Phenotypic characterization of alveolar monocyte recruitment
in acute respiratory distress syndrome
Simone
Rosseau,
Peter
Hammerl,
Ulrich
Maus,
Hans-Dieter
Walmrath,
Hartwig
Schütte,
Friedrich
Grimminger,
Werner
Seeger, and
Jürgen
Lohmeyer
Department of Internal Medicine, Justus-Liebig-University, 35385 Giessen, Germany
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ABSTRACT |
In 49 acute respiratory
distress syndrome (ARDS) patients, the phenotype of alveolar
macrophages (AMs) was analyzed by flow cytometry. Bronchoalveolar
lavage (BAL) was performed within 24 h after intubation and on
days 3-5, 9-12,
and 18-21 of mechanical ventilation. The
27E10high/CD11bhigh/CD71low/
25F9low/HLA DRlow/RM3/1low AM
population in the first BAL indicated extensive monocyte influx into the alveolar compartment. There was no evidence of increased local
AM proliferation as assessed by nuclear Ki67 staining. Sequential BAL
revealed two distinct patient groups. In one, a decrease in 27E10 and
CD11b and an increase in CD71, 25F9, HLA DR, and RM3/1 suggested a
reduction in monocyte influx and maturation of recruited cells
into AMs, whereas the second group displayed sustained monocyte recruitment. In the first BAL from all patients, monocyte
chemoattractant protein (MCP)-1 was increased, and AMs displayed
elevated MCP-1 gene expression. In sequential BALs, a decrease in MCP-1
coincided with the disappearance of monocyte-like AMs, whereas
persistent upregulation of MCP-1 paralleled ongoing monocyte influx. A
highly significant correlation between BAL fluid MCP-1 concentration, the predominance of monocyte-like AMs, and the severity of respiratory failure was noted.
alveolar macrophages; monocyte chemoattractant protein-1; fluorescence-activated cell sorting; cell surface molecules
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INTRODUCTION |
THE
RECRUITMENT of leukocyte populations is one of the fundamental
mechanisms involved in inflammatory processes. Neutrophil influx into
the alveolar space has long been known in acute respiratory distress
syndrome (ARDS) and severe pneumonia (10, 14,
31), but excessive expansion of the alveolar mononuclear
phagocyte population in ARDS has also been reported (35).
It has recently been suggested that local proliferation causes
expansion of the alveolar macrophage (AM) population in inflammatory
lung disease (5), but there is growing evidence that
enlargement of the AM pool in sarcoidosis (18),
hypersensitivity pneumonitis (20), idiopathic lung
fibrosis (ILF) (22), and human immunodeficiency virus
(HIV)-related pulmonary disease (42) is attributed to the
recruitment of peripheral blood monocytes into the lung. These AM
precursors were identified by their immature monocyte-like immunophenotype, and augmented release of proinflammatory mediators was
ascribed to these cells (3, 22,
43).
Monocyte chemoattractant protein (MCP)-1 belongs to the supergene
family of C-C chemokines located on chromosome 17 and has specificity
for the recruitment of mononuclear leukocytes (24). Elevated MCP-1 levels in bronchoalveolar lavage (BAL) fluids have been
demonstrated in patients with sarcoidosis, ILF (9),
hypersensitivity pneumonitis (38), and ARDS
(16). In addition to its role as a chemoattractant, MCP-1
may participate in the pathogenesis of monocyte-mediated lung injury by
activating mononuclear phagocytes through the generation of
receptor-mediated calcium influx (33), increasing the
release of reactive oxygen species (41), and modulating
the adhesion properties of these cells (40). MCP-1 expression in monocytes is induced by transendothelial migration (39) and interaction with extracellular matrix proteins
(27). Thus one might speculate that extravasated monocytes
themselves trigger sustained monocyte recruitment and promote ongoing inflammation.
In the present study, we employed fluorescence-activated cell-sorting
(FACS) analysis for detailed immunophenotyping of the alveolar space
mononuclear phagocyte population in acute lung injury. Sequential BAL
was performed in patients with ARDS induced by sepsis, pancreatitis, or
pneumonia. We analyzed the expression of monocyte differentiation,
maturation, and activation markers; BAL fluid MCP-1 levels; and AM
MCP-1 gene expression and inquired about a correlation between alveolar
monocyte recruitment and MCP-1 levels and lung injury in these patients.
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MATERIALS AND METHODS |
Study population.
Patients included in the present study were recruited from the
intensive care unit of the Department of Internal Medicine, Justus-Liebig-University (Giessen, Germany). Healthy volunteers and
mechanically ventilated patients suffering from cardiogenic lung edema
(CLE) served as control groups. The study was approved by the local
ethics committee, and written informed consent was obtained from all
participants or the patient's closest relatives.
All patients were mechanically ventilated; the inspired O2
fraction (FIO2) and respirator settings,
including positive end-expiratory pressure, were chosen according to
the requirements of pulmonary insufficiency. General therapeutic
approaches included nutrition, volume substitution, and antibiotics.
Vasoactive or inotropic drugs were administered according to
hemodynamic variables as determined by right heart catheterization. In
all patients, the first BAL was performed within 24 h after
intubation. The second BAL was performed on days
3-5, the third on days
9-12, and the fourth on days
18-21, when possible, after the onset of
mechanical ventilation. General exclusion criteria for entry into this
study were cancer, interstitial lung disease, and HIV infection.
