoptic bronchoscopy for the preoperative evaluation of an esophageal cancer (n = 4), for the search of lung cancer in patients with chronic bronchitis (n = 3), and for suspected hemoptysis (n = 3). In all cases, fiberoptic bronchoscopy was normal.BAL procedure.
The BAL was done as previously described (27). All
patients were intubated and mechanically ventilated when the BAL was performed. Briefly, six aliquots of 20 ml of sterile saline solution were injected through the bronchoscope wedged in a pathological lung
segment and gently aspirated manually. The first aliquot, representative of a bronchial lavage, was discarded, and the other aliquots were pooled and filtered on sterile gauze. Ten milliliters of
the BAL fluid were immediately processed for a bacteriological direct
examination and culture. The diagnosis threshold for lung infection was
bacterial growth 104 colony-forming units/ml
(4). The remainder of each BAL (at least 10 ml) was kept
on ice and handled rapidly in our laboratory.
Purification and culture of blood and alveolar neutrophils. Alveolar neutrophils could be purified from 20 BAL samples. Blood neutrophils were available from those patients and were studied in parallel. Blood neutrophils from patients and healthy volunteers were isolated by sedimentation in medium containing 9% Dextran T-500 (Pharmacia, Uppsala, Sweden) and 38% Radioselectan (Schering, Lys-lez Lannoy, France). The leukocyte-rich suspension was then centrifuged on Ficoll-Paque medium (Pharmacia). The cell pellet was washed with PBS, and erythrocytes were removed by hypotonic lysis.
Blood neutrophils from the leukocyte-rich suspension and alveolar neutrophils from the BAL fluid were then further purified by incubation with pan antihuman HLA class II-coated magnetic beads (Dynal, Oslo, Norway), as previously described (10). The final cell population was >99% neutrophils, and cell viability was >98% as assessed by the Trypan blue exclusion test. Neutrophils were then resuspended in RPMI 1640 culture medium (Sigma, St. Louis, MO) supplemented with 2 mM glutamine, antibiotics, and 10% heat-inactivated FCS (Biowittacker, Gagny, France). Neutrophils were cultured at 37°C with 5% CO2 in 24-well tissue culture plates (Costar, Cambridge, MA) for 20 h (107 neutrophils · mlHGF and urea assay. HGF was quantified by using a commercial ELISA kit (Quantikine; R&D Systems, Abington, UK) following the manufacturer's instructions. Both HGF and pro-HGF were measured in this assay. The detection limit was 40 pg/ml.
BAL and plasma urea concentrations were measured on an Hitachi 911 autoanalyzer (Roche Diagnostics) to estimate the amount of epithelial lining fluid (ELF) according to Rennard et al. (23).Immunocytochemical detection of HGF on blood and alveolar polymorphonuclear neutrophils. Blood smears and BAL cytocentrifuge smears from patients were air-dried for 24 h, fixed in 4% formaldehyde in PBS for 20 min, and permeabilized by incubation in a PBS containing 0.1% Triton X-100 at room temperature for 30 min [according to Sorensen et al. (26)]. Smears were first incubated with normal horse serum for 30 min to block nonspecific binding. Binding of primary antibody was performed during a 1-h incubation at room temperature using anti-human-HGF monoclonal antibodies (12.5 µg/ml; MAb294, clone 24612.111; R&D Systems) diluted in PBS containing 0.3% gelatine. The slides were then consecutively incubated with a biotinylated antibody and alkaline phosphatase-labeled streptavidin following the manufacturer's instructions (Vectastain ABC kit; Vector Laboratories, Burlingame, UK). Staining was completed by incubation with a substrate chromogen solution (Fast red substrate solution; Dako, Carpinteria, CA). After being washed, the slides were counterstained in Mayer's hematoxylin and mounted and analyzed with light microscopy. Positive staining developed as a red-colored reaction product. Smears incubated with a nonspecific mouse immunoglobulin of the same isotype (10 µg/ml; R&D Systems) served as a negative control.
Statistical analysis. All results are expressed as medians with 25th-75th percentiles in parentheses. Statistical analysis was done with Sigmastat (Jandel Scientifics), with statistical significance defined at P = 0.05. Differences between the data obtained from different cultures of a given patient were analyzed with Wilcoxon's paired nonparametric test. The significance of differences between patients and healthy volunteers was determined with the Mann-Whitney U-test. For correlation between nonnormally distributed variables, we used the Spearman's rank order test. For statistical analysis, concentrations of HGF below the detection limit were assigned to the value of the detection limit (40 pg/ml).
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RESULTS |
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HGF secretion by blood neutrophils.
