1 Department of Internal Medicine, Division of Pneumology, 4 Division of Pathology, Justus Liebig University, 35392 Giessen; 3 Medical Policlinic, University of Munich, 81366 Munich, Germany; 2 Department of Medicine, Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine, Nashville 37232; and Department of Veterans Affairs, Nashville, Tennessee 37212
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intratracheal instillation of
the monocyte chemoattractant JE/monocyte chemoattractant protein
(MCP)-1 in mice was recently shown to cause increased alveolar
monocyte accumulation in the absence of lung inflammation, whereas
combined JE/MCP-1/lipopolysaccharide (LPS) challenge provoked acute
lung inflammation with early alveolar neutrophil and delayed alveolar
monocyte influx. We evaluated the role of resident alveolar
macrophages (rAM) in these leukocyte recruitment events and related
phenomena of lung inflammation. Depletion of rAM by pretreatment of
mice with liposomal clodronate did not affect the JE/MCP-1-driven
alveolar monocyte accumulation, despite the observation that rAM
constitutively expressed the JE/MCP-1 receptor CCR2, as analyzed by
flow cytometry and immunohistochemistry. In contrast, depletion of rAM
largely suppressed alveolar cytokine release as well as neutrophil and
monocyte recruitment profiles upon combined JE/MCP-1/LPS treatment.
Despite this strongly attenuated alveolar inflammatory response,
increased lung permeability was still observed in rAM-depleted mice
undergoing JE/MCP-1/LPS challenge. Lung leakage was abrogated by
codepletion of circulating neutrophils or administration of anti-CD18.
Collectively, rAM are not involved in JE/MCP-1-driven alveolar monocyte
recruitment in noninflamed lungs but largely contribute to the alveolar
cytokine response and enhanced early neutrophil and delayed monocyte
influx under inflammatory conditions (JE/MCP-1/LPS deposition). Loss of
lung barrier function observed under these conditions is rAM
independent but involves circulating neutrophils via
2-integrin engagement.
monocyte; neutrophil; vascular permeability; lung; inflammation; depletion
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
WHEN FORMULATING IMMUNE MODULATORY strategies targeting the initiation of pulmonary host responses to airborne inflammatory agents, one needs to consider the complex cross-talk among various cellular components of the alveolar microenvironment, their secretory products, and leukocyte populations recruited from the vascular bed on inflammatory challenge. Resident alveolar macrophages (rAM) are strategically located to initiate, propagate, and modulate early lung inflammatory events, and studies employing liposomal clodronate-mediated macrophage depletion in mice have shown that macrophages are important effector cells of pulmonary immune responses, both innate and adaptive (10, 20). In lipopolysaccharide (LPS)-induced pneumonia, depletion of alveolar macrophages is associated with reduced alveolar cytokine liberation and alveolar neutrophil accumulation (1, 19). Contrarily, in Pseudomonas or Klebsiella pneumonia, depletion of alveolar macrophages attenuates early lung cytokine responses but ultimately leads to increased alveolar neutrophil traffic without improvement of late-stage overall pulmonary infection or even survival (2, 3, 9). This indicates that the sequence of inflammatory events developing during LPS- vs. bacterially induced pneumonia is differentially dependent on and thus affected by alveolar macrophages.
A putative contribution of alveolar macrophages to the recruitment of monocytes to the alveolar space is only poorly defined. Monocytes corecruited with neutrophils at the onset of LPS-induced lung inflammation were recently shown to exhibit increased expression of the LPS/LPS-binding protein receptor CD14 compared with circulating monocytes and have been suggested to contribute to the aggravation of inflammatory sequelae (12). Moreover, strongly elevated numbers of newly recruited monocytes within the bronchoalveolar space of patients with septic acute respiratory distress syndrome (ARDS), together with heavily increased levels of the monocyte chemoattractant protein (MCP)-1, have recently been correlated with poor patient outcome (17a). Finally, an animal model mimicking key pathophysiological features of ARDS, including early alveolar neutrophil and strongly increased alveolar monocyte recruitment, together with loss of lung barrier integrity, was most recently described to develop in response to combined JE/MCP-1-plus-Escherichia coli LPS challenge in mice (14).
