The evaluation of monocytes recruited into the alveolar space under both physiological and inflammatory conditions is hampered by difficulties in discriminating these cells from resident alveolar macrophages (rAMs). Using the intravenous injected fluorescent dye PKH26, which accumulated in rAMs without labeling blood leukocytes, we developed a technique that permits the identification, isolation, and functional analysis of monocytes recruited into lung alveoli of mice. Alveolar deposition of murine JE, the homologue of human monocyte chemoattractant protein (MCP)-1 (JE/MCP-1), in mice provoked an alveolar influx of monocytes that were recovered by bronchoalveolar lavage and separated from PKH26-stained rAMs by flow cytometry. Alveolar recruited monocytes showed a blood monocytic phenotype as assessed by cell surface expression of F4/80, CD11a, CD11b, CD18, CD49d, and CD62L. In contrast, CD14 was markedly upregulated on alveolar recruited monocytes together with increased tumor necrosis factor-α message, discriminating this monocyte population from peripheral blood monocytes and rAMs. Thus monocytes recruited into the alveolar air space of mice in response to JE/MCP-1 keep phenotypic features of blood monocytes but upregulate CD14 and are “primed” for enhanced responsiveness to endotoxin with increased cytokine expression.
- monocyte chemoattractant protein-1
- alveolar macrophage
- fluorescent dye
- flow cytometry
in the past few years, it has been well documented that alveolar deposition of endotoxin [lipopolysaccharide (LPS) of gram-negative bacteria] provokes a significant influx of monocytes into the interstitium and the alveolar compartment of rodents along with the well-known recruitment of neutrophils (20, 32). Moreover, transgenic mouse models have confirmed a causal relationship between the increased expression of the monocyte chemotactic factor monocyte chemoattractant protein-1 (MCP-1) and the accumulation of monocytes and lymphocytes at various extravascular sites (10, 12, 15, 21). Overexpression of transgenic human MCP-1 by type II alveolar epithelial cells caused a substantial accumulation of monocytes within the bronchoalveolar space of mice (15), demonstrating that chemokines such as MCP-1 alone, in the absence of further stimuli like endotoxin, are sufficient to elicit a monocytic influx into the alveolar air spaces. Given their capacity to elaborate inflammatory cytokines, reactive oxygen species, or proteolytic enzymes (29,33), monocyte accumulation has been implicated in several inflammatory diseases in a variety of organ systems (1, 4, 14,18, 30).
Notwithstanding this progress, the pathophysiological role of monocyte accumulation in the lung in acute and chronic pulmonary inflammation is currently largely unknown, although these cells are accessible by bronchoalveolar lavage (BAL). This fact is mainly attributable to the difficulties in discriminating “freshly” recruited monocytes from resident alveolar macrophages (rAMs), by far the predominant alveolar leukocyte population under baseline conditions, from comigrating polymorphonuclear neutrophils, and, possibly, from lymphocytes. In the present study, we developed a novel fluorescence-activated cell sorting (FACS)-based technique that allows the clear discrimination, isolation, and characterization of monocytes recruited into the bronchoalveolar space. Employing this technique, we investigated the monocyte influx into the alveolar compartment of mice in response to regional MCP-1 deposition. In essence, freshly recruited monocytes were found to retain several phenotypic markers of peripheral blood (PB) monocytes but displayed a strong upregulation of CD14 along with an increased tumor necrosis factor (TNF)-α message and a markedly enhanced readiness to liberate this cytokine in response to endotoxin challenge. Such “priming” during the recruitment process, discriminating these cells from both the PB monocytes and the rAMs, may be relevant for pulmonary host defense mechanisms and inflammatory events under conditions of alveolar microbial challenge.
MATERIALS AND METHODS
BALB/c female mice weighing 18–21 g (age 10–12 wk) were purchased from Charles River (Sulzfeld, Germany) and used in all experiments.
The red fluorescent dye PKH26-PCL and diluent B solution were purchased from Zynaxis (Malvern, CA; distributed by Sigma, Deisenhofen, Germany). Murine JE, the homologue to the human MCP-1 gene product (JE/MCP-1) (30, 31), was purchased as a recombinant protein preparation (rJE/MCP-1) from R&D Systems (Wiesbaden, Germany) as were murine macrophage inflammatory protein (MIP)-1α and regulated on activation normal T cell expressed and secreted (RANTES), which were used in selected experiments instead of rJE/MCP-1. The rJE/MCP-1 as well as the fluorescent dye PKH26 and the diluent B solution were endotoxin free as analyzed by amebocyte lysate assay (detection limit <10 pg/ml; COATEST, Chromogenix, Mölndal, Sweden).