The ability of blood neutrophils from 10 control subjects and
ventilated patients to produce HGF was tested. The results are summarized in Fig. 1. Unstimulated blood
neutrophils from control subjects secreted HGF in vitro. Mean HGF
concentration in culture supernatants was 823 (666)
pg · ml1 · 10
7 neutrophils
(median and 25th-75th percentile). With LPS stimulation, the HGF
concentration doubled to 1,730 (1,684-2,316)
pg · ml
1 · 10
7 neutrophils
(P = 0.001). Similarly, unstimulated blood neutrophils from patients who secreted HGF in vitro totaled 956 (655-2,140) pg · ml
1 · 10
7
neutrophils (P = 0.4 vs. control subjects). With
LPS stimulation, HGF release by neutrophils also doubled to 2,767 (2,165-3,688) pg · ml
1 · 10
7 neutrophils
and was higher than HGF release by LPS-stimulated neutrophils from
control subjects (P < 0.0001).
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HGF secretion by alveolar neutrophils.
The analysis of BAL cells from the ventilated critically ill patients
showed a prominent neutrophilic alveolitis (Table
3). The ability of alveolar neutrophils
to secrete HGF could be measured only for ventilated patients, since
the BAL fluid in controls contained virtually no neutrophils. Alveolar
neutrophils released HGF in vitro [653 (397-1,209)
pg · ml1 · 10
7 neutrophils
(Fig. 1)]. Interestingly, LPS stimulation did not significantly
increase the HGF release from alveolar neutrophils [762
(434-1,305)
pg · ml
1 · 10
7
neutrophils]. For each patient, HGF release by alveolar neutrophils was significantly lower than that from blood neutrophils, either unstimulated (P = 0.02) or LPS stimulated
(P < 0.0001). HGF release by alveolar neutrophils did
not correlate with BAL HGF concentrations nor with HGF secretion by
blood neutrophils.
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Alveolar neutrophils contain less HGF than blood neutrophils.
Immunocytochemistry showed a positive granular cytoplasmic
staining of blood and BAL neutrophils (Fig.
2). Alveolar macrophages stained weakly
positive. Blood lymphocytes were negative (Fig. 2A). We
hypothesized that low HGF release by alveolar neutrophils and
hyporesponsiveness to LPS could be the result of prior degranulation (17). To test this hypothesis, we estimated the HGF
contents of neutrophils through the sum of extracellular and
intracellular HGF in neutrophils cultured for 15 min in basal
conditions. Total HGF was higher in blood neutrophils [3,370
(2,826-5,239)
pg · ml1 · 10
7 neutrophils,
n = 10 samples] than in alveolar neutrophils [1,401 (895-1,969)
pg · ml
1 · 10
7 neutrophils,
n = 10 samples, P = 0.02].
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Plasma and alveolar HGF concentrations. HGF was detected in the plasma of both normal volunteers and patients. The plasma HGF level in patients [2,267 (1,568-3,981) pg/ml] was significantly higher than that of controls [464 (410) pg/ml; P < 0.0001].
HGF was detected in all of the BAL supernatants from patients [1,209 (516-2,605) pg/ml], whereas it was undetectable in all of the BAL from nonventilated controls. BAL HGF concentration was not different in patients with or without lung infection. To compare lung and plasma HGF levels, we estimated the volume of ELF by using the urea dilution method, according to Rennard et al. (23), and we calculated the HGF concentration in the ELF. HGF concentration in ELF [13,773 (7,033- 25,151) pg/ml] was higher than in plasma [2,793 (1,682-4,671) pg/ml, P < 0.0001], thus suggesting some intra-alveolar production of HGF.Correlation between HGF concentration and biological or clinical
parameters.
Plasma HGF and BAL HGF concentration did not correlate with any of the
clinical scores (LIS, SAPS II, OSF,
PaO2/FIO2).
Interestingly, however, HGF concentration in BAL supernatant was
positively correlated with the absolute number of neutrophils in BAL
fluid (P = 0.01, R = 0.58, Spearman's
rank correlation test; Fig. 3).
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DISCUSSION |
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This study demonstrates for the first time that blood and alveolar neutrophils from ventilated critically ill patients with acute respiratory failure participate in the local and systemic production of HGF, a growth factor involved in the regulation of type 2 pneumocyte proliferation and alveolar repair. Moreover, the alveolar neutrophils obtained from the ventilated patients are hyporesponsive to LPS in terms of HGF release, compared with blood neutrophils.
The results indicate some local production of HGF in the alveolar space, since HGF concentration in the ELF was higher than in plasma. Yamanouchi et al. (32) found that alveolar HGF concentrations were higher than plasma HGF concentrations in patients with pulmonary fibrosis. These results are in agreement with those of Verghese et al. (29), who also reported that HGF concentrations in the pulmonary edema fluid were seven times higher than plasma HGF concentrations in patients with acute alveolar injury.