BAL was performed in 16 patients with ARDS after sepsis or pancreatitis
and in 33 patients with pneumonia-induced ARDS (ARDS/PNEUMONIA; Table
1). The general criteria for the
diagnosis of ARDS were roentgenographically diffuse and bilateral
alveolar infiltrates, pulmonary capillary wedge pressure < 16 mmHg, and the absence of acute or chronic left heart failure. First and
second BALs were performed in 49 patients, 39 patients underwent a
third BAL, and only 18 patients underwent a fourth BAL due to prior
extubation or death.
Eight mechanically ventilated CLE patients underwent bronchoscopy and
BAL within 24 h after intubation. Sequential BAL was not performed
because all CLE patients were extubated 72 h after the onset of
respirator therapy.
Fifteen healthy volunteers underwent a single bronchoscopy and BAL. The
participants had no history of cardiac or pulmonary disease, were free
of respiratory symptoms, did not take any medication, and displayed
normal lung function when tested.
BAL.
Bronchoscopy and BAL were performed by means of a fiber-optic
bronchoscope, with 10 aliquots of 20 ml of warm sterile saline being
infused into one segment and removed by gentle suction (35-60% recovery in ARDS patients, 65-85% recovery in healthy volunteers, and 78-113% recovery in patients with CLE). Lavage fluid was
filtered through sterile gauze, collected on ice, and immediately
centrifuged at 200 g at 4°C for 10 min to sediment BAL
fluid cells. The cells were counted with a hemocytometer, viability was
assessed by trypan blue exclusion, and differential cell counting was
performed in Pappenheim-stained cytocentrifuge preparations.
Supernatant aliquots were frozen in liquid nitrogen and stored at
80°C for subsequent cytokine measurements.
Immunofluorescence staining of BAL fluid cells.
Immunofluorescence labeling of BAL fluid cells was performed as
previously described (25). Briefly, 2 × 105 cells were distributed to each well of flexible
round-bottom microtiter plates (Falcon, Heidelberg, Germany) and washed
with PBS (Sigma, Munich, Germany) containing 0.1% BSA (Sigma) and
0.02% sodium azide (NaN3; Merck, Darmstadt, Germany).
Before the addition of monoclonal antibodies, 20 µl of a human
immunoglobulin preparation (Behring, Marburg, Germany) were added to
block AM FcIgG receptors. Saturating amounts of antibodies
directed against CD14 (My4, Coulter Immunology, Hialeah, FL), CD71
(OKT9, Ortho, Raritan, NJ), HLA DR (L243, Becton Dickinson, San Jose,
CA), 25F9 (45), 27E10 (3), RM3/1 (Dianova,
Hamburg, Germany; 46), CD11b (M522; 26), MHC I (positive control,
W6/32.HL, generously provided by A. Ziegler, Humboldt University,
Berlin, Germany; 1), or isotype controls (negative control;
Dianova) were added and incubated for 30 min at 4°C. The plates were
washed twice with PBS and incubated with biotin-labeled
F(ab')2 fragments of sheep anti-mouse immunoglobulin antibodies (Biozol, Munich, Germany) for 30 min on ice. In a further 30-min incubation step, biotinylated secondary antibodies were developed with streptavidin-coupled phycoerythrine-cyanine 5-tandem conjugate (TriColor, Medac, Hamburg, Germany). This technique circumvents the problem of autofluorescence imposed by the AMs. After
two final washes, the cells were resuspended in PBS and kept on ice
until flow cytometric analysis.
Quantification of proliferating AMs.
The monoclonal antibody Ki67 (Dianova) was used for the quantification
of proliferating AMs in ARDS patients and healthy control subjects.
Immunocytochemical staining was performed on cytospins of BAL fluid
cells with an immunoalkaline phosphatase technique [alkaline
phosphatase-anti-alkaline phosphatase (APAAP)] as previously described
(12). Briefly, cytospins were incubated with the Ki67 antibody followed by rabbit anti-mouse Ig (DAKO, Glostrup, Denmark), APAAP complexes, and the alkaline phosphatase substrate (Dianova). Counterstain was performed with Gill's hematoxylin (Sigma). Finally, the percentage of positive cells was estimated from 200 AMs.
Flow cytometric phenotyping of AMs.
Flow cytometry was performed with the use of a FACStarPLUS
flow cytometer from Becton Dickinson equipped with a 5-W argon-ion laser operating at 488 nm and 200 mW. AMs were gated by dual light scatter and autofluorescence characteristics. The data on forward- and
right-angle light scatter, green autofluorescence, and TriColor fluorescence intensity were recorded for 104 cells on the
FACStarPLUS data-handling system and further analyzed with
PC-LYSYS research software (Becton Dickinson). Specific TriColor
fluorescence distribution of the AMs is expressed as the fluorescence
intensity index [fluorescence intensity index of MAb = (MFI of
MAb
MFI of isotype)/(MFI of W6/32.HL
MFI of isotype),
where MFI is the mean fluorescence intensity corrected for the negative
control and signal-to-noise ratio values and MAb is the monoclonal
antibody].
Flow sorting of AMs.