Rat anti-mouse antibodies (Abs) specific for F4/80, CD18, CD45, and CD49d (very late activating antigen-4) and fluorescein isothiocyanate (FITC)-conjugated goat anti-rat F(ab′)2 Abs were obtained from Serotec (Munich, Germany). Antibodies specific for murine CD4, CD11a (lymphocyte function-associated antigen-1), CD11b (Mac-1), CD14, and CD62L (L-selectin) as well as FcBlock (a mixture of CD16/ CD32) were from PharMingen (Hamburg, Germany). The secondary goat anti-rat F(ab′)2 alkaline phosphatase-conjugated Ab used for immunocytochemistry was obtained from Biotrend (Cologne, Germany). The Vector red substrate kit was purchased from Vector Laboratories (Burlingame, CA).
Treatment of Animals
For in vivo fluorescent labeling of rAMs, BALB/c mice were anesthetized with xylazine hydrochloride (2.5 mg/kg im; Rompun, Bayer, Germany) and ketamine hydrochloride (50 mg/kg im; Ketavet, Pharmacia & Upjohn). The highly aliphatic fluorescent dye PKH26-PCL (1 mM stock solution in ethanol) was diluted under sterile conditions with diluent B to the given in vivo concentrations by calculating a dilution factor of 20 and was then slowly injected intravenously in a total volume of 100 μl into mice via the tail vein with a 29-gauge sterile cannula.
For in vitro fluorescent labeling of PB leukocytes, blood was collected from donor mice into EDTA-containing tubes as described inCollection of blood samples and BAL and subjected to lysis of the red blood cells followed by two washing steps in RPMI 1640 medium. PB leukocytes were resuspended in 1 ml of diluent B solution, and red fluorescent staining was performed by adding 1 ml of a 20 μM PKH26 solution, yielding a 10 μM final concentration of the dye. Fluorescent staining was allowed to proceed for 5 min at room temperature and was then terminated by the addition of 2 ml of mouse serum. PB leukocytes were washed two times in RPMI 1640 medium supplemented with 20% mouse serum and filtered through sterile 70- and 20-μm nylon meshes. Successful red fluorescent staining of PB leukocyte populations was verified by FACS analysis of sample aliquots. PKH26-labeled leukocytes (∼4 × 107 /mouse) were injected into recipient mice via the tail vein in a total volume of 200 μl.
Intratracheal instillation of murine rJE/MCP-1.
Twenty-four hours after the intravenous injection of PKH26, BALB/c mice were anesthetized as described in PKH26 labeling, and the neck fur above the trachea was shaved followed by disinfection of the skin. A small incision was made, and the underlying connective tissue was bluntly dissected to expose the trachea. A 26-gauge Abbocath (Abbott, Wiesbaden, Germany) was inserted into the trachea, and murine rJE/MCP-1 [50 μg/80 μl PBS containing 0.1% human serum albumin (HSA)] was slowly instilled into the lungs. Subsequently, the catheter was removed, and the skin was sutured. Sham-operated control mice received 80 μl of PBS-0.1% HSA. In a series of experiments, mice received an intratracheal instillation of murine recombinant MIP-1α or murine recombinant RANTES (50 μg/mouse; R&D Systems) instead of rJE/MCP-1. In selected experiments, recipient mice received an intravenous injection of PKH26-prelabeled PB leukocytes from donor mice followed by a 15-min delayed intratracheal instillation of rJE/MCP-1. Mice were allowed to recover from anesthesia and were then returned to their cages, with free access to food and water.
Collection of blood samples and BAL.
Blood samples and BAL fluid were collected 48 h after rJE/MCP-1 administration except for those experiments in which alveolar monocytes were analyzed 72 and 96 h after rJE/MCP-1 instillation. The mice received an overdose of ether, and the abdominal cavity was rapidly opened to expose the vena cava. Blood was drawn into a 23-gauge cannula connected to a 1-ml insulin syringe that was filled with 200 μl of NaCl-EDTA as an anticoagulant. The lysis of red blood cells was performed in a total volume of 10 ml of an ammonium chloride solution (pH 7.2; Merck, Darmstadt, Germany) for 5 min at room temperature. After a wash at 1,400 rpm for 9 min at 4°C, the cell pellet was resuspended in 0.5 ml of PBS containing 10% FCS and immediately placed on ice.
