Pulmonary Surfactant Protein A Up-Regulates Activity of the Mannose Receptor, a Pattern Recognition Receptor Expressed on Human Macrophages

Buffers, reagents, and media

Dulbecco’s PBS with and without Ca²⁺ and Mg²⁺ ions (Life Technologies, Grand Island, NY) and RPMI 1640 medium with l-glutamine (RPMI) (Life Technologies) were purchased. RPMI medium was used alone or with 20 mM HEPES buffer (pH 7.2; Sigma-Aldrich, St. Louis, MO) and 1 mg/ml human serum albumin (HSA) (Calbiochem, La Jolla, CA). Polymyxin B sulfate (PMB) was purchased from Sigma-Aldrich. Lipoarabinomannan (LAM) from the Erdman strain of M. tb was provided by Dr. P. J. Brennan and colleagues (Colorado State University, Ft. Collins, CO; National Institutes of Health, National Institute of Allergy and Infectious Diseases Contract 25147).

Antibodies

Mouse anti-human mAb against CR1 (anti-CD35; clone J3D3), CR4 (anti-CD11c; clone BU15), and CR3 (anti-CD11b; clone Bear 1) were purchased from Immunotech (Westbrook, ME). Bear 1 recognizes an epitope on the α-chain of CR3 (20), BU15 recognizes the α-chain of CR4 (21), and J3D3 recognizes CR1 (22). Mouse mAb IgG1 from Immunotech was used as the isotypic control for the above mAb. Purified and PE-labeled mouse anti-human MR and its purified and PE-conjugated subtypic control mAb, mouse IgG1κ, were purchased from BD PharMingen (San Diego, CA). Polyclonal rabbit anti-human MRs and rabbit anti-mouse MRs were provided by Dr. P. Stahl (Washington University, St. Louis, MO). A polyclonal rabbit anti-human Ab against SP-A was used (12). Normal rabbit serum (NRS) was used as one control for the experiments involving polyclonal Ab. FITC- or PE-conjugated goat anti-rabbit Ab and FITC- or PE-conjugated goat anti-mouse Ab were used as secondary Abs and were purchased from Cappel (West Chester, PA).

SP-A proteins

The SP-A proteins used in this study were developed and purified as previously described (23) (Fig. 1⇓). In brief, bronchoaveolar lavage was used to obtain APP-SP-A from healthy volunteers (native human SP-A) (24). Native rat SP-A was purified from silica-pretreated Sprague Dawley rat lungs (25, 26). Recombinant rat SP-A (which is deficient in hydroxyproline content and hence designated SP-A^hyp) was produced from SF-9 insect cells after infection with a recombinant baculovirus containing a 1.6-kb cDNA for rat SP-A (23). Recombinant rat SP-A proteins devoid of oligosaccharides at one or both of the consensus sequences for N-linked glycosylation were generated by amino acid substitutions at the Asn (SP-A^hyp,thr1) or Asn¹⁸⁷ site (SP-A^hyp,ser187) or at both sites (SP-A^{hyp,thr1,ser187}) (23). Carbohydrate-deficient recombinant SP-A proteins retain structural and biologic functions, including oligomerization, aggregation of phospholipid liposomes, binding to immobilized carbohydrate, inhibition of lipid secretion from type II cells, and competition for receptor occupancy on type II cells (23). The synthesis of the mutant recombinant rat SP-A protein containing a nested deletion of the proximal collagen-like region (Gly⁸–Gly⁴⁴) (TM2), truncation of the protein at the neck region resulting in a protein lacking the collagen and the NH₂-terminal regions (TM1-2-3), and an amino acid substitution at the Asn¹⁸⁷ site (TM1-2-3^ser187) were made as previously described (27, 28). Purity of the SP-A preparation was assessed by SDS-PAGE. Bacterial endotoxin levels were determined using the Limulus amebocyte lysate kit (BioWhittaker, Walkersville, MD). Endotoxin levels in SP-A preparations ranged from undetectable to 300 pg/μg protein, with an average of 15 pg of endotoxin per microgram of protein.

