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The Journal of Immunology, 2001, 166: 6376-6382.
Copyright © 2001 by The American Association of Immunologists

Surfactant Protein A Suppresses Lipopolysaccharide-Induced IL-10 Production by Murine Macrophages1

Laurent Salez*, Viviane Balloy*, Nico van Rooijen{dagger}, Mai Lebastard{ddagger}, Lhousseine Touqui*, Francis X. McCormack§ and Michel Chignard2,*

* Unité de Pharmacologie Cellulaire, Unité Associée Institut Pasteur/Institut National de la Santé et de la Recherche Médicale, Paris, France; {dagger} Department of Cell Biology, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands; {ddagger} Unité d’Immunophysiologie et Parasitisme Intracellulaire, Institut Pasteur, Paris, France; and § Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Cincinnati, Cincinnati OH 45267


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Upon LPS exposure, mononuclear phagocytes produce TNF-{alpha} and IL-10, two cytokines with pro- and anti-inflammatory activities, respectively. We previously described that murine resident alveolar macrophages, which play a central role in the immunosurveillance of the lung alveoli, do not synthesize IL-10 in vivo or in vitro when exposed to LPS. In the present report we demonstrate that during lung inflammation induced by the intranasal administration of LPS, bronchoalveolar cells collected between days 3 and 5 are able to synthesize IL-10 when exposed to LPS. We also show that depletion of resident alveolar macrophages by an intratracheal instillation of liposome-encapsulated clodronate is followed by subsequent replenishment of the airspaces by mononuclear phagocytes. This is accompanied by the transient competence of cells for IL-10 production. The cell capacity to produce IL-10 is evident up to 3 days and then decreases. This led us to hypothesize that the alveolar environment contains a down-regulator of LPS-induced IL-10 synthesis by recently emigrating mononuclear phagocytes. We show that the surfactant protein A, an airspace protein that has known immunomodulatory activities, dramatically inhibits LPS-induced IL-10 formation by bone marrow-derived macrophages. These data show a difference between resident and inflammatory macrophages with respect to IL-10 synthesis. Moreover, this study highlights for the first time the inhibitory role of surfactant protein A in the anti-inflammatory activity of macrophages through inhibition of IL-10 production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Gram-negative sepsis is one of the most common causes of the acute respiratory distress syndrome (ARDS),3 a lung injury that is caused in part by a cytokine-mediated inflammatory process (1, 2). Administration of LPS, a component of the wall of Gram-negative bacteria, to laboratory animals initiates many responses that mimic the clinical signs of ARDS, including the migration of polymorphonuclear neutrophils from the microcirculation into the alveolar spaces (3). LPS stimulates monocytes/macrophages to produce different mediators and particularly TNF-{alpha}, a cytokine that indirectly recruits polymorphonuclear neutrophils into the inflammatory site (4, 5). However, LPS-activated mononuclear phagocytes also produce IL-10 (6, 7), an anti-inflammatory cytokine (8, 9). IL-10 deactivates macrophages (10, 11), inhibits TNF-{alpha} formation (12), and reduces cellular recruitment at the lung level after LPS challenge through a negative feedback mechanism (13, 14). We previously reported that resident alveolar macrophages fail to produce IL-10 in vivo and in vitro upon LPS stimulation (15), a property that could partly contribute to the pulmonary complications of Gram-negative sepsis.

Surfactant protein A (SP-A) is a component of pulmonary surfactant, a lipoproteinaceous film that lowers the surface tension at the air-liquid interface of the lung. It is a member of the collectin family (16, 17, 18), which is composed of proteins that contain both a collagen-like domain and a calcium-dependent carbohydrate binding domain. SP-A plays an important role in the innate immunity of the lung, particularly through effects on phagocyte functions (17, 18). However, conflicting data from in vitro studies have generated considerable controversy regarding whether SP-A is primarily an anti-inflammatory or a pro-inflammatory immunomodulatory molecule. For instance, incubation of SP-A with alveolar macrophages has been reported to stimulate or inhibit oxygen radical production (19, 20, 21) and to stimulate or inhibit NO production (22, 23). SP-A has been shown to inhibit TNF-{alpha} synthesis induced by LPS-stimulated cells (24), but can also independently stimulate TNF-{alpha} production (25). Moreover, SP-A binds to LPS (26) and to the surface of a variety of bacteria (27, 28), viruses (29), and fungi (30) and enhances the phagocytosis of some micro-organisms (31), but inhibits (32) or does not affect the phagocytosis of others (33).