For BAL, the trachea was exposed, and a small incision was made to insert a shortened 21-gauge cannula that was firmly fixed and then connected to a 1-ml insulin syringe filled with 300 μl of PBS-5 mM EDTA (pH 7.2). BAL was performed with 300-μl aliquots until an initial BAL volume of 1.5 ml was recovered. The cells were separated by centrifugation (300 g for 9 min), and the supernatant was used for quantification of TNF-α protein (by ELISA). Subsequently, the BAL was completed with 500-μl aliquots until an additional BAL volume of 4.5 ml was recovered. After the additional BAL volume was spun, the cells were pooled and counted with a hemacytometer.
Flow Cytometry and Cell Sorting
A FACStarPLUS flow cytometer equipped with a large nozzle sort head assembly was used throughout the study. The sorting conditions used to purify monocytes/macrophages have previously been shown to not affect cellular functions (22).
The antigen profile of PB monocytes was analyzed by dual-color flow cytometry. Samples of PB leukocytes were incubated with a FITC-conjugated F4/80 rat anti-mouse Ab specific for monocytic cells (11, 23) and phycoerythrin-conjugated rat anti-mouse Abs specific for CD11a, CD11b, CD14, CD18, CD49d, and CD62L. Incubation was performed for 30 min on ice followed by two washing steps. Flow cytometry of PB monocytes was performed by gating F4/80-positive cells according to their forward scatter (FSC) versus fluorescence 1 (FL1; F488/535) characteristics followed by analysis of phycoerythrin-labeled cells in the fluorescence 2 (FL2; F488/575) channel.
Alveolar recruited monocytes and rAMs were analyzed by single-color flow cytometry. After BAL, the cells were immediately placed on ice and after centrifugation at 1,400 rpm at 4°C for 9 min, were resuspended in PBS containing 10% FCS and 0.02% sodium azide. The cells were incubated with 10 μl of FcBlock for 5 min followed by a 30-min incubation with purified rat anti-mouse Abs specific for F4/80, CD11a, CD11b, CD18, CD49d, and CD62L. Staining of cells with the Ab specific for CD14 was performed with purified mouse IgG at a 20-fold excess to inhibit nonspecific reactivity as recommended by the manufacturer. After being washed, the cells were incubated with a FITC-conjugated goat anti-rat Ab for 30 min on ice. After two more washing steps, the cells were immediately analyzed on a FACStarPLUS flow cytometer by gating alveolar recruited monocytes and rAMs according to their different FSC versus FL2 characteristics followed by analysis of FITC-labeled cells in the FL1 channel. The identification of alveolar recruited monocytes and their discrimination from rAMs were based on three criteria. In contrast to rAMs, alveolar recruited monocytes exhibited low red fluorescence characteristics but were specifically stained with the monocyte/macrophage antibody F4/80 (11,23). In addition, Pappenheim-stained cytospin preparations of flow-sorted and thus highly purified alveolar recruited cells demonstrated a monocytic morphology of this population (data not shown).
For cell sorting of PB monocytes, alveolar recruited monocytes, and rAMs, individual sort windows were set according to their different FSC versus FL1 characteristics (PB monocytes) and according to their different fluorescence emission characteristics at 535 ± 30 (FL1) and 575 ± 26 nm (FL2; alveolar recruited monocytes and rAMs). After cell sorting, the purity of the cell preparations was analyzed by1) postsort analysis of sorted cells and 2) differential cell counts of Pappenheim-stained sorted cells. The cell purity of sorted monocytes and alveolar macrophages was always >96%, with cell viabilities of >94% as analyzed by propidium iodide staining followed by FACS analysis (22).