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FIGURE 1.

Schematic of rat and recombinant rat (hyp) SP-As. Native rat SP-A is composed of two isoforms, which are defined the by presence or absence of an interchain disulfide-forming N-terminal extension composed of amino acids Ile-Lys-Cys (IKC). The long and short isoforms are otherwise identical, containing an N-terminal segment (NTS) denoted domain 1, a collagen-like region divided by a midpoint interruption at Gly⁴⁴– into domains 2 and 3, a neck region, and a carbohydrate recognition domain. Sites of N-linked glycosylation (complex branching structure) are found at Asn¹ of the NTS and Asn¹⁸⁷ of the CRD. Recombinant rat SP-A synthesized in insect cells is similar to the native protein, except for simplified mannose-rich glycosylation (Y-shaped structure), incomplete proline hydroxylation, and oligomeric assembly as nonamers rather than octadecamers. Mutant recombinant SP-As devoid of attached carbohydrate at the 1 (hyp, thr1) or 1 and 187 (hyp, thr1, ser187) positions were generated by Asn¹Thr and Asn¹⁸⁷Ser substitutions. Truncated mutant (TM) recombinant SP-As containing deletions of first half of the collagen-like region (TM2), the NTS, and the entire collagen-like region (TM1, 2, 3), or the NTS, the collagen-like region and the carbohdyrate attached to Asn¹⁸⁷ (TM1, 2, 3, ser187) are shown.

Human monocytes and macrophages

Blood was obtained from healthy adult volunteers who were purified protein derivative skin test negative. Mononuclear cells from single donors were isolated from heparinized blood on Ficoll-sodium diatrizoate (Pharmacia Fine Chemical, Piscataway, NJ) and cultured in Teflon wells (Savillex, Minnetonka, MN) for 1 (monocytes) or 5 days (MDMs) in the presence of 20% autologous serum (1.5–2.0 × 10⁶ mononuclear cells/ml) at 37°C (29). On the day of each experiment, PBMCs were removed from Teflon wells and washed extensively, and the monocyte or MDM fraction was purified by adherence.

SP-A^−/− and SP-A^+/+ mice

Breeder pairs of gene-targeted SP-A-deficient mice (SP-A^−/−) and wild-type (SP-A^+/+) controls of the same strain (129J background) were kindly provided by Drs. J. Whitsett and T. Korfhagen (University of Cincinnati, Cincinnati, OH) (30). The mice used in the current study were the progeny of these original breeders and were bred at the University of Iowa (Iowa City, IA). Pathogen-free female and male 8- to 14-wk-old mice were housed under barrier conditions with an environmentally controlled atmosphere. All conditions and handling of the animals were approved by the Animal Care and Use Committee at University of Iowa and followed National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Mice were sacrificed via CO₂ asphyxiation. Resident peritoneal macrophages were obtained by peritoneal lavage with ice-cold HBSS supplemented with 10 mM HEPES (Life Technologies). AMs were obtained by bronchoalveolar lavage with 37°C HBSS supplemented with 10 mM HEPES (31).

Flow cytometry

PBMCs (29) were incubated with SP-A proteins (10 μg/ml) or HSA (control) in Teflon wells for various time periods. After washing, cells were incubated with PE-conjugated mAbs to CR1, CR3, or CR4. PBMCs incubated with the appropriate PE-conjugated subtypic control mAb served as negative controls. Additionally, in the initial experiments, after washing, the cells were incubated with rabbit anti-human MR Ab or NRS followed by PE-conjugated anti-rabbit secondary Ab. Subsequent experiments used PE-conjugated mAb against human MR or the appropriate PE-conjugated subtypic control mAb. As a positive control for up-regulation of the MR, MDMs were incubated with IL-4 (Genzyme, Cambridge, MA) for 20 h before staining (5). To determine whether new protein synthesis was involved in MR up-regulation, 10 μg/ml cycloheximide (CHX) was added to select wells for 60 min before addition of SP-A. To determine whether LPS was contributing to the response, LPS at double the highest level found in the SP-A preparations (15 ng/ml) was added to select wells. Additionally, in some experiments, the LPS neutralizer PMB (5 μg/ml) (32) was added to select wells 30 min before or simultaneous with SP-A. Because the results were the same with both approaches, experiments using PMB were combined.