We presently show that following lung inflammation induced with LPS, murine airways become populated with IL-10-producing cells. We demonstrate that these IL-10-competent cells are emigrating mononuclear phagocytes. However, the IL-10 production is transient, suggesting that a component of the alveolar lining fluid may modify the reactivity of mononuclear phagocytes. We further show that SP-A inhibits LPS-induced IL-10 production by bone marrow-derived macrophages (BMDM), suggesting that SP-A may modulate IL-10 production by mononuclear phagocytes that have been recruited to the alveolar space.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

LPS (Escherichia coli O55:B5) was obtained from Difco (Detroit, MI). M3/84 mAb and control isotype were purchased from PharMingen (San Diego, CA). Rabbit anti-rat secondary Abs and monoclonal rat anti-alkaline phosphatase-anti-alkaline phosphatase (APAAP) complex were obtained from Dako (Trappes, France). Fast blue BB salt, levamisole, naphthol AS-MX, brefeldin A, and Gey’s medium were obtained from Sigma (St. Louis, MO). RPMI 1640 medium and PBS were purchased from Life Technologies (Grand Island, NY). FCS, inactivated at 56°C during 1 h, was obtained from Roche (Mannheim, Germany). FCS contains <10-2 pg/ml of LPS. Clodronate was a gift from Roche. Human SP-A was isolated from the lung washings of a patient with alveolar proteinosis by a modification of the method described by Suwabe et al. (34), which included serial sedimentation of the surfactant pellet in the presence of 1 mM Ca2+, elution with EDTA and adsorption to mannose-Sepharose. The level of LPS associated with the SP-A was 140 pg of LPS/µg of SP-A.

Administration of LPS to mice

Seven-week-old male C57BL/6 mice, weighing 25–30 g, provided by the Center d’Elevage R. Janvier (Le Genest St. Isle, France), were lightly anesthetized by ether inhalation and intranasally inoculated with 330 µg/kg of sonicated LPS dissolved in 50 µl of saline.

Preparation of cells from bronchoalveolar lavage fluid (BALF)

Animals were killed by i.p. administration of a lethal dose of sodium pentobarbitone (Sanofi, Libourne, France). The trachea was cannulated, and bronchoalveolar lavage was performed with a syringe by eight cycles of instillation and aspiration with 0.5 ml of saline (total, 4 ml). Collected cells were counted (Coulter, Luton, U.K.) and centrifuged at 400 x g for 20 min. In one set of experiments cells were resuspended in the culture medium at 1.5 x 106/ml and incubated in hydrophobic tubes with 1 µg/ml of LPS. In another set of experiments, cells were resuspended at concentrations that were based on the expected relative proportions of monocytes to total leukocytes (15). For instance, in control animals nearly 100% of cells recovered by lavage were monocytes/macrophages, while only 45–50% of the cells recovered from animals that were lavaged 4 days after intranasal instillation of LPS were of monocytic lineage. Therefore, cells from control animals were resuspended at 2 x 105/300 µl of culture medium, while cells obtained from animals that had received intranasal administration of LPS 4 days earlier were resuspended at 4 x 105/300 µl of culture medium. Cell suspensions were then dispensed in 96-well tissue culture plates for a 1-h adhesion step. Wells were washed to remove nonadherent cells, and remaining adherent macrophages were immediately incubated with 300 µl of culture medium containing 1 µg/ml of LPS. In all cases, cells were stimulated at 37°C in RPMI 1640 medium supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS (v/v). Conditioned media were collected, centrifuged (400 x g, 10 min, 4°C), and stored at -20°C until assayed for IL-10 and TNF-{alpha} concentrations. In all cases experiments were performed with a pool of cells collected from several mice, as indicated in the figures. At all time points assays were performed in triplicate.