AMs from healthy control subjects and from the first BAL of ARDS
patients were separated by flow sorting with the
FACStarPLUS flow cytometer described in
Flow cytometric phenotyping of AMs. A 100-µm
ceramic nozzle was attached to a large-nozzle sort-head assembly
(MACROsort, Becton Dickinson), which is specially designed for the
separation of large biological particles. The cytometer was adjusted to
a sheath pressure of 8.5 psi and a transducer frequency of 17.164 kHz,
flow rate was 400 events/s, and droplet delay was
15.2-15.7 with two deflected droplets. AMs were separated from granulocytes and lymphocytes by forward- and right-angle light-scatter properties and autofluorescence characteristics. Before
the cells were sorted, the sample line tubing was sterilized with 70%
ethanol and subsequently flushed with sterile normal saline. Unsorted
cell suspensions and sorted AM preparations were maintained on ice
throughout the separation procedure. The sheath fluid was routinely
assayed for endotoxin content by the use of a Limulus lysate
assay (COATEST Endotoxin, Chromogenix, Mölndal, Sweden) and
always ranged <10 pg/ml. These sorting conditions have been shown to
prevent artificial, isolation-induced cytokine production by AMs
(28). FACS separation of AMs obtained from BAL samples
always yielded purities of >96%. The viability of flow-sorted AMs was
consistently >90%.
Ex vivo AM MCP-1 gene expression.
Total cellular RNA from flow-sorted AMs (1 × 106 AMs
each) was isolated with the acid guanidinium
thiocyanate-phenol-chloroform method as previously described
(11). The constituent mRNA was reverse transcribed
according to the instructions of the manufacturer (StrataScript RT-PCR
kit, Stratagene, Heidelberg, Germany) in a final volume of 25 µl. The
synthesis of cDNA was carried out in a GeneAmp PCR System 2400 (Perkin-Elmer, Norwalk, CA) for 50 min at 37°C, and enzyme
inactivation was achieved by heating the reaction to 94°C for 7 min.
Subsequently, the reaction mixture was diluted with RNase-free water to
60 µl and stored at
80°C until used. The PCR was performed in 1×
PCR buffer (Perkin-Elmer), 1 mM each deoxynucleotide triphosphate
(dATP, dCTP, dGTP and dTTP), 1 µM intron-spanning specific primers
(
-actin, 5'-AAAGAACCTGTACGCCAACACAGTGCTGTCT-3' and
5'-CGTCATACTCCTGCTTGCTGATCCACATCTG-3', and MCP-1,
5'-TGAAGCTCGCACTCTCGCCT-3' and 5'-GTGGAGTGAGTGTTCAAGTC-3'; Stratagene),
0.75 U of AmpliTaq DNA polymerase (Perkin-Elmer), and 2 µl
of first-strand cDNA in a total volume of 25 µl. PCR profiles
consisted of initial denaturation at 94°C (1.5 min) followed by 25 (
-actin) or 35 (MCP-1) cycles of denaturation (94°C for 50 s), primer annealing (60°C for 60 s), and primer extension
(72°C for 60 s) in a GeneAmp PCR System 2400. The final
extension was performed at 72°C for 7 min. Aliquots of PCR products
were electrophoresed through 1.8% (wt/vol) NuSieve-agarose gels
stained with ethidium bromide for ~2 h at 75V. Negative controls were
routinely performed by running PCR without a cDNA template to exclude
false-positive amplification products. Positive controls were performed
with cDNA preparations obtained from lipopolysaccharide (LPS)-stimulated AMs. To verify the specificity of PCR amplifications obtained from the above-mentioned procedure, automated DNA sequencing was carried out on the purified cDNA samples according to the instructions of the manufacturer (model 373 A, Applied Biosystems, Darmstadt, Germany). By comparing the resulting DNA sequences with the
corresponding published sequences, we identified PCR products as
expected segments of spliced MCP-1 or
-actin mRNA species. With PCR
conditions optimized for primer and magnesium concentrations and cycle
number, amplification of cDNA samples was verified to be in the
exponential phase of PCR by comparing the amount of input RNA
equivalents with the yield of the MCP-1 and
-actin PCR products. The
specific MCP-1 product was quantified as previously described
(6) with some modifications. Briefly, with
-actin as a
housekeeping gene, input cDNA concentrations of different samples were
adjusted with distilled water to obtain comparable cDNA contents before
PCR amplification. Aliquots of unlabeled MCP-1 and corresponding
-actin PCR products were denatured at 94°C for 5 min before being
blotted onto nylon membranes (32). Membranes were baked at
80°C for 2 h, prehybridized, and hybridized in 10 ml of buffer
composed of 5× saline-sodium citrate, 5× Denhardt's solution, 1%
sodium dodecyl sulfate (Sigma), 50% deionized formamide (Clontech,
Palo Alto, CA), and heat-denatured salmon sperm DNA (Boehringer
Mannheim) at 42°C overnight with respective probes that were labeled
with [
-32P]dCTP by random hexamer priming
(15). Spin column chromatography (Boehringer Mannheim) was
used to remove unincorporated deoxynucleotide triphosphates as well as
small probe fragments. After two washes, quantification of MCP-1 gene
expression levels was performed with the use of a PhosphorImager SF
(Molecular Dynamics, Sunnyvale, CA). The results are expressed as mean
ratios normalized to
-actin signals.
BAL fluid MCP-1 levels.