Histology and Immunohistochemistry
Mice were killed with a lethal dose of ether, the chest was rapidly opened, and the thoracic organs were carefully removed. Lungs were fixed by instilling ice-cold PBS-buffered paraformaldehyde solution (2.5%, pH 7.2) through the trachea at a constant pressure of 20 cmH2O. Fixation was allowed to proceed for 2 h at 8°C; subsequently, tissue samples were paraffin embedded. Additional lungs were embedded in TissueTek and snap-frozen in liquid nitrogen for cryomicrotomy. Sections of 10 μm from all lungs were stained with hematoxylin and eosin and evaluated for evidence of tissue damage and cellular infiltrates.
Lung cryosections were stained with anti-CD45 antibodies with alkaline phosphatase-based immunohistochemistry. Briefly, the sections were fixed for 5 min with 3% paraformaldehyde solution, washed, and preincubated in PBS containing 5% goat serum, 1% BSA, and 0.05% Tween 20 to block nonspecific binding. Overnight incubation with a 1:50 diluted anti-mouse CD45 monoclonal Ab (Serotec) was carried out at 4°C. Incubation with the secondary alkaline phosphatase Ab diluted 1:400 was performed overnight at 4°C. The sections were developed with a Vector red substrate kit for 60 min. Levamisol (2.5 mM) was added to inhibit endogenous alkaline phosphatase activity. Counterstaining of the sections was performed with methyl green. Control staining was performed by omission of the primary antibody and substitution with nonspecific serum at the same dilution. Microscopy was performed with a Leitz Orthoplan bright-field microscope at a ×160 magnification (Leica, Wetzlar, Germany). Positively stained cells were counted in 10 randomly selected microscopic fields, corresponding to a total area of ∼9.5 mm2. Cellular infiltrates were evaluated in four different localizations: within the peribronchial or perivascular tissue, in the alveolar spaces, or within the alveolar septum. (6, 7).
Isolation of Total Cellular RNA and Real-Time RT-PCR
Total cellular RNA was isolated, and reverse transcription was carried out as described recently in detail (22). Quantitation of murine TNF-α mRNA transcripts was performed with real-time RT-PCR as previously published by our group (9). Briefly, a Perkin-Elmer ABI prism 7700 sequence detection system (Perkin-Elmer, Weiterstadt, Germany) was used. The TaqMan PCR reagent kit (Perkin-Elmer) was employed following the recommendations of the manufacturer. PCRs were carefully optimized for primer concentration (ranging between 200 and 900 nM), Mg2+ concentration (5.5–6 mM), and TaqMan probe (200 nM). Both the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase and TNF-α PCR amplifications were performed at 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Among all runs, the calculated efficiencies varied from 0.8 to 0.9. Sequences of intron-spanning primers and TaqMan probes used for real-time RT-PCR are described elsewhere (26). We analyzed TNF-α gene expression in flow-sorted PB monocytes, alveolar monocytes, and rAMs of PKH26 plus MCP-1-treated mice as well as in sorted AMs of PKH26-treated or sham-operated control mice receiving vehicle alone. Sorted rAMs collected from mice receiving an intratracheal instillation of 20 μg of LPS served as positive controls.
Cell Culture Experiments
To analyze whether CD14-positive alveolar monocytes were more susceptible to LPS challenge than CD14-negative PB monocytes of PKH26 plus MCP-1-treated mice, we compared their LPS-inducible TNF-α release. F4/80-positive blood monocytes and alveolar monocytes collected from the same animals were flow sorted to high purity and cultured in 24-well plates (Costar, Bodenheim, Germany) in RPMI 1640 medium-10% FCS at a density of 2 × 104 cells/ml. The cells were stimulated with 1 ng/ml of Salmonella abortus equi endotoxin for 4 h. Cell-free culture supernatants were collected and stored at −86°C until ELISA analysis.
The quantitation of murine TNF-α protein from culture supernatants of unstimulated or LPS-stimulated blood monocytes and alveolar monocytes and rJE/MCP-1 levels of BAL fluids from PKH26 plus MCP-1-treated mice was performed with commercially available ELISA kits following the instructions of the manufacturer (R&D Systems).
The data are means ± SD from at least four independent experiments. Significance between treatment groups was estimated by Mann-Whitney U-test. Differences were assumed to be significant when P values were <0.05.