In the current study, we evaluated the role of rAM in the process of
alveolar monocyte recruitment in response to the monocyte chemoattractant JE/MCP-1 in either the absence (noninflammatory conditions) or the presence of E. coli LPS (acute lung
inflammatory conditions). In response to sole JE/MCP-1 challenge, rAM
were not found to be involved in the alveolar monocyte recruitment process. In the presence of JE/MCP-1 plus E. coli LPS,
however, selective depletion of alveolar macrophages dramatically
affected alveolar inflammatory responses reflected by profoundly
reduced bronchoalveolar lavage (BAL) levels of tumor necrosis factor
(TNF)- and macrophage inflammatory protein (MIP)-2 and suppression
of alveolar neutrophil and monocyte recruitment. In contrast, induction of lung barrier failure was not influenced by rAM depletion but was
blocked by interfering with neutrophil migration. Thus distinct functions during lung inflammatory sequelae, but not during monocyte recruitment in noninflamed lung tissue, are attributable to rAM.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals. Female BALB/c mice (18-21 g) were purchased from Charles River (Sulzfeld, Germany) and used throughout the study between 8 and 12 wk of age.
Reagents. JE, the murine homolog to the human MCP-1 gene product (JE/MCP-1), was purchased from R&D Systems (Wiesbaden, Germany) as a recombinant protein preparation. The recombinant (r) JE/MCP-1 was LPS-free as analyzed by amebocyte lysate assay (detection limit <10 pg/ml, COATEST; Chromogenix, Mölndal, Sweden). Clodronate (dichloromethylene-diphosphonate, Cl2MDP) was obtained from Sigma (St. Louis, MO). The red fluorescent dye PKH26, as well as diluent B solution, was obtained from Zynaxis (Malvern, distributed by Sigma, Deisenhofen, Germany). FITC-labeled albumin was obtained from Sigma. For flow cytometric detection of CCR2 expression on rAM and circulating monocytes, we used a rat anti-mouse anti-CCR2 monoclonal antibody (MAb) most recently described in detail (clone MC-21, isotype rat IgG2b) (11). For detection of CCR2 in mouse lung histological sections, we used a goat polyclonal antibody cross-reacting with murine chemokine receptor CCR2 (C-20) purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The secondary antibody was an anti-goat alkaline-phosphatase-labeled F(ab)2 fragment purchased from Biotrend (Cologne, Germany). The VectorRed substrate kit was from Vector Laboratories (Burlingame, CA). All other biochemicals were from Merck (Darmstadt, Germany) and Sigma (Deisenhofen). Hybridoma-producing function-blocking anti-murine CD18 antibody (2E6, isotype IgG2a) has been described (16) and was obtained from American Type Culture Collection (Rockville, MD). Nonimmune hamster IgG was used as control. Rat anti-murine Gr-1 MAb (clone RB6-8C5, isotype rat IgG2b) was purchased from BD Biosciences (Heidelberg, Germany). The antibody is a complement-fixing isotype and widely used for depletion of circulating polymorphonuclear neutrophils (PMN) (15, 21).
Preparation of liposome-encapsulated dichloromethylene-diphosphonate. For liposomal encapsulation of clodronate, 8 mg of cholesterol were added to 86 mg of egg phosphatidylcholine, and the chloroform phase was evaporated under helium until a white film remained. Further removal of the chloroform phase was performed under low vacuum in a Speedvac Savant concentrator. The clodronate solution was made by dissolving 1.2 g of dichloromethylene diphosphonic acid in 5 ml of sterile phosphate-buffered saline (PBS). Five milliliters of the clodronate solution were added to the liposomes and mixed thoroughly. Empty liposomes were made by the addition of sterile PBS alone. This solution was sonicated and ultracentrifuged at 10,000 g for 1 h at 4°C. The liposomal pellets were then removed and resuspended in PBS, followed by ultracentrifugation at 10,000 g for 1 h at 4°C. Subsequently, liposomes were resuspended in 5 ml of sterile PBS, stored at 4°C, and used within 48 h. The final concentration of the liposomal clodronate suspension was 5 mg/ml.
Treatment of animals. A single dose of liposomal clodronate was administered to mice via intratracheal instillation (IT) routes. Control mice received empty (PBS containing) liposomes. Briefly, mice were anesthetized with tetrazoline hydrochloride and ketamine, and tracheas were exposed by surgical resection. IT of liposomal clodronate (100 µl) was performed under stereomicroscopic control (L10; Leica, Wetzlar, Germany) using a 29-gauge Abbocath, which was inserted into the trachea. After instillation, the neck wound was closed with sterile sutures. After 24-36 h of liposomal clodronate pretreatment of mice for efficient depletion of the rAM pool, mice were challenged with IT of rJE/MCP-1 (50 µg/mouse) in either the absence or presence of E. coli LPS (10 ng/mouse), using the same instillation technique as described for the liposomal clodronate, corresponding to most recent reports (12, 14).