MCP-1 protein in BAL samples was measured with the ELISA technique.
Maxisorp microtiter plates (Nunc, Wiebaden, Germany) were coated
overnight at 4°C with polyclonal goat antibodies to human MCP-1 (R&D
Systems, Abingdon, UK) followed by three washing steps with PBS
containing 0.05% Tween 20 (Sigma). Fifty-microliter samples of BAL
fluid were dispensed into the wells and incubated for 2 h at room
temperature. After being washed, application of a mouse monoclonal
antibody directed against MCP-1 (R&D Systems) was followed by
sequential incubation with a biotinylated donkey anti-mouse Ig antibody
(Dianova), avidin and biotinylated horseradish peroxidase (DAKO), and
the substrate 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
(ABTS; Sigma). Serial dilutions of human recombinant MCP-1 provided a
standard curve for each individual ELISA. Plates were read at 405 nm
with an ELISA photometer. Quantification of each cytokine was
performed in triplicate, with detection ranges of 10-1,000 pg/ml.
MCP-1 bioassay.
Human monocytes were isolated by a combination of Ficoll (Sigma)
density gradient centrifugation and counterflow elutriation (Beckman
Instruments, Palo Alto, CA). The isolated monocytes (purity 90-95%) were radiolabeled with 5 µCi of 111In (10 mCi/ml of 111InCl3; Amersham, Braunschweig,
Germany) tropolon (Fluka Chemical, Hauppauge, NY) as previously
described (13). Human pulmonary artery endothelial
cells (5 × 104, passages 4 and
5; Clonetics, San Diego, CA) were seeded onto 5-µm pore
size Transwell inserts (diameter 6.5 mm; Costar, Cambridge, MA) and
grown to confluence in 4-5 days. BAL supernatants from healthy
control subjects and ARDS patients were added to the lower compartments
in the absence and presence of a neutralizing mouse anti-human MCP-1
antibody (R&D Systems), and 1 × 106 radiolabeled
monocytes were added to the filter inserts of Transwell chambers. After
120 min, the monocytes from the lower compartment were collected and
counted in a gamma counter (Packard Canberra, Frankfurt, Germany), and
the number of monocytes that migrated through the endothelial barrier
is expressed as a percentage of counts in the lower compartment in
relation to the counts initially added to the upper compartment.
Statistical analysis.
Differences among the study groups were analyzed with the
Kruskal-Wallis test followed by Dunn's multiple contrast hypothesis or
the Wilcoxon-Mann-Whitney test considering Bonferroni's correction. For correlation of AM maturity and immaturity markers with BAL fluid
MCP-1 concentration and oxygenation index [arterial
PO2 (PaO2)/FIO2], the data
were log transformed to achieve normal distribution as assessed by the
Kolmogorov-Smirnov test. P values < 0.05 were
considered to represent a significant difference. Statistical
procedures were performed with the SPSS for MS Windows analysis system.
 |
RESULTS |
AM phenotype and MCP-1 levels in healthy control subjects, patients
with CLE, and in first BAL of ARDS patients.
In the first BAL after the onset of mechanical ventilation, a
significant increase in total cell count and a massive neutrophil influx were noted in all patients with ARDS (Table
2). Compared with that in the healthy
control subjects, the percentage of AMs was decreased in ARDS patients,
but absolute cell counts revealed a significant expansion of the AM
population (Table 2). AM and neutrophil counts in patients with ARDS
after sepsis or pancreatitis were not significantly different from the
cell counts in ARDS/PNEUMONIA patients. In CLE patients, absolute cell
counts were not different from those in healthy control subjects, but
neutrophil granulocytes were significantly increased (Table 2).
The immunophenotype of AMs from healthy control subjects,
patients with CLE, and the first BAL from ARDS patients is presented in
Fig. 1. In ARDS patients, expression of
CD14 and CD11b on AMs was markedly increased and of CD71, HLA DR, and
the marker of mature tissue macrophages, 25F9, was significantly
decreased. This AM phenotype resembled the expression pattern of
peripheral blood monocytes, suggesting monocyte recruitment into the
alveolar compartment in ARDS. In addition, 27E10, recognizing
inflammatory acute-phase monocytes/macrophages (3), was
highly elevated, whereas expression of RM3/1, a monocyte/macrophage
marker associated with the downregulatory phase of the inflammatory
process (46), was hardly expressed. AMs presenting this
"immature" phenotype (27E10high, CD11bhigh,
CD71low, HLA DRlow, 25F9low, and
RM3/1low paralleled by CD14high) displayed
lower autofluorescence intensity and were much smaller than
"mature" macrophages (27E10low, CD11blow,
CD71high, HLA DRhigh, 25F9high, and
RM3/1high paralleled by CD14low), again
suggesting monocyte origin of this AM subpopulation (Fig. 2). The AM phenotype in patients with
ARDS after sepsis or pancreatitis did not significantly differ from the
AM phenotype in patients with ARDS caused by pneumonia (Fig. 1), and
the AM phenotype in CLE patients closely resembled the AM phenotype in
healthy control subjects.

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Fig. 1.