Dose- and Time-Dependent Accumulation of PKH26 in rAMs
rAMs from sham-operated mice receiving PBS-HSA vehicle alone exhibited low red autofluorescence intensity measured at 575 nm (FL2; Fig. 1 A). After PKH26 was intravenously injected, the lipophilic red fluorescent dye dose and time dependently accumulated in rAMs, thereby consistently increasing their red fluorescence intensity. Highest values were observed at concentrations of 15 μM (calculated for the intravascular space; Fig.1, D–F) at 24–72 h (Fig. 1,G–L). The observed increase in FL2 emission by rAMs was correlated with the intracellular uptake of the dye as analyzed by fluorescence microscopy (data not shown). Interestingly, intravenous injection of PKH26 labeled rAMs without staining PB monocytes (see Fig. 5, left).
Recruitment of Monocytes Into the Bronchoalveolar Space and Their Discrimination From rAMs
rAMs collected from untreated mice exhibited low red fluorescence but increased green autofluorescence (FL1) characteristics as shown in Fig. 2 A. The effect of an intravenous injection of PKH26 (15 μM) on FL2 emission characteristics of rAMs was used to discriminate rAMs and recruited alveolar cells because PKH26 strongly accumulated in rAMs, leading to heavily increased red FL2 emission characteristics (Fig.2 B). Forty-eight hours after the intratracheal instillation of rJE/MCP-1, a strong accumulation of monocytes and, to a much lesser extent, of CD4-positive lymphocytes (FACS analysis not shown in detail) was observed within the lungs of mice together with significantly increased total BAL fluid cell numbers (Fig.2 C, Table 1). Importantly, newly recruited monocytes and rAMs could easily be discriminated by their strongly different red FL2 emission characteristics (Fig.2 C). Incubation of BAL fluid cells recovered from PKH26 plus MCP-1-treated mice with the monocyte/macrophage-specific monoclonal Ab F4/80 specifically stained newly recruited alveolar monocytes and rAMs but not lymphocytes (Fig. 2 D).
To demonstrate that alveolar accumulating monocytes were recruited from the intravascular compartment, PB leukocytes were isolated from donor mice, stained with PKH26 in vitro, and intravenously injected into recipient mice. Mice that received intravenous PKH26-positive PB leukocytes without intratracheal instillation of rJE/MCP-1 or intratracheal instillation of rJE/MCP-1 without an intravenous injection of PKH26-positive PB leukocytes exhibited basal FL2 emission characteristics of BAL fluid-derived cells (Fig.2 E). In contrast, recipient mice that received intravenous PKH26-positive PB leukocytes simultaneously with intratracheal instillation of rJE/MCP-1 showed a significant increase in BAL fluid-derived leukocytes with high FL2 emission characteristics, thus demonstrating the recruitment of PKH26-prelabeled leukocytes from the intravascular compartment (Fig. 2 E).
We also compared the monocyte-recruiting capacities of rJE/MCP-1 with other leukocyte-recruiting chemoattractants such as MIP-1α and RANTES (Fig. 2 F). On intratracheal instillation of 50 μg of each of these chemoattractants into the lungs of mice, distinct BAL fluid leukocyte profiles related to the respective chemokine used were observed. In contrast to rJE/MCP-1, intratracheal instillation of MIP-1α provoked predominantly neutrophil recruitment into the alveolar compartment, with alveolar recruited monocytes accounting for only ∼6%. The intratracheal instillation of RANTES into the lungs of mice primarily induced the recruitment of lymphocytes but not of monocytes (∼2%) into the alveolar compartment. These data clearly demonstrate a major potential of rJE/MCP-1 in the recruitment of PB monocytes to the alveolar air space under in vivo conditions.
Lung Histology of Mice Receiving PKH26 or PKH26 Plus rJE/MCP-1
The histological examination of paraffin-embedded lung sections of mice receiving intravenous PKH26 (Fig.3 B) revealed a cellular architecture comparable to that of control mice receiving vehicle alone (Fig. 3 A). Lung sections of mice receiving both intravenous PKH26 and intratracheal rJE/MCP-1 or PKH26 alone were evaluated for the distribution of recruited leukocytes within the peribronchial or perivascular tissue, in the alveolar spaces, or within the alveolar septum. Interestingly, in PKH26 plus MCP-1-treated mice, the highest leukocyte numbers were located in the alveolar spaces (P < 0.01; Fig.4 B), with significantly decreased leukocyte numbers within the alveolar septum (P < 0.05; Fig. 4 D), compared with mice receiving intravenous PKH26 alone. In contrast, leukocyte numbers in the peribronchial (Fig. 4 A) and perivascular (Fig.4 C) spaces did not differ between mice receiving PKH26 plus MCP-1 and mice receiving PKH26 alone. These data demonstrate that intratracheal instillation of rJE/MCP-1 primarily elicits a monocytic recruitment to the alveolar space.