Depletion of circulating neutrophils was achieved by intraperitoneal injections of carefully titrated Gr-1 MAb (~7.5 µg/mouse, diluted in 100 µl PBS/0.1% mouse serum) using aseptic conditions. Successful selective depletion of circulating neutrophils was determined by evaluation of differential cell counts of Pappenheim-stained blood smears and ranged between ~85 and 90%. For inhibition experiments using function-blocking anti-CD18 MAb, mice were anesthetized and received intravenous injections of 100 µg of MAb in a volume of 100 µl (PBS/0.1% mouse serum) via lateral tail veins 15 min before IT procedures, as recently outlined in detail (13, 14). Analysis of lung barrier function was performed by assessment of FITC-albumin leakage into the alveolar space, as described in detail (14). Briefly, mice received intravenous injections of FITC-labeled albumin (1.5 mg/mouse, Sigma) via lateral tail veins 1 h before death. Undiluted BAL fluid and serum samples (diluted 1:10 and 1:100 in PBS, pH 7.4) were measured with a spectrometer (FL 600; Bio-Tek, Winooski, VT; absorbance, 488 nm; emission, 520 ± 20 nm). The lung permeability index is defined as the ratio of fluorescence signals of undiluted BAL fluid samples to fluorescence signals of 1:100 diluted serum samples.Isolation of peripheral blood leukocytes and alveolar macrophages. Mice were killed with an overdose of isoflurane (Forene; Abbott, Wiesbaden, Germany). Isolation of peripheral blood leukocytes from EDTA-anticoagulated blood and BAL for isolation of rAM or alveolar-recruited neutrophils and monocytes from macrophage-depleted and JE/MCP-1 with or without LPS-challenged mice was performed as recently described in detail (12, 14). Quantitation of alveolar-recruited monocytes recovered from the lungs of macrophage-depleted, JE/MCP-1-challenged mice and alveolar-recruited neutrophils and monocytes recovered from the lungs of macrophage-depleted and JE/MCP-1 plus LPS-challenged mice was done on differential cell counts of Pappenheim-stained cytocentrifuge preparations, using overall morphological criteria, including differences in cell size and shape of nuclei and subsequent multiplication of those values with the respective absolute BAL cell counts.
Immunofluorescence analysis. Single-color immunofluorescence staining was used to analyze expression of the JE/MCP-1 receptor CCR2 and the monocyte/macrophage marker F4/80 (7) on the cell surface of rAM from untreated mice. Briefly, ~5 × 105 cells were preincubated with Fc-Block (10 µl; BD Biosciences) for blockade of FcIgG receptors in flexible microtiter plates (BD Biosciences) on ice. Negative controls were incubated with isotype-matched control IgG (PharMingen, Wiesbaden, Germany). Cells were incubated with either anti-CCR2 MAb (11) or anti-F4/80 MAb (Serotec) for 30 min on ice, washed three times in PBS (supplemented with 5% mouse serum/0.2% Na azide), followed by incubation with biotinylated F(ab')2 fragments for 30 min on ice. Subsequently, cells were washed three times, and phycoerythrine (PE)-conjugated streptavidin (BD Biosciences) was added to the wells for 15 min on ice in the dark.
Dual-color immunofluorescence staining was used to simultaneously analyze CCR2 expression on F4/80-positive peripheral blood monocytes. Briefly, blood leukocytes were incubated with anti-CCR2 MAb, washed, and incubated with secondary biotinylated F(ab')2 fragments for 30 min on ice, followed by incubation of cells with PE-conjugated streptavidin and FITC-conjugated anti-F4/80 MAb. After 15 min, cells were washed twice and subjected to flow cytometric analysis.Flow cytometry. All samples were analyzed on a FACStarPlus flow cytometer (BD Biosciences) equipped with an argon ion laser operating at an 488-nm excitation wavelength and a laser output of 200 mW. The optical system of the flow cytometer was adjusted daily, using standardized fluorescent Calibrite beads (BD Biosciences).