Measurement of immunoreactive IL-10 content of supernatants by immunoenzyme assay

Concentrations of IL-10 were determined as previously described (15). Briefly, the solid phase immunoenzyme assay (35, 36) is an immunometric assay for murine IL-10 that uses the same monoclonal anti-murine (JES-2A5) Ab for both capture and revelation steps. The assay relies on the reaction of the thiol groups of mAb Fab' with maleimido groups previously introduced into acetylcholinesterase (AchE) as previously described (37). Anti-IL-10 mAb was isolated from the ascetic fluids of mice injected with anti-IL-10-producing hybridoma cells, and purification was achieved by affinity chromatography on protein G column (HiTrap affinity columns; Pharmacia Biotech, Uppsala, Sweden) after precipitation by ammonium sulfate as described previously (38). Assays were performed in 96-well microtiter plates (MaxiSorp; Nunc, Roskilde, Denmark) coated with 10 µg/ml purified anti-IL-10 mAb (JES-2A5). For the immunological capture, 100 µl of IL-10 standards (15.6–2000 pg/ml) or samples were added to coated plates for 18 h at 4°C. This was followed by epitope immobilization and epitope release. Thus, after washing the plates (10 mM phosphate buffer, pH 7.4, and 0.1% Tween 20), a 0.25% glutaraldehyde solution (100 µl) was added to each well, and the reaction was allowed to proceed for 5 min at 20°C while stirring. Wells were then washed, and 100 µl/well of a 10 mg/ml borane-trimethylamine complex solution containing 1 N HCl was added for an additional 5 min while shaking. Finally, after a washing step the binding of labeled Ab was performed by adding 100 µl/well of the JES-2A5-AchE conjugate at the concentration of 10 Ellman U/ml for 18 h at 4°C. For measurements of the solid phase bound enzyme activity, plates were extensively washed, and solid phase bound AchE activity was determined colorimetrically by adding 200 µl of Ellman’s medium. Absorbance was read at 405 nm. The lower limit of detection of this assay is ~10 pg IL-10/ml of sample.

Measurement of immunoreactive TNF-{alpha} content of supernatants by enzyme immunometric assay

Levels of TNF-{alpha} in the BALF and cell supernatants were also determined by an enzyme immunometric assay as previously described (15). Rat anti-murine TNF-{alpha} mAbs MP6-XT22 and MP6-XT3 were purified from the ascetic fluids of mice that had been injected with hybridomas (provided by P. Minoprio, Institut Pasteur, Paris, France). The characteristics of these rat anti-murine TNF-{alpha} mAbs were described in detail previously (39), and their purification was performed exactly as described above for the IL-10 mAbs. Immunometric assays were performed in 96-well microtiter plates (MaxiSorp; Nunc), coated with 10 µg/ml anti-TNF-{alpha} mAb, MP6-XT3, as described previously (38). The one-step procedure used for immunometric assays involved the simultaneous addition of 100 µl of TNF-{alpha} standards (7.8–1000 pg/ml) or samples, and 100 µl of the anti-TNF-{alpha} mAb, MP6-XT22-AchE conjugate, at a concentration of 10 Ellman U/ml. The following steps were performed exactly as described for IL-10. The lower limit of detection of this assay is ~15 pg of TNF-{alpha}/ml of sample.

Preparation of mouse BMDM

Mice were euthanized by CO2 inhalation. Using aseptic technique, femurs were collected and placed in dishes containing sterile PBS. The bone marrow was exposed and flushed with sterile PBS using a 2-cc syringe attached to a 26-gauge needle. Medium (2 ml/bone) containing bone marrow cells was transferred at a 50-ml conical centrifuge tube (4°C) containing Gey’s medium (5 ml/ml medium) to lyse red cells. After a 10-min incubation, medium was centrifuged for 10 min at 400 x g. The supernatant was discarded, and pelleted cells were resuspended in RPMI 1640 medium supplemented with 10% FCS and 10% CSF-1-conditioned medium (40) to a final concentration of 1.6 x 106 cells/ml. Fifteen milliliters of medium was transferred to a 100-mm tissue culture plate (TPP; ATGC Biotechnologie, Noisy le Grand, France). Forty-eight hours later, medium was supplemented with 5 ml of the same medium. After 3 days, nonadherent cells were removed with a sterile pipette, transferred to a 50-ml conical tube, and centrifuged for 10 min at 400 x g. The cell pellets were resuspended in 1 ml of RPMI 1640 supplemented with antibiotics and 10% FCS. Medium was flushed through 25-, 27-, and 30-gauge needles, successively, using a 2-ml syringe to separate aggregates and produce single-cell suspensions. Cells were resuspended at the desired concentration of 2 x 105 cells/300 µl in RPMI 1640 supplemented with antibiotics, 10% FCS, and 4% CSF-1-conditioned medium. Aliquots (300 µl) of the cell suspension were then dispensed into 96-well tissue culture plates (TPP). After an overnight incubation in a 5% CO2 humidified air atmosphere at 37°C, wells were washed twice with prewarmed RPMI 1640 and stimulated as indicated in the figure legends.