In experiments using mouse peritoneal or alveolar macrophages, 5 × 10⁵ cells were incubated with rabbit anti-mouse MR (1/200) or NRS (control). After washing, cells were incubated with FITC-conjugated goat anti-rabbit Ig as the secondary Ab.

Cells were fixed in paraformadehyde and were analyzed for mean fluorescence intensity (MFI) and percentage of positive cells (95/5% cutoff) using a FACScan Flow Cytometry System (BD Biosciences, Franklin Lakes, NJ). Macrophages were identified using side scatter vs forward scatter. MFI due to nonspecific binding (using a subtypic control Ab) was subtracted out to provide a specific MFI. The percent change in specific MFI in each experiment was calculated as the percentage change in positive cells.

ELISA to determine possible cross-reactivity between SP-A and Abs against MR

APP-SP-A from 0 to 500 ng/well was adhered to a Costar medium binding ELISA plate (Cambridge, MA) using 0.05 M carbonate/bicarbonate buffer (pH 9.6). After washing, nonspecific binding sites were blocked with 5% BSA in PBS for 60 min at 4°C. Anti-SP-A, polyclonal anti-MR, monoclonal anti-MR, NRS, or isotypic control Ab was added to appropriate wells overnight at 4°C. After washing, the appropriate HRP secondary Ab (Bio-Rad Laboratories, Richmond, CA) was incubated with the monolayers for 2 h at room temperature. The developing reaction (Bio-Rad Laboratories) was stopped with 5% oxalic acid (J. T. Baker Chemical Company, Phillipsburg, NJ). The plate was read at 405 nm on a microplate autoreader (Bio-Tek Instruments, Winooski, VT). The mean ± SD of the absorbance for triplicate wells of each type was calculated. The OD of control wells devoid of Ag or primary Ab (typically ≤0.10) was subtracted out from each test group.

Immunofluorescence microscopy

MDMs in monolayer culture on glass coverslips were incubated with APP-SP-A (10 μg/ml) or HSA for 1 h at 37°C and then were washed, incubated for an additional 10 min at 37°C, and then cooled to 4°C. The cells were next fixed with 1% paraformaldehyde for 5 min, washed, and incubated with monoclonal anti-MR (1/500) in 0.2% BSA in PBS without Ca²⁺ and Mg²⁺ ions for 1 h, followed by FITC-conjugated goat anti-mouse Ab (1/100; Cappel) for 1 h. In certain experiments, lysosome-associated membrane protein-1 Ab, which stains intracellular late endosomal and lysosomal compartments (33), served as a control to confirm a nonpermeabilized cell membrane. To determine the possible effect of LPS contamination of the SP-A preparation, certain MDM monolayers were incubated with either 5 μg/ml PMB before SP-A or 15 ng/ml LPS.

The coverslips were mounted on glass slides and examined by confocal, scanning laser microscope (Zeiss, Thornwood, NY). Fluorescence intensity of each cell section was quantitated using the line intensity option of the Laser Sharp Reference Program (Bio-Rad Laboratories). A line was placed through the cell membrane of each cell at four points, corresponding to 12, 3, 6, and 9 o’clock on the cell, and MFI of that area of the cell was calculated using the software program. The values presented represent specific MFIs in which the MFI of the subtypic control slides has been subtracted out. MFI was determined for 20 cells per coverslip in each test group, three coverslips per donor, and a minimum of two donors were used for each treatment.