Expression of monocyte markers (CD14 and CD11b),
macrophage differentiation antigens (25F9, HLA DR, and CD71), and
inflammatory acute (27E10)- and late (RM3/1)-phase markers on alveolar
macrophages (AMs) from healthy volunteers, patients with cardiogenic
lung edema (CLE), and first bronchoalveolar lavage (BAL) of patients
with acute respiratory distress syndrome (ARDS) after sepsis or
pancreatitis or ARDS caused by pneumonia (ARDS/PNEUMONIA). AMs were
gated by dual light-scatter characteristics and autofluorescence
properties. Fluorescence-activated cell-sorting (FACS) setting included
cell-by-cell compensation for autofluorescence. Results are means ± SE of fluorescence intensity index corrected for negative control
and signal-to-noise ratio values; n, no. of subjects.
Significant difference compared with healthy control subjects:
* P < 0.01; ** P < 0.001.
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Fig. 2.
Flow cytometric identification of 2 different macrophage
populations in the 1st BAL fluid from a patient with ARDS. BAL was
performed 4 h after onset of mechanical ventilation. Macrophage
subpopulations [monocytes (MONO) and AMs] were gated by
autofluorescence characteristics and CD14 intensity (dot plot analysis;
top) and were further analyzed for forward-scatter
properties (histogram; bottom). Small CD14high
immature AMs exhibiting lower autofluorescence intensity (MONO) and
large, highly autofluorescent CD14low mature AMs were
discriminated. PMN, neutrophil granulocytes; LY, lymphocytes.
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Nuclear staining of Ki67 antigen for quantification of AM
proliferation, which might contribute to the expansion of the AM population in ARDS, revealed only 0.5-1.5% positive AMs both in healthy control subjects and in the first BAL from ARDS patients (not significant).
Compared with those in healthy control subjects, MCP-1 levels were
elevated by more than one order of magnitude in the first BAL fluid
from ARDS patients, with no significant difference between ARDS induced
by sepsis or pancreatitis and ARDS with pneumonia as the underlying
disease (Fig. 3). Correction of BAL fluid
MCP-1 data according to the urea method was performed routinely to
obtain concentrations in the assumed volume of alveolar lining fluid. This resulted in
12-fold (mean; range 4- to 27-fold) higher cytokine concentrations. However, statistical analysis of these urea-corrected data did not reveal any previously unobserved differences (data not
shown). Monocyte-specific chemotactic activity in the first BAL fluid
from ARDS patients was significantly upregulated compared with that in
healthy control subjects (Fig. 3), with no significant difference
between ARDS and ARDS/PNEUMONIA patients. This monocyte-specific chemotactic activity was nearly completely reduced to control levels by
a neutralizing antibody against human MCP-1 (Fig. 3). BAL fluid MCP-1
levels in CLE patients were not significantly different compared with
those in healthy control subjects (77 ± 59 pg/ml;
P = 0.32; n = 8).

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Fig. 3.
Top: monocyte chemoattractant protein (MCP)-1
levels in BAL supernatant from healthy volunteers (n = 15) and in 1st BAL fluid from ARDS (n = 16) and
ARDS/PNEUMONIA (n = 33) patients. Data are means ± SE. * P < 0.001 compared with healthy
volunteers. Middle: monocyte-specific chemotactic activity
in BAL fluid from healthy volunteers (n = 15) and in
1st BAL fluid from ARDS (n = 16) and ARDS/PNEUMONIA
(n = 33) patients. Monocytes (1 × 106) transmigrated monolayers of human pulmonary artery
endothelial cells in the presence of BAL supernatant or BAL supernatant
plus saturating amounts of a neutralizing antibody against human MCP-1.
Data are means ± SE. * P < 0.01 compared with
healthy volunteers. Bottom: MCP-1-to- -actin ratio in AMs
from healthy control subjects and 1st BAL fluid from patients with ARDS
(n = 12) and ARDS/PNEUMONIA (n = 27).
MCP-1 gene expression of flow-sorted AMs was analyzed with
semiquantitative RT-PCR. Data are means ± SE.
* P < 0.001 compared with healthy volunteers.
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Flow-sorted AMs in the first BAL from ARDS patients showed strong
upregulation of MCP-1 gene expression compared with that in healthy
control subjects. The MCP-1-to-
-actin ratio differed significantly
from the control value, without a statistical difference between ARDS
and ARDS/PNEUMONIA patients (Fig. 3).
AM phenotype and MCP-1 levels in sequential BALs from ARDS
patients.
AM absolute cell counts increased, whereas neutrophil cell counts
slightly decreased in sequential BAL from ARDS patients (Table
3), with no significant difference
between ARDS and ARDS/PNEUMONIA patients. Immunophenotyping of AMs in
the course of sequential BALs revealed two distinct patient groups. The
first subgroup (subgroup A; Figs.
4 and 5; a
representative FACS analysis is presented in Fig.
6) displayed pronounced downregulation of
27E10 and CD11b and significant upregulation of CD71, HLA DR, and 25F9, i.e., a transition of the predominance of immature to mature
phenotype. In addition, expression of RM3/1, the monocyte/macrophage
marker associated with the downregulatory phase of the inflammatory
process, was markedly increased. A biphasic course was noted for CD14. It was significantly decreased on AMs obtained from the second BAL
compared with those from the first BAL, healthy control subjects, and
CLE patients. In the following lavages, CD14 was again upregulated but
did not surpass the expression level of AM from healthy control subjects or CLE patients. The second subgroup (subgroup B;
Figs. 4 and 5) exhibited prolonged predominance of the immature
phenotype in the course of sequential BALs, indicated by a continuously elevated expression of 27E10 and CD11b on AMs and the absence of CD71,
25F9, RM3/1, and HLA DR upregulation on these cells. CD14 was
persistently downregulated in these subgroups.