Single-color flow cytometry of CCR2 or F4/80 expression by rAM was done by gating on forward-scatter vs. side-scatter characteristics, followed by analysis of F4/80 or CCR2 cell surface expression in the fluorescence 2 channel (F488/575). Dual-color flow cytometry of CCR2 expression by F4/80-positive peripheral blood monocytes was done by gating F4/80-positive leukocytes on forward-scatter vs. F4/80 fluorescence 1 (FITC) characteristics, followed by analysis of CCR2 cell surface expression in the fluorescence 2 channel (F488/575).Histology and immunohistochemistry. Mice were killed with an overdose of isoflurane, and cryomicrotomy of mouse lungs (10 µm sections) was done as recently described (4). Briefly, after a short fixation, sections were preincubated to block nonspecific binding. Incubation with the anti-CCR2 antibody was performed overnight at 4°C. After incubation with the secondary alkaline phosphatase-conjugated antibody, the color reaction was developed with a VectorRed substrate kit. Levamisole (2.5 mM) was added to inhibit endogenous alkaline phosphatase activity. Counterstaining of the sections was performed with methyl green. Control staining was done by incubation with nonspecific serum instead of primary antibody.
Statistics. All data are given as means ± SE. Statistical significance between treatment groups was estimated by Mann-Whitney's U-test. Differences were assumed to be statistically significant when P values were < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of alveolar macrophage depletion on alveolar monocyte
recruitment elicited by intratracheal JE/MCP-1 instillation.
Both flow cytometric (Fig. 1) as well as
immunohistochemical analysis (Fig. 2)
clearly demonstrated CCR2 expression on alveolar macrophages, albeit at
lower levels than on peripheral blood monocytes, indicating the
potential of alveolar macrophages for specifically binding the CCR2
ligand JE/MCP-1. This agent is known to elicit alveolar monocyte
recruitment upon deposition within the alveolar space
(12). To determine whether alveolar macrophages are indeed involved in this JE/MCP-1-induced alveolar monocyte recruitment, we
specifically depleted alveolar macrophages by IT of liposomal clodronate. In mice pretreated for 24-36 h with liposomal
clodronate (100 µl), the pool of rAM was found to be depleted by
85 ± 5% compared with alveolar macrophage counts in BAL fluids
recovered from control mice receiving PBS-containing (empty) liposomes
(100 µl) (means ± SE; n = 7; P < 0.05 vs. control). Importantly, peak alveolar monocyte recruitment
rates at 48 h post-JE/MCP-1 challenge in mice either pretreated
with liposomal clodronate or receiving PBS-containing empty
liposomes were in the same order of magnitude (Fig.
3). Thus rAM depletion by pretreatment
with liposomal clodronate did not affect JE/MCP-1-driven alveolar
monocyte recruitment.
|
|
|
Effect of alveolar macrophage depletion on intra-alveolar cytokine
liberation and alveolar neutrophil and monocyte recruitment profiles in
response to intra-alveolar deposition of JE/MCP-1 plus E. coli LPS.
We next questioned whether selective depletion of alveolar
macrophages affects the liberation of proinflammatory cytokines TNF-
and MIP-2 and subsequent alveolar neutrophil and monocyte traffic after
combined JE/MCP-1/LPS challenge. IT of liposomal clodronate into the
lungs of mice per se did not provoke TNF-
or MIP-2 release, as shown
in Fig. 4 (values at 0 h).
Alveolar macrophage-depleted mice challenged intratracheally
with JE/MCP-1/LPS for various time periods had drastically reduced peak
TNF-
and MIP-2 BAL fluid levels, which was particularly evident
after 6 h, compared with nonmacrophage-depleted mice (Fig. 4).
Moreover, as shown in Fig. 5A,
macrophage-depleted mice challenged intratracheally with combined
JE/MCP-1/LPS demonstrated significantly reduced alveolar neutrophil
recruitment at 12-48 h posttreatment compared with
nonmacrophage-depleted mice undergoing combined JE/MCP-1/LPS challenge
(Fig. 5A, P < 0.05 vs. control). In
parallel, alveolar macrophage-depleted mice challenged intratracheally
with combined JE/MCP-1/LPS also demonstrated significantly attenuated
alveolar monocyte recruitment at 12-48 h posttreatment (Fig.
5B; P < 0.05 vs. control).
|
|
Effect of alveolar macrophage depletion on induction of lung
barrier dysfunction in JE/MCP-1/LPS-challenged mice.
To evaluate whether reduced BAL fluid levels of proinflammatory
cytokines such as TNF- and MIP-2, as well as heavily reduced alveolar leukocyte traffic achieved by macrophage depletion, also prevent a lung microvascular permeability increase from occurring in
mice challenged with combined JE/MCP-1/LPS (Fig.