Activated neutrophils can damage the lung through several potential
mechanisms, including the release of proteases, cytokines, and reactive
oxygen species, but recent data also suggest a role for neutrophils in
the modulation of inflammation and tissue repair. In particular, we
found that blood neutrophils release oncostatin M (10), a
potent inducer of several antiproteases, including 1-antitrypsin (3). Blood neutrophils have
been shown to secrete vascular endothelial growth factor, a mediator of
vascular repair (9). In the current study, the results
support a role for neutrophils in the production of HGF in the alveolus
during acute respiratory failure from pneumonia, atelectasis,
contusion, or even severe hydrostatic edema (Table 1). First, we found
for the first time that alveolar and blood neutrophils contain
immunoreactive HGF and have the capacity to secrete immunoreactive HGF
in vitro. Second, BAL neutrophils stained positive with an anti-HGF
antibody, as previously shown for neutrophils in the skin
(17) and the liver (24). Third, there was a
positive correlation between the absolute number of BAL neutrophils and
BAL HGF concentration in patients with acute lung injury; this is a
consistent result, since we obtained similar results in a different
series of patients in a previous study (27). These results
suggest that human alveolar macrophages likely contribute to the
alveolar HGF burden, since alveolar macrophages were weakly
immunostained with an anti-HGF antibody. This conclusion is supported
by Sakai et al. (25), who found that human alveolar
macrophages contained immunoreactive HGF. Moreover, LPS-stimulated
human blood monocytes have been shown to secrete HGF in vitro
(8).
We observed that blood neutrophils from ventilated patients and
from control subjects spontaneously secrete HGF in vitro and that blood
neutrophils from ventilated patients are more reactive to LPS than
neutrophils from control subjects, thus suggesting a preactivation of
blood neutrophils from these patients. Chollet-Martin et al.
(5) previously found that, in the basal state, both whole
blood neutrophils and alveolar neutrophils obtained by BAL from ARDS
patients were activated, as shown by decreased L-selectin CD62L,
increased 2-integrin CD11b expression, and decreased
F-actin content. We also observed that HGF secretion by alveolar
neutrophils was not increased with LPS stimulation, whereas blood
neutrophils could be further stimulated to secrete HGF. Our group has
previously described the local LPS hyporesponsiveness of alveolar
macrophages during bacterial pneumonia (6). However, in
the current study, pulmonary infection is not relevant to alveolar
neutrophil hyporeactivity since HGF secretion by alveolar neutrophils
was not different in patients with or without pneumonia.
Immunohistochemistry results, as well as the evaluation of the sum of
intracellular and extracellular HGF, suggest that the
hyporesponsiveness of alveolar neutrophils could be because of their
previous degranulation in vivo, as suggested originally by Martin et
al. (15).
At this time, we do not know if the HGF secreted by neutrophils is biologically active since the assay that we used recognized both pro-HGF (inactive) and HGF, which is the biologically active form of the protein obtained by cleavage at a specific site. However, it should be noted that alveolar HGF has been shown by Verghese et al. (29) to be biologically active in patients with acute alveolar injury. We believe that HGF secretion by neutrophils is biologically significant. Indeed, during acute lung injury and pneumonia, there is a massive influx of neutrophils into the alveoli, and neutrophils will release HGF in the immediate vicinity of epithelial cells, the main cellular target of HGF. There may be local deleterious effects of HGF. Indeed, HGF inhibits the rate-limiting enzyme in de novo phosphatidylcholine synthesis and is capable of significantly inhibiting the synthesis and secretion of the phosphatidylcholines of pulmonary surfactant (30). However, HGF release by neutrophils in the airspaces of the lung may be beneficial in terms of alveolar repair, as has been reported for liver repair (28, 33). In different animal models of lung injury, intratracheal or intravenous HGF administration can decrease the extent of pulmonary lesions and improve the survival of the animals (21, 22, 31), even when given after the insult (31). HGF may exert its protective action through the following different pathways: stimulation of proliferation (16) and migration (12) of type 2 pneumocytes, inhibition of their apoptosis (7), and stimulation of angiogenesis (1).
In conclusion, these results demonstrate that both blood and alveolar neutrophils contribute to the production of HGF in the lung in ventilated critically ill patients with acute respiratory failure from infectious or noninfectious causes. We speculate that, in addition to their potent proinflammatory effects, neutrophils may have a beneficial effect during alveolar repair.
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ACKNOWLEDGEMENTS |
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We are very grateful to Patricia Mechighel (INSERM U 408) and Véronique Leçon and Francine Hochedez (Laboratoire de Biochimie A, Hôpital Bichat) for help in some of the experiments. J. Moreau provided valuable help in immunocytochemistry experiments.
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FOOTNOTES |
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This work was supported by a grant from the Programme Hospitalier de Recherche Clinique (PHRC 96).
Address for reprint requests and other correspondence: B. Crestani, Service de Pneumologie, Hôpital Bichat, 46 rue Henri Huchard, 75877 Paris Cedex 18, France (E-mail: bruno.crestani{at}bch.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.
10.1152/ajplung.00121.2001
Received 3 April 2001; accepted in final form 10 October 2001.
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