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Fig. 4.
Immunophenotyping of AMs from patients with ARDS after
sepsis or pancreatitis at 4 different time points (BAL number). First
BAL was performed within 24 h after intubation, 2nd BAL was
performed on days 3-5, 3rd BAL on days
9-12, and 4th BAL on days
18-21 after onset of mechanical ventilation. Two
different subgroups of ARDS patients were identified: those with
transient (solid lines) or persistent (dotted lines) changes in the AM
immunophenotype profile. Values of the 3rd and 4th BALs are missing in
the case of prior extubation or death. All single values (expressed as
fluorescence intensity index) of the time course of monocyte markers
(CD14 and CD11b), macrophage differentiation antigens (25F9, HLA DR,
and CD71), and inflammatory acute (27E10)- and late (RM3/1)-phase
markers are given.
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Fig. 5.
Immunophenotyping of AMs in the course of sequential BAL
in patients suffering from ARDS caused by pneumonia. First BAL was
performed within 24 h after intubation, 2nd BAL was performed on
days 3-5, 3rd BAL on days
9-12, and 4th BAL on days
18-21 after onset of mechanical ventilation. Two
different subgroups of ARDS/PNEUMONIA patients were identified: those
with transient (solid lines) or persistent (dotted lines) changes in
the AM immunophenotype profile. Values of 3rd and 4th BALs are missing
in the case of prior extubation or death. All single values (expressed
as fluorescence intensity index) of the time course of monocyte markers
(CD14 and CD11b), macrophage differentiation antigens (25F9, HLA DR,
and CD71), and inflammatory acute (27E10)- and late (RM3/1)-phase
markers are given.
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Fig. 6.
A representative FACS analysis of AMs from 1st (BAL I)
and 3rd (BAL III) BALs of a patient with transient changes in the AM
immunophenotype profile is given (abscissa: green autofluorescence,
ordinate: specific TriColor fluorescence of different antibodies). The
progressive disappearance of immature monocyte-like AMs
(27E10high/CD11bhigh/CD71low/HLA
DRlow/25F9low/RM3/1low) with
upregulation of CD14 expression in 1st BAL concomitant with the
increasing predominance of the mature AM phenotype
(27E10low/CD11blow/CD71high/HLA
DRhigh/25F9high/RM3/1high), with
downregulation of CD14 expression in the 3rd BAL, is obvious.
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Nuclear staining of Ki67 antigen for quantification of AM proliferation
revealed 0.3-1.1% positive AMs in sequential BALs from ARDS
patients (not significant compared with that in healthy control subjects).
MCP-1 levels in sequential BALs did, however, differ markedly between
the subgroups, with a sustained versus transient AM "immaturity"
profile. Patients were ascribed to the two subgroups depending on the
course of CD11b, 27E10, CD71, HLA DR, RM3/1, and 25F9 expression by AMs
as displayed in Figs. 4 and 5. Patients displaying progressive
macrophage maturation exhibited a steady decrease in MCP-1 levels on
repetitive BAL (Fig. 7, subgroups A), whereas patients with ongoing predominance of immaturity
markers demonstrated sustained or even increased BAL fluid MCP-1 levels (Fig. 7, subgroups B). When analyzed for all ARDS patients
and lavages (n = 155), BAL fluid MCP-1 levels were
significantly correlated to the AM surface expression of 27E10 (Fig.
8). This correlation was also
found at individual days of sequential BAL. In the first BAL, MCP-1 and
27E10 correlated, with r = 0.56 (P < 0.01; n = 49 samples); in the second BAL,
r = 0.61 (P < 0.01; n = 49 samples); in the third BAL, r = 0.53 (P < 0.05; n = 39 samples); and in the
fourth BAL, r = 0.55 (P < 0.05;
n = 18 samples).

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Fig. 7.
MCP-1 protein levels in sequential BAL fluids from ARDS
(top) and ARDS/PNEUMONIA (bottom) patients. Two
different subgroups of ARDS and ARDS/PNEUMONIA patients were
identified: those with transient (ARDS-A and ARDS/PNEUMONIA-A) or
persistent (ARDS-B and ARDS/PNEUMONIA-B) changes in the AM
immunophenotype profile as described in RESULTS. ARDS-A:
n = 11 subjects for 1st and 2nd BALs, 9 subjects for
3rd BAL, and 6 subjects for 4th BAL; ARDS-B: n = 5 subjects for 1st, 2nd, and 3rd BALs and 1 subject for 4th BAL;
ARDS/PNEUMONIA-A: n = 26 subjects for 1st and 2nd BALs,
18 subjects for 3rd BAL, and 9 subjects for 4th BAL; ARDS/PNEUMONIA-B:
n = 7 subjects for 1st, 2nd, and 3rd BALs and 2 subjects for 4th BAL. Data are means ± SE. * P < 0.01 compared with values of the respective subgroup A.
|
|

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|
Fig. 8.