6A), we assessed lung barrier
function in clodronate- and empty liposome-treated mice. Of note,
depletion of rAM by liposome-encapsulated clodronate per se did not
provoke induction of lung permeability compared with sham-treated
control mice (0-h time points in Fig. 6, A and B). Surprisingly, alveolar macrophage depletion before
inflammatory challenge did not prevent lung barrier dysfunction within
the 48-h observation period (Fig. 6B). Notably, however,
when mice were made neutropenic in parallel to depletion of alveolar
macrophages or received intravenous injections of function-blocking
anti-CD18 MAb before the challenge with JE/MCP-1 plus LPS, their lung
barrier function was nearly fully preserved (Fig. 6, C and
D), suggesting an alveolar macrophage-independent but CD18-
and PMN-mediated process promoting increased microvascular
permeability.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we evaluated the contribution of rAM
to pulmonary responses elicited by IT of JE/MCP-1 in either the absence
or presence of E. coli LPS. Although alveolar macrophages were found, by both flow cytometry and immunohistochemistry, to constitutively express the JE/MCP-1 receptor CCR2 and thus might represent a potential target for JE/MCP-1, liposomal
clodronate-mediated depletion of alveolar macrophages did not affect
solely JE/MCP-1-driven alveolar monocyte accumulation. In contrast,
depletion of alveolar macrophages strongly attenuated the
intra-alveolar release of TNF- and MIP-2 and also significantly
suppressed the alveolar neutrophil and monocyte recruitment profiles
elicited by combined JE/MCP-1/LPS challenge. However, this attenuated
inflammatory response after alveolar macrophage depletion did not
protect mice from an increase in lung vascular permeability, whereas
the latter was drastically reduced by either simultaneous depletion of
circulating neutrophils or systemic administration of anti-CD18 MAb
before combined JE/MCP-1/LPS challenge.
Alveolar macrophages as resident phagocytes located at the air-tissue interface of the lung are strategically positioned to respond to the local appearance of microbial or inflammatory agents, thus contributing to the initiation and modulation of lung host defense and inflammation. Interestingly, using a highly sensitive flow cytometric approach for detection of low-level chemokine receptor expression, we found that rAM collected from untreated mice express the major JE/MCP-1 receptor CCR2, albeit at lower levels than circulating monocytes collected from the same animals. Moreover, our immunohistochemical studies also identified rAM along with bronchial epithelial cells of large and small airways as CCR2-expressing cells within the alveolar compartment in situ. Thus we speculated that the constitutive expression of CCR2 by rAM might indicate direct involvement of these phagocytes in the JE/MCP-1-driven alveolar monocyte recruitment process. Alternatively, alveolar deposited or locally liberated low-molecular-weight chemokine JE/MCP-1 might directly pass the epi-/endothelial barrier to bind to and chemoattract circulating CCR2-expressing monocytes from the vascular into the alveolar compartment.
Evaluation of the role of CCR2-expressing alveolar macrophages in the pulmonary responses to JE/MCP-1 in either the absence or presence of E. coli LPS was accomplished by IT of a single dose of liposomal clodronate. This way, a depletion of the rAM pool of ~85% was achieved, corresponding well to previously published data (3, 8, 20, 23). However, this substantial rAM depletion did not affect the JE/MCP-1-driven alveolar monocyte accumulation, supporting the view that, despite their CCR2 expression, these phagocytes are not directly involved in the monocyte recruitment process in noninflamed lungs. This finding does not exclude the possibility that the constitutive CCR2 expression of the alveolar macrophages may well enable these cells to respond with directed migration to locally released JE/MCP-1, as may be secreted by alveolar epithelial cells and/or alveolar-recruited neutrophils during, e.g., pulmonary inflammation (18, 22), thus promoting the accumulation of rAM at the focus of alveolar inflammation.
Although depletion of alveolar macrophages did not affect alveolar
monocyte recruitment under noninflammatory conditions, depletion of the
resident phagocytes dramatically altered the overall pulmonary
inflammatory response developing on combined JE/MCP-1/LPS treatment of
mice (14). We observed strongly reduced BAL fluid TNF-
and MIP-2 levels in macrophage-depleted mice undergoing this challenge,
which suggests that the alveolar macrophages are centrally involved in
the cytokine release response elicited by alveolar LPS. Moreover,
depletion of alveolar macrophages also greatly attenuated the early
alveolar neutrophil accumulation reported recently to peak at ~12 h
after combined JE/MCP-1/LPS challenge (14). These data are
well in line with previous observations in models of LPS only-induced
lung inflammation, where depletion of alveolar macrophages was also
associated with strongly reduced lung neutrophil recruitment profiles
(1, 19). Thus alveolar macrophages apparently function to
promote rapid neutrophil influx into the alveolar compartment under
conditions of acute inflammation.