Preparation of liposome-encapsulated clodronate and depletion of alveolar macrophages

Liposomes composed of phosphatidylcholine and cholesterol (molar ratio, 6/1), with or without added dichloromethylene diphosphonate (clodronate), were produced as previously described (41). Briefly, 86 mg of phosphatidylcholine and 8 mg of cholesterol were dissolved in 10 ml of chloroform and dried to a film by low vacuum rotary evaporation. The lipids were rehydrated in 10 ml of PBS or in a solution of 2.5 g of clodronate in 10 ml of PBS and incubated at room temperature. The liposome suspension was then diluted in 100 ml of PBS and centrifuged at 100,000 x g for 30 min to remove free clodronate, after which liposomes were resuspended in 4 ml of PBS.

Mice were anesthetized by an i.m. injection of 1 mg of ketamine and 0.2 mg of xylazine. Alveolar macrophage depletion was achieved by the intratracheal instillation of 50 µl of a liposome-encapsulated clodronate suspension (42). After different time intervals, BAL was performed as described above.

Immunocytochemistry on BALF cells

Freshly stimulated BALF cells were applied to glass slides using a Shandon cytocentrifuge (Shandon Scientific, Chechire, U.K.). Slides were immediately fixed and permeabilized with acetone for 10 min. Intracellular staining of IL-10 was performed using mAb (JES-2A5) or control isotype (GL113) with the avidin-biotin peroxidase complex Vectastain Elite kit (Vector, Burlingame, CA), exactly as described by the manufacturer. After IL-10 staining, the same slides were used for establishing the identity of IL-10-producing cells by double immunocytochemical staining. The presence of Mac-3+ cells was detected using the APAAP procedure. Briefly, slides were incubated in phosphate-free buffer (Tris-HCl, pH 7.6) for 30 min, and mAbs for Mac-3 (M3/84) or isotype IgG1 at 1/100 dilution were incubated with cells in the same buffer for 1 h. The slides were then washed three times for 10 min. Rabbit anti-rat secondary Abs were then incubated at 1/100 dilution for 30 min, and the slides were again washed three times with buffer. The next incubation was performed with the APPAP complex (monoclonal rat anti-APAAP) for 1 h in the same buffer. After three lavages the slides were developed with fast blue BB salt prepared in Tris-HCl, pH 8.2, with levamisole and naphthol AS-MX. The slides were finally washed with tap water and examined with an optical microscope.

Statistical analysis

Results were expressed as the mean ± SEM for the indicated number of independently performed experiments. Comparisons between values were analyzed by Student’s t test for unpaired data, and p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPS-induced IL-10 production by bronchoalveolar cells collected at different time intervals after induction of lung inflammation with LPS

At various time intervals after intranasal instillation of 330 µg/kg LPS (50 µl), BALF were collected, and the total cell population was kept in suspension. Cells (1.5 x 106/ml) were stimulated in vitro with 1 µg/ml of LPS, and immunoreactive IL-10 was assayed in the supernatants after a 6-h incubation. As shown in Fig. 1Go, IL-10 production was highest in cells collected between days 3 and 5 and decreased significantly by day 6. In the absence of LPS challenge, IL-10 was not detected at any time point (data not shown).



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  		FIGURE 1. LPS-induced IL-10 production by bronchoalveolar cells collected at different time intervals after induction of lung inflammation. At different time intervals after intranasal instillation of a single dose of 330 µg/kg of LPS, BALF (1.5 x 106 cells/ml) were incubated in suspension with 1 µg/ml of LPS. After a 6-h incubation, IL-10 was assayed in culture supernatants. Results are expressed as the mean ± SEM obtained from six distinct animals for each time point.