Correlation of BAL fluid MCP-1 protein levels
with the expression of 27E10 on AMs (top) and correlation of
oxygenation index (arterial
PO2/inspired O2
fraction) with 27E10 expression on AMs (bottom) analyzed for
all ARDS patients and all lavages (n = 155). Values
were log transformed to achieve normal distribution as assessed by the
Kolmogorov-Smirnov test.
|
|
Correlation with the clinical data.
In ARDS patients, the expression of AM immaturity markers correlated
inversely with lung function given as
PaO2/FIO2
(shown for 27E10 in Fig. 8; CD11b: r =
0.62;
P < 0.01; n = 155 samples), and the
expression of "maturity" markers correlated positively with the
oxygenation index (CD71: r = 0.66, P < 0.01; 25F9: r = 0.71, P < 0.01; RM3/1:
r = 0.59, P < 0.01, HLA DR:
r = 0.64, P < 0.01; n = 155 samples). When individual days of sequential BAL were analyzed,
the correlation of the immaturity marker 27E10 with
PaO2/FIO2 was
r =
0.52 (P < 0.05;
n = 49 samples) in the first BAL, r =
0.6 (P < 0.05; n = 49 samples) in
the second BAL, r =
0.55 (P < 0.05;
n = 39 samples) in the third BAL, and r =
0.54 (P < 0.05; n = 18 samples) in
the fourth BAL. The correlation of
PaO2/FIO2
with the maturity marker CD71 was r = 0.53 (P < 0.05; n = 49 samples) in
the first BAL, r = 0.62 (P < 0.05;
n = 49 samples) in the second BAL, r = 0.49 (P < 0.05; n = 39 samples) in the third BAL, and r = 0.53 (P < 0.05;
n = 18 samples) in the fourth BAL. The overall survival
in ARDS patients was 63.3% (31 of 49 patients). In the subgroup with a
progressive transition to mature AMs and a decrease in MCP-1 levels in
sequential BAL, 28 of 37 patients (75.7%) survived. The survival rate
was only 25% (3 of 12) in patients with a sustained predominance of
immaturity markers on AMs and elevated MCP-1 levels.
 |
DISCUSSION |
The present study shows that in addition to the well-known
phenomenon of neutrophil recruitment, there is an early profound expansion of the sessile pool of AMs, with an impressive shift from a
mature to an immature cell type in both ARDS after sepsis or
pancreatitis and ARDS caused by severe pneumonia. Immunophenotyping of
the alveolar mononuclear phagocytes, together with the demonstration of
a lack of increased local proliferation, strongly suggests that a rapid
influx of monocytes from the vascular compartment represents the
predominant underlying event. In addition, evidence is presented that
upregulation and secretion of MCP-1 in the alveolar compartment may be
centrally linked to this process. Interestingly, a state of sustained
monocyte influx was found to be significantly correlated with the
severity of respiratory failure.
As anticipated for sessile mature macrophages, the alveolar mononuclear
phagocyte population of healthy control subjects was characterized by a
27E10low/CD11blow/CD71high/HLA
DRhigh/25F9high/RM3/1high phenotype
profile (4, 18, 20,
30, 45). This contrasted sharply with the
profile of
27E10high/CD11bhigh/CD71low/HLA
DRlow/25F9low/RM3/1low, which was
consistently noted in the early lavage fluids of ARDS patients and
represents a monocyte-like immunophenotype. In parallel with the
observation of an overall increase in BAL fluid total AM numbers, these
findings suggest that the rapid expansion of the AM population was
predominantly caused by an influx of monocytes. This view is further
supported by the analysis of forward scatter properties, demonstrating
smaller average cell size in the initial lavage fluids from ARDS
patients and by the lower autofluorescence characteristics of these
cells. The monocyte-like population of AMs in ARDS patients revealed
exceptional upregulation of the calprotectin complex 27E10, a
heterodimer of the calcium-binding proteins MRP8/MRP14, which are only
present on inflammatory acute-phase macrophages (3) and
which have previously been shown to be upregulated on AMs in pneumonia
(36). No evidence for increased local proliferation of AMs
in ARDS patients was obtained as judged by nuclear Ki67 staining. Such
local AM replication has been suggested as an alternative mechanism
that may increase AM numbers in the alveolar space (5).
Because all patients with ARDS were lavaged under conditions of
mechanical ventilation in contrast to healthy control subjects, one
might argue that this therapeutic intervention rather than the
underlying lung inflammatory disease might be responsible for the
alveolar monocyte influx. Because the AM phenotype of mechanically
ventilated patients suffering from CLE closely resembled the AM
phenotype of healthy control subjects, respirator therapy as well as
lung edema formation per se may not be responsible for the marked
alveolar monocyte recruitment in ARDS patients.
Performance of sequential BAL over 2-3 wk after the onset of
respirator therapy showed the appearance of two distinct groups of ARDS
patients. The larger subgroup was characterized by a progressive disappearance of the monocyte-like alveolar mononuclear phagocyte population concomitant with an increasing predominance of the mature AM
phenotype. In addition, enhanced expression of RM3/1, a marker present
on inflammatory late-phase monocytes/macrophages (46), was
noted. These findings clearly suggest a decrease in monocyte influx
into the alveolar space in these patients. In contrast, the other
subgroup displayed evidence for sustained monocyte recruitment, with
persistent predominance of the immature AM phenotype profile.