Immunophenotypic Profile of PB Monocytes, Alveolar Recruited Monocytes, and rAMs From Mice Receiving PKH26 Plus MCP-1
We analyzed the expression of selected surface molecules such as F4/80, CD11a, CD11b, CD14, CD18, CD49d, and CD62L, some of which are known to be functionally relevant for the monocyte transmigration process into the bronchoalveolar space (17, 20). Figure5 shows that PB monocytes as well as alveolar recruited monocytes and rAMs were stained with the monocyte/macrophage-specific monoclonal Ab F4/80. Likewise, CD11a and CD18 were expressed on all three cell populations, whereas CD11b, CD49d, and CD62L were detectable on both PB monocytes and alveolar monocytes but not on rAMs (Fig. 5). Interestingly, a strong CD14 expression was consistently found on newly recruited alveolar monocytes (n = 15 experiments), whereas PB monocytes and rAMs were not stained by the anti-CD14 Ab. The expression of the antigen markers CD11b, CD14, CD49d, and CD62L detected on alveolar monocytes but not on rAMs 48 h after intratracheal instillation of rJE/MCP-1 was nearly unchanged 72 and 96 h after rJE/MCP-1 administration (data not shown).
Proinflammatory Cytokine Expression by Flow-Sorted Alveolar Monocytes and rAMs From Mice Receiving PKH26 Plus MCP-1
The increased CD14 expression on alveolar recruited monocytes raised the question of whether these cells also expressed further activation-associated immediate-early gene products such as TNF-α in vivo. Therefore, PB monocytes, alveolar monocytes, and rAMs recovered from the same PKH26 plus MCP-1-treated mice were sorted to high purity (shown for rAMs and alveolar monocytes in Fig.6) and together with rAMs of PKH26-treated or sham-operated mice were subjected to real-time RT-PCR quantitation of TNF-α gene expression (Fig.7). Interestingly, alveolar recruited monocytes expressed slightly but significantly increased TNF-α mRNA levels compared with those in PB monocytes and rAMs of the same animal, suggesting that CD14 expression on alveolar monocytes of PKH26/MCP-1-treated mice is associated with slightly elevated TNF-α expression in vivo.
To evaluate the functional relevance of increased CD14 expression, flow-sorted CD14-positive alveolar monocytes and PB monocytes (consistently CD14 negative) were challenged with LPS in vitro, and TNF-α release was analyzed (Fig. 8). Importantly, alveolar monocytes showed an approximately fourfold increased TNF-α secretory response compared with PB monocytes, suggesting that monocytes recruited into the bronchoalveolar space in response to rJE/MCP-1 are rendered more susceptible to LPS stimulation.
In essence, freshly recruited monocytes were found to retain several phenotypic markers of PB monocytes but displayed strong upregulation of CD14 along with an increased TNF-α message and a markedly enhanced readiness to liberate this cytokine in response to endotoxin challenge. Such priming during the recruitment process may be relevant for pulmonary host defense mechanisms and inflammatory events under conditions of alveolar microbial challenge.