Iodination of mannose-BSA

Mannose-BSA was iodinated by the lactoperoxidase method in the core radioisotope faculty at the Veterans Affairs Medical Center (Iowa City, IA). Specific activity was typically 2–4 mCi/μg mannose-BSA protein, with 95% of total counts being TCA precipitable. The preparation was used within 3 wk of iodination (29).

Uptake of ¹²⁵I-labeled mannose-BSA by monocyte-derived macrophages

PBMCs in Teflon wells (cells is suspension) were incubated with 10 μg/ml APP-SP-A or HSA for 60 min at 37°C. After washing, cells were incubated in PBS containing Ca²⁺ and Mg²⁺ with or without 2.5 mg/ml mannan for 20 min at 37°C. One microgram of ¹²⁵I-labeled mannose-BSA was then added for 10 min at 37°C. The cells were cooled to 4°C and washed six times, and the final pellet was collected and counted using a Beckman gamma counter (Beckman Coulter, Fullerton, CA). In other experiments, 5-day-old MDMs were placed in monolayer culture on glass coverslips. The monolayers were incubated with 10–20 μg/ml SP-A or HSA for 60 min at 37°C. After washing, separate wells were incubated with PBS or 4 mg/ml mannan in PBS for 10 min at 37°C. Then, 1 μg of ¹²⁵I-labeled mannose BSA was incubated with the monolayers for 10 min at 37°C. The monolayers were cooled to 4°C and the glass coverslips were transferred to a new 24-well tissue culture pate and were washed eight times. The cells were lysed with 1% SDS and the lysates were counted in a Beckman gamma counter. MR functional activity was defined as mannan-inhibitable uptake of ¹²⁵I-labeled mannose-BSA. As a positive control, MDMs in Teflon wells were incubated with IL-4 (Genzyme) for 20 h before analysis.

Preparation of LAM-coated microspheres

LAM-coated microspheres were prepared as described (34). Briefly, polybead polystyrene microspheres (Polysciences, Warrington, PA) (2.0 × 10⁸) that were 1 μm in diameter were washed two times in 0.05 M carbonate-bicarbonate buffer (pH 9.6) in presiliconized polypropylene tubes (National Scientific Supply, San Rafael, CA) and then incubated with 50 μg of Erdman M.tb strain LAM or buffer (control) for 1 h at 37°C on an Adams Nutator. The microspheres were then washed twice and incubated in 5% HSA in PBS (2 h at 37°C) to block nonspecific binding sites. Lastly, the microspheres were washed, resuspended in 0.5% HSA, and used in the adherence assay described below.

Phagocytosis of LAM- and HSA-coated microspheres by SP-A-treated macrophages

MDMs (2 × 10⁵) were plated on a substrate of SP-A or HSA (control) for 90 min (12). After washing, monolayers were incubated with LAM-coated or HSA-coated microspheres (2.0 × 10⁷) in RPMI + HEPES + HSA at 37°C for 60 min. After 60 min, the MDMs were washed to remove nonadherent microspheres and were fixed in 10% formalin. In certain wells, monolayers were preincubated with 2.5 mg/ml mannan for 20 min to inhibit MR activity (29). The mean number (±SD) of microspheres per MDM on each of duplicate or triplicate coverslips was determined by counting a minimum of 200 consecutive MDMs per coverslip by phase contrast microscopy.

Statistics

A two-tailed Student’s t test was used for analyzing differences between specific test groups and control groups in each experiment.