Interestingly, these immunophenotype features were highly significantly
correlated with the severity of disease, and a tendency toward higher
mortality with presumed ongoing influx of monocytes was found in the subgroup.
In contrast to the other immunophenotype markers, the course of CD14
expression on AMs in sequential BAL was more complex. Clearly elevated
in the initial BAL, CD14 expression sharply dropped and subsequently
increased again toward normal values in the patients with a
predominance of AM maturation, whereas the CD14 levels remained low in
the second to fourth BAL fluids from patients with presumed ongoing
monocyte influx. The expression of the LPS/LPS-binding protein receptor
on myeloic cells seems to be subject to a complex regulatory mechanism
because monocyte maturation either increased or decreased CD14
expression depending on the microenvironment of the cell
(19, 44). Such complex regulation was also
suggested to underlie the observation of both enhanced and depressed AM CD14 expression in sarcoidosis (18, 20,
37). Without being able to elucidate the regulation of
CD14 in the present study in more detail, the fact that the control
values of CD14 expression, with a progressive appearance of mature AMs,
were noted in later BAL samples from ARDS patients further supports the
notion of a normalization of the alveolar mononuclear phagocyte
population in these subgroups.
Elevated MCP-1 levels in BAL fluid have been previously demonstrated in
patients with sarcoidosis (9), ILF (9), and ARDS (16). The present study offers strong evidence of a
major role for MCP-1 in alveolar monocyte recruitment under the
currently investigated conditions of ARDS. First, in parallel with the
appearance of immature monocyte-like AMs in the alveolar compartment,
the initial BAL fluid MCP-1 levels were increased in all patients with
acute inflammatory lung injury, and the BAL fluid of ARDS patients
displayed increased monocyte chemotactic activity. This monocyte-specific chemotactic activity was significantly reduced by a
neutralizing anti-MCP-1 antibody. BAL fluid MCP-1 concentrations were
also measured in patients requiring mechanical ventilation due to CLE,
and MCP-1 levels in these patients were not different from those in
healthy control subjects, suggesting that lung inflammation rather than
respirator therapy or lung edema formation caused the alveolar space
MCP-1 response. Second, the patient subgroup presenting a decrease in
monocyte-like AMs in sequential BAL exhibited a marked decline in MCP-1
to near control values, whereas the subgroup displaying evidence for
sustained monocyte influx showed continuously elevated or even further
increased BAL fluid MCP-1 levels. Third, when analyzed for all ARDS
patients independent of the affiliation to the different subgroups,
there was a highly significant correlation between a monocyte-like
immunophenotype of the AM population and BAL fluid MCP-1
concentrations. Fourth, concomitant with elevated MCP-1 protein levels
in the initial BAL, flow-sorted AMs from ARDS patients showed
extensively upregulated MCP-1 gene expression. These data, obtained
under clinical conditions, are well in-line with investigations in
experimental lung injury, suggesting that the induction of MCP-1 in AMs
may be a major contributor to the recruitment of peripheral blood
monocytes into the alveolar compartment (7,
8, 21). MCP-1 has previously been shown to be
sufficient for the transmigration of monocytes across the alveolocapillary barrier in a transgenic mouse model (17),
which, of course, does not deny a substantial contribution of other
monocyte-specific chemokines (23). Peripheral blood
monocytes do not express MCP-1 message constitutively, but this is
strongly induced by transendothelial migration (39) and
interaction with extracellular matrix proteins (27). Taken
altogether, one might thus speculate that extravasated monocytes
themselves, in a positive feedback loop, trigger further alveolar
monocyte influx by MCP-1 synthesis. Monocytes and macrophages may not,
however, be the only cells responsible for the regulation of the
alveolar space MCP-1 response in acute lung injury. Epithelial cells
have been shown to secrete MCP-1 constitutively (2,
23) and in response to the macrophage-derived
early-response cytokines TNF-
and interleukin-1
(34), and they showed a polar secretion of this chemokine
into the apical compartment for initiating or maintaining a chemotactic
gradient (29).
In conclusion, evidence is presented that the early expansion of the
alveolar mononuclear phagocyte population observed in patients with
ARDS is mainly due to a rapid influx of monocytes from the vascular
compartment. Upregulation of MCP-1 synthesis and its secretion into the
alveolar space are suggested to be centrally involved in this process.
These events were found to be significantly correlated with the
severity of respiratory failure. Further studies are encouraged to
elucidate the mechanisms responsible for MCP-1 regulation and the on-
and off-switching of the monocyte recruitment response in more detail.
 |
ACKNOWLEDGEMENTS |
We thank M. Lohmeyer, R. Maus, and G. Wahler for excellent
technical assistance and Dr. R. L. Snipes for proofreading the manuscript.
 |
FOOTNOTES |
This work was supported by the Deutsche Forschungsgemeinschaft,
Klinische Forschergruppe "Respiratorische Insuffizienz"
(Justus-Liebig-University, Giessen, Germany).
Address for reprint requests and other correspondence
and present address of S. Rosseau: Charité-Humboldt Univ. Berlin,
Dept. of Internal Medicine and Infectious Diseases, Campus Virchow, Augustenburger Platz 1, 13353 Berlin, Germany (E-mail:
simone.rosseau{at}charite.de).
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. §1734 solely to indicate this fact.
Received 17 August 1999; accepted in final form 2 February 2000.
 |
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