Interestingly, the alveolar macrophage-depleted mice also demonstrated
a significant reduction of the alveolar monocyte recruitment response
to JE/MCP-1-plus-E. coli LPS challenge. As discussed, this
contrasts to the finding that clodronate-mediated depletion of
macrophages did not affect the peak alveolar monocyte accumulation provoked by sole JE/MCP-1 instillation, mimicking noninflammatory conditions. Thus it appears that rAM largely contribute to the overall
inflammatory response to LPS that underlies the monocyte recruitment-enhancing function of the resident phagocytes under these
conditions. The rAM-dependent pathways orchestrating the local cytokine
response and the strong promotion of early neutrophil and delayed
monocyte influx in response to alveolar JE/MCP-1/LPS deposition do,
however, still need to be defined in detail. In this context, several
previous observations are of interest. Previous studies in vitro showed
that alveolar macrophage-derived secretory products like TNF- can
activate alveolar epithelial cells to release chemokines such as
interleukin-8 and MCP-1, which may then promote both alveolar
neutrophil and monocyte recruitment (18). Recent studies
from our laboratory showed that stimulation of primary isolates of
human alveolar epithelial cells with TNF-
provoked a directed apical
MCP-1 secretion together with a directed migratory response of
monocytes across the alveolar epithelial layer (17). These
data support the concept that rAM-dependent alveolar cytokine release
may employ local epithelial cells to promote directed alveolar
leukocyte traffic under conditions of inflammation. This may hold true
for both neutrophil and monocyte recruitment being considered as
independent events. However, interdependencies between these two types
of alveolar leukocyte flux may also be relevant, and two observations
may support this assertion. First, alveolar-recruited and -activated
neutrophils are well known to be capable of releasing MCP-1 within the
alveolar air space (22). Second, transient neutropenia, as
well as selective inhibition of neutrophil chemotaxis in BALB/c mice,
was most recently shown to drastically attenuate the alveolar monocyte
influx in response to JE/MCP-1 plus E. coli LPS, implying a
potential role of corecruited neutrophils in the alveolar monocyte
traffic (U. Maus, K. v. Grote, W. A. Kuziel, M. Mack, E. J. Miller, J. Cihak, M. Stangassinger, R. Maus, D. Schlöndorff, W. Seeger, and J. Lohmeyer; unpublished observations).
An unexpected finding of the present study was the observation that,
despite heavily reduced alveolar cytokine release, as well as strongly
suppressed leukocyte recruitment patterns, rAM-depleted mice still
exhibited a marked loss of lung barrier function at 6 and 12 h
post-JE/MCP-1/LPS challenge. Importantly, alveolar macrophage-depleted
control mice did not exhibit such an increase in lung permeability,
thus excluding a direct effect of liposomal clodronate on lung barrier
integrity. However, when macrophage-depleted mice were additionally
made neutropenic before intrabronchial instillation of combined
JE/MCP-1/LPS, lung barrier failure was largely prevented, which was
also true when mice were systemically treated with function-blocking
anti-CD18 MAb, which were most recently shown to be relevant for the
alveolar recruitment of both neutrophils and monocytes
(13). These data clearly demonstrate that 1)
induction of lung barrier failure is not necessarily dependent on rAM;
2) a 2-integrin-dependent step is obviously
required for the loss of barrier integrity, probably involving
leukocyte-endothelial interactions. This finding corresponds well to
recently published observations from Gao and coworkers
(5), who demonstrated that pulmonary microvascular
permeability induced by E. coli in mice was solely the
result of neutrophil engagement of CD18 integrins; and 3)
most probably the circulating neutrophil is a major culprit in
provoking increased lung vascular permeability in response to alveolar
JE/MCP-1/LPS challenge, even when its migratory response to the
alveolar compartment is largely suppressed due to rAM depletion. In
this context, it is important to note that Gautum et al.
(6) have most recently proposed neutrophil-derived
heparin-binding protein (CAP37, azurocidin) as a missing link in
neutrophil-evoked alteration of vascular permeability, which notably
exerts its effects on endothelial cell permeability on engagement of
neutrophil
2-integrins. Certainly, whether this mode of
action is also operative in the described model of acute lung
inflammation still deserves further investigation.