SP-A up-regulates expression of the MR on human macrophages

SP-A up-regulates the phagocytosis of M. tb rapidly (within 60 min) via a direct interaction with the macrophage, indicating that SP-A influences macrophage receptor activity (12). We used flow cytometry to determine whether SP-A up-regulates the expression of the MR on MDMs. The results of a typical experiment are displayed as histograms (Fig. 2⇓) and cumulative data are presented in Table I⇓. The MR was constitutively expressed on MDMs incubated with HSA (control) (Fig. 2⇓A). After exposure to SP-A for 60 min, expression of the MR (based on both percent change in MFI and percent positive cells) was enhanced (p < 0.05) when compared with the control (Fig. 2⇓A and Table I⇓). IL-4 up-regulated MR expression as a positive control (5) (Fig. 2⇓C and Table I⇓). SP-A effects can be influenced by LPS contamination (35, 36). Incubation of macrophages with PMB and then SP-A did not affect the SP-A-induced up-regulation of MR expression (p > 0.1) (Table I⇓), and PMB alone had no effect on MR expression (p > 0.05) (data not shown). Additionally, incubation with LPS alone did not increase MR expression (p > 0.1) (Table I⇓).

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FIGURE 2.

MR but not CR4 expression is up-regulated after exposure to SP-A. In this representative experiment (n = 6), 5-day-old PBMCs in Teflon wells were incubated with APP-SP-A or HSA (control) for 1 h. After washing, the SP-A-treated and control MDMs were incubated with either polyclonal rabbit anti-human MR Ab or NRS (as a negative control) followed by PE-conjugated anti-rabbit Ig (A). Additionally, PBMCs were incubated with PE-conjugated CR4 or subtypic control mAb (B). As a positive control for the MR, PBMCs were incubated with IL-4 before staining for the MR (C). The stained cells were analyzed by flow cytometry; the average of duplicate samples is shown. In each histogram, the thin, broken line represents the fluorescence intensity of HSA-treated cells incubated with the appropriate control Ab (SP-A-treated cells incubated with subtypic control Ab gave the same results; data not shown). The dotted line represents the fluorescence intensity of HSA-treated cells stained for the MR or CR4, and the thick, solid line represents the fluorescence intensity of APP-SP-A- or IL-4-treated cells stained for the MR or CR4.

To determine whether SP-A selectively up-regulated MR expression or was capable of up-regulating other receptors involved in phagocytosis, we assessed the surface expression of CRs after SP-A incubation by flow cytometry. CR4 was constitutively expressed on MDMs (Fig. 2⇑B). Incubation with SP-A did not significantly alter expression of CR4 (Fig. 2⇑B and Table I⇑) or CR1 and CR3 (Table I⇑).

We next assessed the location of the MR on control or APP-SP-A-treated monocytes or MDMs by confocal microscopy (Fig. 3⇓ and Table II⇓). As reported (3), monocytes did not express MR (Fig. 3⇓A), and exposure to SP-A for 1 h did not induce MR expression on monocytes (p > 0.1) (Fig. 3⇓B). In contrast, MDMs expressed a low level of MR constitutively (Fig. 3⇓C and Table II⇓), and SP-A markedly increased the expression of MR on MDMs (Fig. 3⇓D and Table II⇓). Similar to the flow cytometry data, incubation of monolayers with LPS or with PMB before SP-A had no effect on MR expression in control-treated or SP-A-treated cells, respectively (Table II⇓). At any given time, 20–25% of total MR protein exists on the cell surface, with the majority residing in intracellular pools (37). For confocal microscopy experiments, MDMs were fixed with paraformaldehyde and thus were nonpermeabilized. Consistent with this, fixed MDMs incubated with Ab to lysosome-associated membrane protein-1, an intracellular late endosomal and lysosomal marker (33), revealed no significant staining (data not shown). Therefore, the MR expression being measured in these experiments represented cell surface protein only.

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FIGURE 3.

MDMs treated with APP-SP-A demonstrate enhanced expression of the MR. One-day-old monocytes (A and B) or 5-day-old MDMs (C and D) in monolayer culture were incubated with HSA (A and C) or APP-SP-A (10 μg/ml) (B and D) for 1 h. After washing, cells were fixed in paraformaldehyde and sequentially stained with mouse anti-human MR mAb or subtypic control mAb (data not shown) followed by a secondary FITC-anti-mouse Ab. Glass coverslips were mounted and visualized by confocal microscopy. A representative experiment is presented (n = 2–6).