 
Identification of the monocyte/macrophage population as the IL-10-producing bronchoalveolar cells collected from inflamed lungs

The cellular source of IL-10 production by mononuclear phagocytes was assessed by two different approaches. First, BALF cells collected 4 days after LPS instillation were enriched in mononuclear phagocytes by adhesion and exposed to 1 µg/ml of LPS in vitro for 6 h. As a control, BALF cells collected from naive mice were similarly processed in parallel. Stimulation of adherent cells collected 4 days after the induction of acute lung inflammation, but not adherent cells recovered from the naive mice, resulted in the secretion of IL-10 into the extracellular medium (Fig. 2Go). Interestingly, TNF-{alpha} production by day 4 cells was significantly reduced under these experimental conditions compared with the production by resident alveolar macrophages recovered from the naive mice. As a second method to assess the source of IL-10 production in the lung, the BALF cell suspensions were challenged with 1 µg/ml of LPS and double immunostained for Mac-3 and intracellular IL-10. Staining revealed that all IL-10-positive cells were also Mac-3 positive (Fig. 3Go). Specificity was verified by staining with control isotype Ab, which revealed no signal (data not shown). Taken together, the two approaches converged to prove that monocytes/macrophages are the competent cells producing IL-10 in the airspaces after an instillation of LPS.



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  		FIGURE 2. LPS-induced IL-10 and TNF-{alpha} production by bronchoalveolar adherent cells collected from control and inflamed lungs. BALF were collected from naive mice (control) and mice that had received 4 days before an instillation of 330 µg/kg LPS (inflamed), and total recovered cells were adhered to 96-well tissue culture plates for 1 h. Wells were then washed and immediately stimulated (2 x 105/well) with 1 µg/ml of LPS over 6 h. Culture supernatants were assayed for IL-10 and TNF-{alpha} concentrations. Results are expressed as the mean ± SEM obtained from three distinct experiments, each performed with a pool of cells collected from three mice.

 


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  		FIGURE 3. Immunocytochemical identification of the monocyte/macrophage population as the IL-10-producing bronchoalveolar cells collected from inflamed lungs. BALF were collected 4 days after instillation of 330 µg/kg LPS, and cells were adjusted to 1.5 x 106/ml and stimulated with 1 µg/ml of LPS in the presence of 10 mg/ml brefeldin A over 4 h. Total cells were centrifuged on slides and immunostained for Mac-3 (blue) and/or intracellular IL-10 (red). Magnification: A–D, x100; E and F, x600. This result is representative of three separate experiments.

 
LPS-induced IL-10 production by bronchoalveolar cells collected at different time intervals after alveolar macrophage depletion

Positive staining for Mac-3 reveals the monocytic lineage of IL-10-producing cells, but cannot discriminate between IL-10 production by resident macrophages following a change of phenotype due to a possible effect of tolerance (43) or by circulating monocytes that have been newly recruited into the airspaces (44). In a previous study we observed that intranasal LPS instillation produces a progressive expansion of the murine airspace mononuclear cell population for up to 4 days (15). Comparison of these kinetics with the kinetics of IL-10 production shown in Fig. 1Go suggested to us that the newly recruited mononuclear phagocytes could be the source of IL-10 production. To test this hypothesis, alveolar macrophage depletion and subsequent repopulation of the airspaces with monocytes recruited from the circulating monocyte pool were achieved by intratracheal instillation of liposome-encapsulated clodronate (45, 46). Cellular analysis of BALF collected at daily intervals after clodronate administration revealed that 99% of resident alveolar macrophages were depleted at 24 h. When similar experiments were performed with PBS-containing liposomes, no changes in alveolar macrophage counts were observed (data not shown). Macrophage depletion was followed by a progressive recruitment of new cells. At each time point, collected cells in suspension were stimulated with 1 µg/ml of LPS, and IL-10 formation was assayed in the supernatants. IL-10 production was absent when LPS was omitted (data not shown). Time-course studies of the increase in cells in the BALF and IL-10 production indicated a correlation between days 1 and 3 (Fig. 4Go), suggesting that the newly recruited monocytes were the origin of IL-10 synthesis upon LPS challenge. The observation that IL-10 production decreased after day 3 and almost vanished by day 7 suggested that the recently arrived monocytes lose the capacity to synthesize IL-10 with time and progressively behave like resident alveolar macrophages.