The IT route used here as an experimental approach to deliver clodronate into the lungs of mice yielded an ~85% depletion of alveolar macrophages and was less effective than the inhalative route reported earlier in other studies with depletion rates amounting to 95% (8, 9). Although we cannot fully exclude the possibility that residual alveolar macrophages still might have been participating in the inflammatory response to combined JE/MCP-1 plus LPS, the lack of any attenuation of lung barrier dysfunction after depletion of ~85% alveolar macrophages argues strongly against a major contribution of residual alveolar macrophages to the lung barrier failure observed in the present study.
In conclusion, rAM are not involved in JE/MCP-1-driven alveolar monocyte recruitment in noninflamed lungs. They are, however, centrally enrolled in the alveolar cytokine response and the enhanced early neutrophil and delayed monocyte traffic into the alveolar compartment occurring under inflammatory conditions due to alveolar JE/MCP-1/LPS deposition. Interestingly, the loss of lung barrier function observed under these conditions is rAM independent but involves circulating neutrophils in a CD18-dependent fashion.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: U. A. Maus, Dept. of Internal Medicine, Div. of Pneumology, Justus-Liebig-Univ., Klinikstr. 36, Giessen 35392, Germany (E-mail: Ulrich.A.Maus{at}med.uni-giessen.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. Section 1734 solely to indicate this fact.
First published January 18, 2002;10.1152/ajplung.00453.2001
Received 26 November 2001; accepted in final form 10 January 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Berg, T,
Lee ST,
Thepen T,
Lee CY,
and
Tsan MF.
Depletion of alveolar macrophages by liposome-encapsulated dichlormethylene diphosphonate.
J Appl Physiol
74:
2812-2819,
1993[Abstract].
2.
Broug-Holub, E,
Toews GB,
van Iwaarden JF,
Strieter RM,
Kunkel SL,
Paine R, III,
and
Standiford TJ.
Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival.
Infect Immun
65:
1139-1146,
1997[Abstract].
3.
Cheung, D,
Halsey K,
and
Speert DP.
Role of pulmonary alveolar macrophages in defense of the lung against Pseudomonas aeruginosa.
Infect Immun
68:
4585-4592,
2000
4.
Ermert, L,
Ermert M,
Duncker H-R,
Grimminger F,
and
Seeger W.
In situ localization and regulation of thromboxane A2 synthase in normal and LPS-primed lungs.
Am J Physiol Lung Cell Mol Physiol
278:
L744-L753,
2000
5.
Gao, XP,
Xu N,
Sekosan M,
Mehta D,
Ma SY,
Rahman A,
and
Malik AB.
Differential role of CD18 integrins in mediating lung neutrophil sequestration and increased microvascular permeability induced by Escherichia coli in mice.
J Immunol
167:
2895-2901,
2001
6.
Gautum, N,
Olofsson AM,
Herwald H,
Iversen LF,
Lundgren-Åkerlund E,
Hedqvist P,
Arfors KE,
Flodgaard H,
and
Lindbom L.
Heparin-binding protein (HBP/CAP37): a missing link in neutrophil-evoked alteration of vascular permeability.
Nat Med
7:
1123-1127,
2001[ISI][Medline].
7.
Gordon, S,
Lawson L,
Rabinowitz S,
Crocker PR,
Morris L,
and
Perry VH.
Antigen markers of macrophage differentiation in murine tissues.
Curr Top Microbiol Immunol
181:
1-37,
1992[Medline].
8.
Hashimoto, S,
Pittet JF,
Hong K,
Folkesson H,
Bagby G,
Kobzik L,
Frevert C,
Watanabe K,
Tsurufuji S,
and
Wiener-Kronish J.
Depletion of alveolar macrophages decreases neutrophil chemotaxis to Pseudomonas airspace infections.
Am J Physiol Lung Cell Mol Physiol
270:
L819-L928,
1996
9.
Kooguchi, K,
Hashimoto S,
Kobayashi A,
Kitamura Y,
Kudoh I,
Wiener-Kronisch J,
and
Sawa T.
Role of alveolar macrophages in initiation and regulation of inflammation in Pseudomonas aeruginosa pneumonia.
Infect Immun
66:
3164-3169,
1998
10.