The described technique for the recruitment and discrimination of monocytes from rAMs is based on two steps: 1) in vivo labeling of rAMs by intravenous injection of the red fluorescent dye PKH26 to increase their red fluorescence intensity and 2) intratracheal instillation of murine rJE/MCP-1 to attract monocytes into the bronchoalveolar space. Importantly, the fluorescent dye PKH26, which has previously been employed for in vivo labeling of resident peritoneal macrophages (24, 25), did not substantially stain PB leukocytes. Therefore, monocytes recruited into the alveolar air spaces in response to rJE/MCP-1 could easily be discriminated from rAMs being preloaded with PKH26 by the difference in red fluorescence characteristics. The labeling of the rAMs with PKH26 displayed dose- and time-dependent characteristics, with sufficient labeling of rAMs for discrimination purposes being observed at in vivo concentrations of 12–15 μM PKH26. This labeling process, presenting to be stable for >120 h in the present study, is known to occur via incorporation of the aliphatic phagocyte linker molecule of PKH26 into the lipid bilayer portion of the plasma membrane (2, 16). In addition, cytoplasmic accumulation due to phagocytosis of dye aggregates and endocytosis of labeled plasma membranes have been reported (25). However, although this principal mode of action explains the successful in vivo labeling of resident peritoneal macrophages after intraperitoneal injection of PKH fluorochromes (24, 25), the precise mechanism underlying the present finding that rAMs but not PB leukocytes accumulated the fluorescent dye after intravenous administration is not yet known. Actually, based on several observations, we excluded that PKH26 accumulation by rAMs might affect macrophage functions. First, we found that PKH26 per se did not induce a TNF-α message in rAMs, but when PKH26-treated mice were challenged with an intratracheal instillation of S. abortus equiendotoxin, drastically elevated TNF-α mRNA levels were found in rAMs preloaded with PKH26 in vivo, indicating that PKH26 did not render these cells hyporesponsive to LPS challenge in vivo. Second, the rAM antigen profiles of PKH26-treated and nontreated animals were essentially the same, indicating that PKH26 does not alter the expression level of the cell surface molecules analyzed. Importantly, histological examination of lung sections from PKH26-treated mice showed an architecture comparable to that of control mice, in line with the absence of respiratory distress in these mice. This excludes that some rough damage of the pulmonary circulation, representing the first vasculature to be passed by the dye after intravenous injection, might underlie the distribution of PKH26 into the alveolar space, thereby being taken up by the rAMs.
The dose-response analysis of intratracheally administered murine rJE/MCP-1 showed that only concentrations of >10 μg rJE/MCP-1/mouse induced a detectable recruitment of monocytes into the alveolar air space (data not shown). Twenty-four hours after intratracheal instillation of 50 μg rJE/MCP-1/mouse, we measured a significant alveolar accumulation of monocytes and, to a much lesser extent, of CD4-positive lymphocytes. By 48 h, BAL fluid cell numbers of ∼1.6 × 106 cells/mouse, including >25% of newly recruited monocytes, were observed, without an increase thereafter. We did not test higher doses of rJE/MCP-1 for alveolar monocyte accumulation, but a further enhancement of the recruitment response on higher dosage may be anticipated given the findings of Gunn et al. (15), who recovered > 5 × 106total BAL fluid cells from mice overexpressing human MCP-1 in type II alveolar epithelial cells. Importantly, morphometric analysis showed that the alveolar space was the only compartment in which the number of CD45-positive cells significantly increased in response to alveolar rJE/MCP-1 deposition, with the peribronchial and perivascular compartments being unchanged and the alveolar septum even displaying a decrease in cell numbers. This observation is well compatible with the notion that alveolar rJE/MCP-1 challenge provoked monocyte transmigration through both the endothelial and alveolar-epithelial barriers and that further to the intravascular monocyte pool, interstitial (septal) cells were additionally recruited into the alveolar space by this maneuver. In addition to the morphometric analysis, our data demonstrating that in vitro PKH26-prelabeled PB leukocytes from donor mice transferred to recipient mice were recovered from the alveolar air space of recipient mice after intratracheal instillation of rJE/MCP-1 further support the assertion that alveolar recruited monocytes are mainly derived from the intravascular compartment. These findings are in line with a previously published work (20) in which radiolabeled monocytes from donor rats were injected intravenously into recipient rats and recovered from the alveolar air space of recipient rats after intratracheal instillation of LPS. Moreover, these studies indicate that circulating leukocytes may be recruited across the endothelial and epithelial barriers into the alveolar air space under both noninflammatory conditions (present study) and highly inflammatory conditions (20). However, monocyte recruitment in response to rJE/MCP-1 apparently differs from the pattern observed subsequent to alveolar LPS deposition where predominant interstitial monocyte accumulation was observed, with significantly lower portions of cells recruited into the alveolar air space (20).
Alveolar recruited monocytes still expressed the adhesion molecules CD11a, CD11b, CD49d, and CD62L that were also detected on PB monocytes but with the exception of CD11a, not on rAMs (11). These cell surface molecules have previously been shown to be relevant for the monocyte recruitment to various inflammatory sites in vivo (17, 20, 28). Unexpectedly, these antigen markers were also detected on alveolar monocytes recovered 72 and 96 h after bronchial instillation of rJE/MCP-1, indicating that in our model alveolar recruited monocytes retain a monocyte-like phenotype without spontaneously differentiating into rAMs for the time span investigated. These findings, based on immunologic criteria, extend the data from Gunn et al. (15), who also observed a lack of rapid monocyte-to-macrophage transition in the alveolar space as based on morphological variables. The presently employed technique may turn out to be suitable for analyzing the differentiation of monocytes to rAMs in the alveolar compartment in more detail.