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  		FIGURE 4. LPS-induced IL-10 production by bronchoalveolar cells collected at different time intervals after alveolar macrophage depletion. Mouse lungs were lavaged at different time intervals after instillation (50 µl) of a liposome-encapsulated clodronate suspension. Total cell counts were determined by optical microscopy. The remaining BALF was centrifuged, and total recovered cells, resuspended in culture medium (100 µl), were stimulated in vitro with 1 µg/ml of LPS for 6 h. Supernatants were assayed for immunoreactive IL-10. Results are expressed as the mean ± SEM obtained from 10 (cell counts) and five (IL-10 assays) mice for each time point.

 
SP-A inhibition of LPS-induced IL-10 production by BMDM

We thus hypothesized that a local agent(s) present in the airspace microenvironment modulated the progressive extinction of LPS-induced IL-10 production by emigrating monocytes. SP-A is a candidate for this role based on accumulating evidence for its immunomodulatory properties. To assess the role of SP-A in modulation of IL-10 production, we tested the effect of SP-A on LPS-induced IL-10 production by BMDM prepared from naive mice. Fig. 5Go shows that BMDM were able to produce IL-10 and TNF-{alpha} upon LPS stimulation. TNF-{alpha} production was characterized by a progressive increase up to 6 h, followed by a decrease down to basal levels by 24 h. IL-10 production was slower and continuously increased for at least 24 h (data not shown). Preincubation of the BMDM with SP-A had a differential effect on the production of IL-10 and TNF-{alpha}. In this experiment BMDM were pretreated with SP-A for 24 h and then challenged with LPS for 6 h. Fig. 5GoA shows that at a concentration of 10 µg/ml, SP-A inhibited LPS-induced IL-10 production by 52% (p = 0.011; n = 3), without affecting TNF-{alpha} production (Fig. 5GoB). SP-A exposure without subsequent LPS stimulation did not induce TNF-{alpha} or IL-10 production from the BMDM (data not shown). Complementary experiments showed concentration-dependent inhibition of LPS-induced (1 µg/ml) IL-10 production by SP-A to a maximum of 79% for the highest SP-A concentration tested (30 µg/ml; Fig. 6Go). Similarly, the concentration-dependent stimulation of IL-10 production from BMDM with LPS at concentrations of 0.01, 0.1, and 1 µg/ml was inhibited by SP-A at each LPS concentration tested (Fig. 7Go). However, TNF-{alpha} production was not affected at any LPS or SP-A concentration tested (data not shown).



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  		FIGURE 5. Effect of SP-A pretreatment on LPS-induced IL-10 (A) and TNF-{alpha} (B) production by BMDM. Adherent cells were incubated with 10 µg/ml of SP-A for 24 h. The incubation medium was then removed and replaced by fresh medium without SP-A, followed by addition of 1 µg/ml of LPS for 6 h. Extracellular media were collected and assayed for IL-10 and TNF-{alpha} concentrations. Results are expressed as the mean ± SEM of experimental points performed in triplicate with a pool of cells collected from three mice. This experiment is representative of three. *, p < 0.05.

 


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  		FIGURE 6. Concentration-dependent inhibition of LPS-induced IL-10 production from BMDM by SP-A. Cells were incubated with various concentrations of SP-A for 24 h. The incubation medium was then removed and replaced by fresh medium without SP-A, followed by addition of 1 µg/ml of LPS for 6 h. Extracellular medium was collected and assayed for IL-10 concentration. Results are expressed as the mean ± SEM of experimental points performed in triplicate with a pool of cells collected from three mice. This experiment is representative of three. *, p < 0.05.