Leemans, JC,
Juffermans NP,
Florquin S,
van Roijen N,
Vervoordeldonk MJ,
Verbon A,
van Deventer SJH,
and
van der Poll TJ.
Depletion of alveolar macrophages exerts protective effects in pulmonary tuberculosis in mice.
J Immunol
166:
4604-4611,
2001
11.
Mack, M,
Cihak J,
Simonis C,
Luckow B,
Proudfoot AEI,
Plachý J,
Brühl H,
Frink M,
Anders HJ,
Vielhauer V,
Pfirstinger J,
Stangassinger M,
and
Schlöndorff D.
Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice.
J Immunol
166:
4697-4704,
2001
12.
Maus, U,
Herold S,
Muth H,
Maus R,
Ermert L,
Ermert M,
Weissmann N,
Rosseau S,
Seeger W,
Grimminger F,
and
Lohmeyer J.
Monocytes recruited into the alveolar air space of mice show a monocytic phenotype but upregulate CD14.
Am J Physiol Lung Cell Mol Physiol
280:
L58-L68,
2001
13.
Maus, U,
Huwe J,
Ermert L,
Ermert M,
Seeger W,
and
Lohmeyer J.
Molecular pathways of monocyte emigration into the alveolar air space of intact mice.
Am J Respir Crit Care Med
165:
95-100,
2002
14.
Maus, U,
Huwe J,
Maus R,
Seeger W,
and
Lohmeyer J.
Alveolar JE/MCP-1 and endotoxin synergize to provoke lung cytokine upregulation, sequential neutrophil and monocyte influx and vascular leakage in mice.
Am J Respir Crit Care Med
164:
406-411,
2001
15.
Mehrad, B,
Moore TA,
and
Standiford TJ.
Macrophage inflammatory protein-1 is a critical mediator of host defense against invasive pulmonary aspergillosis in neutropenic hosts.
J Immunol
165:
962-968,
2000
16.
Metlay, JP,
Witmer-Pack MD,
Agger R,
Crowley MT,
Lawless D,
and
Steinman RM.
The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies.
J Exp Med
171:
1753-1771,
1990[Abstract].
17.
Rosseau, S,
Selhorst J,
Wiechmann K,
Leissner K,
Maus U,
Mayer K,
Grimminger F,
Seeger W,
and
Lohmeyer J.
Monocyte migration through the alveolar epithelial barrier: adhesion molecule mechanisms and impact of chemokines.
J Immunol
164:
427-435,
2000
17a.
Rosseau, S,
Hammerl P,
Maus U,
Walmrath HD,
Schütte H,
Grimminger F,
Seeger W,
and
Lohmeyer J.
Phenotypic characterization of alveolar monocyte recruitment in acute respiratory distress syndrome.
Am J Physiol Lung Cell Mol Physiol
279:
L25-L35,
2000
18.
Standiford, TJ,
Kunkel SL,
Phan SJ,
Rollins BJ,
and
Strieter RM.
Alveolar macrophage-derived cytokines induce monocyte chemoattractant protein-1 expression from human alveolar type II-like epithelial cells.
J Biol Chem
266:
9912-9917,
1991
19.
Tang, G,
White JE,
Lumb PD,
Lawrence DA,
and
Tsan MF.
Role of endogenous cytokines in endotoxin- and interleukin-1-induced pulmonary inflammatory response and oxygen tolerance.
Am J Respir Cell Mol Biol
12:
339-344,
1995[Abstract].
20.
Thepen, T,
van Rooijen N,
and
Kraal G.
Alveolar macrophage elimination in vivo is associated with an increase in pulmonary immune response in mice.
J Exp Med
170:
499-509,
1989[Abstract].
21.
Vassiloyanakopoulos, AP,
Okamoto S,
and
Fierer J.
The crucial role of polymorphonuclear leukocytes in resistance to Salmonella dublin infections in genetically susceptible and resistant mice.
Proc Natl Acad Sci USA
95:
7676-7681,
1998
22.
Yamamoto, T,
Kajikawa O,
Martin TR,
Sharar SR,
Harlan JM,
and
Winn RK.
The role of leukocyte emigration and IL-8 on the development of lipopolysaccharide-induced lung injury in rabbits.
J Immunol
161:
5704-5711,
1998
23.
Zhang-Hoover, J,
Sutton A,
van Rooijen N,
and
Stein-Streilein J.
A critical role for alveolar macrophages in elicitation of pulmonary immune fibrosis.
Immunology
101:
501-511,
2000[ISI][Medline].