An important finding of the present study was the observation that newly recruited alveolar monocytes, but not PB monocytes or rAMs, express significant quantities of CD14 on the cell surface. CD14 is a glycosylphosphatidylinositol-linked cell surface receptor centrally involved in LPS recognition by myeloid and nonmyeloid cells (8,27, 34). Its expression is rapidly upregulated in rodents in response to inflammatory activation by LPS or cytokines such as TNF-α or interleukin-1β (8, 27). Interestingly, the currently observed CD14 upregulation on newly recruited monocytes in our model was found to be associated with a significantly increased mRNA expression of the immediate-early cytokine TNF-α. Moreover, CD14-positive alveolar monocytes were markedly more responsive to LPS challenge than the (CD14-negative) PB monocytes as evidenced by their fourfold increased TNF-α release on stimulation with endotoxin. We suggest that the alveolar monocytes are primed rather than fully activated, considering the fact that CD14-positive alveolar monocytes still expressed L-selectin, a marker that is known to be shed on inflammatory activation of cells by stimuli such as LPS, TNF-α, or C5a (13, 19). It seems reasonable to suppose that such priming of monocytes entering the alveolar compartment may contribute to pulmonary host defense mechanisms and inflammatory events under conditions of lung infection.
The mechanisms underlying the upregulation of CD14 and TNF-α in the alveolar recruited monocytes are beyond the scope of the current investigation. Some direct impact of PKH26 on the monocytes may be excluded because the PB monocytes were similarly exposed to this dye. Moreover, these changes may not be attributable to a direct influence of rJE/MCP-1 because incubation of isolated PB monocytes with this agent did not reproduce the characteristic features of monocytes entering the alveolar compartment in response to this chemokine (Maus U, Herold, Maus R, Seeger, and Lohmeyer, unpublished data). In contrast, it is quite conceivable that the upregulation of CD14 expression and the enhanced responsiveness to LPS are linked to the transmigration process of monocytes through the endothelial and epithelial barriers, an event that was recently shown to influence not only the barrier cells but also transmigrating monocytes (5). Alternatively, such changes might be evoked by components of the alveolar microenvironment, e.g., surfactant compounds, whereas the rAMs are no longer responsive to such components. To exclude that the observed leukocyte recruitment pattern associated with intratracheal instillation of rJE/MCP-1 merely reflects pleiotropic C-C chemokine characteristics rather than rJE/MCP-1-specific features, the chemoattractants MIP-1α and RANTES, both known for their leukocyte-recruiting capacities (2a, 3, 31a), were delivered into the lungs of mice. Whereas MIP-1α induced a significant accumulation of polymorphonuclear neutrophils (>30%) within the alveolar compartment, a predominant accumulation of CD4-positive lymphocytes was found to be induced on intratracheal instillation of RANTES into the lungs of mice. Thus none of these chemotactic agents were found to reproduce the characteristic feature of rJE/MCP-1 to induce the accumulation of monocytes within the alveolar air spaces of mice.
In conclusion, we established a novel technique that clearly discriminates bronchoalveolar recruited monocytes from rAMs. We demonstrate for the first time that monocytes 1) are recruited into the bronchoalveolar compartment of mice in response to local deposition of exogenous rJE/MCP-1 and 2) retain a PB monocytic phenotype for at least 96 h after entering the alveolar space but 3) show a marked upregulation of CD14 expression along with an enhanced responsiveness to endotoxin challenge in vitro. These findings may be relevant for understanding pulmonary host defense and inflammatory mechanisms.
We thank G. Mansouri for excellent technical assistance.
This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 547.
This work is part of the MD thesis of S. Herold.
Address for reprint requests and other correspondence: U. Maus, Dept. of Internal Medicine, Justus-Liebeg-University, Klinikstrasse 36, Giessen 35392, Germany (E-mail:).
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.
- Copyright © 2001 the American Physiological Society