 


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  		FIGURE 7. SP-A inhibition of BMDM IL-10 production induced by different concentrations of LPS. BMDM were incubated with 10 µg/ml of SP-A for 24 h. The incubation medium was then removed and replaced by fresh medium without SP-A, followed by addition of different concentrations of LPS for 6 h. Extracellular medium was collected and assayed for IL-10 concentration. Results are expressed as the mean ± SEM of experimental points performed in triplicate with a pool of cells collected from three mice. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Innate host defense is characterized by a fine balance between an effective inflammatory response and the maintenance of tissue integrity. Monocytes/macrophages play a prominent role in this equilibrium, as they produce and release a wide variety of biologically active products (47). TNF-{alpha} and IL-10 are prototypical examples of pro- and anti-inflammatory molecules that regulate inflammatory processes. We have previously reported that the balance between TNF-{alpha} and IL-10 produced by alveolar macrophages is deranged compared with that of other monocytic cells, in that resident alveolar macrophages synthesize TNF-{alpha}, but not IL-10, upon LPS challenge (15). The present study shows that, in fact, newly recruited airway monocytes are capable of transient IL-10 production. This was deduced from two sets of experiments dealing with LPS-induced acute lung inflammation and with, as the main direct informative data, the depletion of resident alveolar macrophages. It has been well demonstrated that the intratracheal administration of LPS in rats leads to blood monocyte recruitment to the airspaces (44), and that alveolar macrophage depletion in rats (45) and mice (46) is followed by repopulation of the alveolar spaces with monocytes. We demonstrate here that the emigrating phagocytes are transiently capable of IL-10 production upon LPS stimulation. The observation that IL-10 production is extinguished with time in the airspace suggests that a local agent(s) present in the alveoli may be responsible for the change in the macrophage phenotype. This idea is supported by several reports that the alveolar microenvironment influences macrophage cytokine and growth factor production (48, 49). In other studies comparison of the function of blood monocytes to resident alveolar macrophages following challenge with LPS or whole bacteria has revealed that the two populations bear different phenotypes. For example, Toosi et al. (50) showed a differential response of human monocytes and alveolar macrophages in terms of TGF-{beta} production. They speculated that SP-A has a marked importance in this behavior.

Indeed, SP-A is an abundant protein in the alveolar lining fluid (estimated concentration, 0.3–1.8 mg/ml) (16) and has been shown to influence the functions of phagocytes (16, 17, 18). However, the primary role of SP-A in modulation of inflammation in the alveolar space remains controversial. This is due in part to conflicting reports that SP-A increases and decreases the expression of cell surface molecules involved in macrophage activation and phagocytosis (51, 52) and increases and decreases the production of proinflammatory or anti-inflammatory cytokines (16, 17, 18). For instance, although SP-A has been reported to directly up-regulate the secretion of TNF-{alpha} by monocytic cells (25), it indirectly down-regulates this synthesis when triggered by LPS (24). This led us to hypothesize that SP-A was a factor susceptible to modulate the production of IL-10 by emigrating mononuclear phagocytes.

Our results depict an evident proinflammatory activity of SP-A mediated through inhibition of IL-10 production, an important anti-inflammatory cytokine. Moreover, these data indicate that TNF-{alpha} and IL-10 are regulated by different intracellular pathways. Indeed, it is known that most proinflammatory cytokine genes are regulated by NF-{kappa}B, while IL-10 synthesis appears to occur through NF-{kappa}B-independent pathways (53), perhaps through the Sp1 family (54).

In conclusion, SP-A was found to suppress the synthesis of the anti-inflammatory cytokine IL-10 by macrophages in response to LPS, without affecting TNF-{alpha} production. The regulation of IL-10 production from alveolar macrophages by SP-A may play a role in the pathogenesis of inflammatory lung diseases such as ARDS or other forms of acute and chronic lung injury.


   Acknowledgments
 
We thank Dr. Geneviève Milon for her critical review of the manuscript, and Dr. José Roberto Lapa e Silva for excellent assistance with immunostainings.


   Footnotes
 
1 This work was supported by grants from Ministère de la Recherche, de l’Enseignement, et de la Technologie and Fondation pour la Recherche Médicale. Back

2 Address correspondence and reprint requests to Dr. Michel Chignard, Unité de Pharmacologie Cellulaire, Unité Associée Institut Pasteur/Institut National de la Santé et de la Recherche Médicale 485, 25 rue du Dr. Roux, 75015 Paris, France. Back

3 Abbreviations used in this paper: ARDS, acute respiratory distress syndrome; SP-A, surfactant protein A; BMDM, bone marrow-derived macrophages; BALF, bronchoalveolar lavage fluid; AchE, acetylcholinesterase; APAAP, alkaline phosphatase-anti-alkaline phosphatase. Back

Received for publication November 7, 2000. Accepted for publication March 6, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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