We next determined whether the increased MR expression was due to outward movement of the intracellular pools or to new protein synthesis by incubating MDMs with CHX before SP-A to prevent protein synthesis. Pretreatment with CHX had only a small effect on the magnitude of SP-A-induced MR up-regulation (Table I⇑), supporting the notion that MR up-regulation was dependent primarily upon recycling of preformed MR rather than on newly synthesized MR.

To rule out cross-reactivity of anti-MR for SP-A as previously reported (38), an SP-A ELISA was performed. Polyclonal anti-human Ab against SP-A recognized APP-SP-A (500 ng/well) with an OD₄₀₅ of 1.2 ± 0.2 (n = 3). Neither the polyclonal anti-human MR Ab nor the monoclonal anti-human MR recognized APP-SP-A (OD₄₀₅ of 0.062 ± 0.003 and 0.069 ± 0.005, respectively; n = 3). Additionally, APP-SP-A was examined by Western blotting using the Abs mentioned above. Again, the polyclonal anti-human SP-A Ab recognized SP-A and the anti-MR Ab did not (data not shown). To rule out the possibility that SP-A remained on the surface of the MDMs after incubation and washing in our assays and was being recognized by the anti-MR Ab, we performed confocal microscopy experiments as above using a polyclonal Ab to SP-A as the primary Ab instead of the anti-MR Ab. No specific detection of SP-A on the cell surface was found (data not shown). These data are consistent with the results in our earlier study (12) that showed that SP-A is internalized rapidly by the MDMs.

The source of the SP-A affects the degree of MR expression

To test whether SP-A proteins from different sources were equally capable of up-regulating MR expression, we incubated MDMs with APP, native human or rat SP-A, as well as recombinant SP-A (SP-A^hyp) and measured MR expression compared with control MDMs using flow cytometry (Table I⇑). All four sources of SP-A examined significantly increased MR expression compared with control cells. Incubation with APP-SP-A consistently induced numerically higher levels of MR expression than did the other proteins, but the APP-SP-A-induced increase was only significantly higher (p < 0.05) than the level induced by SP-A^hyp.

The carbohydrate- and collagen-like domains of SP-A are both involved in SP-A-induced MR expression

We next explored the structural components of SP-A that mediate the enhanced MR response. To study the potential influence of the carbohydrate moieties of SP-A, we used recombinant SP-A proteins devoid of oligosaccharides at one (Asn¹⁸⁷) site (SP-A^hyp,ser187) or both (Asn¹ and Asn¹⁸⁷) sites (SP-A^{hyp,thr1,ser187}) of the consensus sequence sites for glycosylation. The absence of carbohydrates on the variant proteins used in these studies has been demonstrated previously (23). MDMs were incubated with variant SP-A proteins and MR expression was measured using flow cytometry (Table I⇑). Although not statistically significant, addition of the SP-A carbohydrate variant proteins resulted in a 60% increase in MFI and a 45–61% increase in positive cells relative to control MDMs. Incubation of MDMs with SP-A protein devoid of N-linked carbohydrates at the Asn¹⁸⁷ site (SP-A^hyp,ser187) resulted in MR expression similar to that seen with SP-A devoid of carbohydrate (SP-A^{hyp,thr1,ser187}) and was particularly evident for the percent change in positive cells. These results provide evidence that glycosylation is important for optimal up-regulation of MR expression.

In parallel experiments, we investigated the importance of the collagen-like region of SP-A for up-regulation of MR expression (Table I⇑). SP-A-truncated proteins lacking the first half of the collagen region but still possessing both carbohydrate attachment sites (TM2), lacking the collagen region and the N-terminal segment but still possessing the carbohydrate attachment site at Asn¹⁸⁷ (TM1-2-3), or lacking the collagen region, the N terminus, and both sugar attachment sites (TM1-2-3^ser187) were added to MDMs. The truncated proteins were capable of increasing MR expression to a small extent, but less than that of APP-SP-A or SP-A^hyp. The level of MR expression achieved was similar among truncated proteins. Taken together, these studies demonstrate a critical role for both the carbohydrates and collagen region of SP-A in mediating its effect.

SP-A enhances the uptake of ¹²⁵I-labeled mannose-BSA by MDMs

To assess whether the MR newly induced by SP-A was functional, we studied mannan-inhibitable pinocytosis of ¹²⁵I-labeled mannose-BSA, an assay of MR recycling (39). When APP-SP-A (10 μg/ml) was added to MDMs in suspension, the uptake of mannose-BSA was increased from 0.55 ± 0.13 to 1.64 ± 0.61 ng (198 ± 35% increase, n = 4, p < 0.05) and was comparable to the increase seen with IL-4 (0.55 ± 0.13 to 1.16 ± 0.37 ng for control vs IL-4, respectively; 110% increase, n = 6, p < 0.05). The addition of APP-SP-A (10 μg/ml) to MDM monolayers also enhanced the uptake of ¹²⁵I-labeled mannose-BSA compared with control cells. However, the percent increase was lower (37 ± 15% increase in mannose-BSA uptake, mean ± SEM, n = 4) than that recorded for MDMs in suspension, suggesting that the state of the cell (adherent vs nonadherent) may be a factor in dictating the magnitude of the response to SP-A.

MDMs in monolayer culture on a substrate of SP-A demonstrate enhanced phagocytosis of LAM-coated microspheres

We have determined that a major capsular lipoglycan, LAM, from M.tb serves as a ligand for the MR during phagocytosis of bacteria (34). Microspheres coated with LAM serve as model phagocytic particles for studies of MR-mediated phagocytosis (34). To determine whether SP-A enhances phagocytosis of LAM microspheres, we studied mannan-inhibitable phagocytosis of LAM microspheres and control microspheres by MDMs in monolayer culture (40) on an SP-A or HSA substrate (Fig. 4⇓). SP-A significantly enhanced the phagocytosis of LAM microspheres in a mannan-inhibitable fashion (168 ± 63% enhancement, mean ± SEM, p < 0.05, n = 8) compared with the HSA substrate (Fig. 4⇓).

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FIGURE 4.

MDMs on a substrate of APP-SP-A demonstrate enhanced phagocytosis of Erdman LAM microspheres. MDMs were plated on a substrate of either HSA or APP-SP-A. After washing, monolayers were incubated with either HSA (control) or Erdman LAM microspheres (2.0 × 10⁷) in the presence or absence (control) of soluble mannan. After washing, MDMs were fixed in paraformaldehyde, and microsphere uptake was enumerated by phase microscopy. Data are the mean ± SD of triplicate coverslips per test group. Within a given substrate, an asterisk indicates values significantly different from HSA microspheres, p < 0.05 by Student’s t test. Representative experiment of n = 8.

AMs from SP-A^−/− mice express less MR than AMs from SP-A^+/+ mice

To determine whether SP-A up-regulates MR expression on AMs in vivo, we studied the expression of the MR on alveolar and peritoneal macrophages isolated from SP-A^−/− and SP-A^+/+ mice using flow cytometry. Alveolar and peritoneal macrophages from both mouse types constitutively expressed MR (Table III⇓). MR expression on peritoneal macrophages did not differ between mouse types. In contrast, the MR expression (reflected by MFI) on AMs from SP-A^−/− mice was only approximately one-half of the MR expression on AMs from SP-A^+/+ mice (Table III⇓), approaching the level of expression seen on peritoneal macrophages. Thus, this result provides evidence that macrophages exposed to SP-A in the alveolus exhibit up-regulated MR expression and is consistent with our findings using human MDMs in in vitro culture.