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* Veterans Affairs Medical Center and Division of Infectious Diseases, Departments of Medicine and Microbiology, Interdisciplinary Immunology Program, University of Iowa, Iowa City, IA 52242;
Lord and Taylor Laboratory for Lung Biochemistry, Program in Cell Biology, Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine and Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80206;
Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Cincinnati, Cincinnati, OH 45267
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Due to their location in the lung, AMs come in contact with the surfactant monolayer. Pulmonary surfactant is a multimolecular complex composed of proteins and lipids and serves to reduce the surface tension of the alveoli, allowing expansion of the lung during inspiration. Surfactant protein A (SP-A), a member of the collectin family, is the most abundant surfactant-associated protein (7). Monomeric SP-A subunits are 28- to 36-kDa (rat) or 35-kDa (human) polypeptides that contain one (human) or two (rat) N-linked oligosaccharide attachment sites, a hydroxyproline-rich collagen-like domain, and carbohydrate recognition domains (CRDs) (8). Subunits of SP-A assemble into trimers and then further associate into 18 mers composed of six trimers through interchain disulfide bond formation at the N terminus and noncovalent interactions between the collagen-like sequences. The resulting "bouquet of tulips" configuration, containing CRDs arranged on stalk-like scaffolding of collagen tails (9), provides a high valency of binding sites for microbe and host cell molecules (8). Human SP-A isolated from patients with the lung disease alveolar proteinosis (APP-SP-A) forms even larger aggregates by self-association of multiple "bouquets" (10, 11). Mutational analyses have been performed on recombinant rat SP-A synthesized in insect cells. Wild-type recombinant rat SP-A produced in this manner retains the functional properties of the natural protein despite simplified, mannose-rich glycosylation and incomplete proline hydroxylation and oligomeric assembly (8). Studies using recombinant proteins with site-directed mutations have revealed that the attached carbohydrate of SP-A is important for SP-A-mediated phagocytosis of Mycobacterium tuberculosis(M.tb) (12) and that the collagen-like domain is required for complete oligomeric assembly and binding to the SP-A receptor on alveolar epithelial cells (8).
Recent studies using SP-A-/- mice provide evidence that SP-A plays an important role in innate immunity (13). SP-A-/- mice are susceptible to infection with a variety of extracellular bacteria (13, 14, 15, 16). SP-A can interact with both microorganisms and leukocytes in vitro (reviewed in Refs. 7 and 17). Our laboratory has demonstrated that SP-A enhances phagocytosis of the intracellular pathogen M.tb by human macrophages and that this response is mediated through a direct interaction between SP-A and the macrophage (12). SP-A-enhanced phagocytosis of M.tb by macrophages, including human AMs, occurs rapidly (within 1–2 h). Because phagocytosis of M.tb is a receptor-mediated event, one mechanism for the effect of SP-A on macrophages may be to enhance the surface expression and/or function of phagocyte receptors (18, 19). In our prior work, inhibitor studies suggested a role for the macrophage MR in SP-A enhancement of phagocytosis (12). Here we examined the effect of SP-A and its distinct structural components on MR expression and function. Our results demonstrate that SP-A up-regulates surface expression of functional MR, but does not alter complement receptor (CR) expression on human macrophages. Both the collagen region and sugars of SP-A are needed to optimize this effect.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 
-chain of CR3 (20), BU15
recognizes the -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 IgG1
, 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-Ahyp) 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-Ahyp,thr1) or
Asn187 site
(SP-Ahyp,ser187) or at both sites
(SP-Ahyp,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
(TM1-2-3ser187) 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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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 x 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 x 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 x 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 x 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 x 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 2A).
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. 2A and Table I). IL-4 up-regulated MR expression as a
positive control (5) (Fig. 2C 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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B). Incubation with SP-A did not significantly alter
expression of CR4 (Fig. 2B 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. 3A), and exposure to SP-A
for 1 h did not induce MR expression on monocytes
(p > 0.1) (Fig. 3B). In contrast, MDMs
expressed a low level of MR constitutively (Fig. 3C and
Table II), and SP-A markedly increased the expression of MR on MDMs
(Fig. 3D 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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), 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-Ahyp) 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-Ahyp.
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-Ahyp,ser187) or both
(Asn1 and Asn187) sites
(SP-Ahyp,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-Ahyp,ser187) resulted in MR expression
similar to that seen with SP-A devoid of carbohydrate
(SP-Ahyp,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
(TM1-2-3ser187) 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-Ahyp. 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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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AMs originate from circulating monocytes that immigrate into the pulmonary environment, where they encounter SP-A, an interaction that may contribute to the unique biological properties of AMs. SP-A is capable of a direct interaction with AMs through binding to cell surface receptor(s), resulting in modulation of macrophage functions such as enhanced chemotaxis, modified respiratory burst, and increased phagocytosis of microbes and apoptotic cells (7, 42). Here we demonstrate that the interaction between SP-A and human macrophages results in increased expression of the MR.
MR, a member of the C-type lectin super family (4), binds with high affinity to glycoconjugates containing mannose, fucose, or N-acetyl glucosamine. MR is not present on monocytes, but is constitutively present on macrophages including MDMs and dendritic cells (2, 3). AMs express particularly high levels of functional MR. In the current study, SP-A increased MDM expression of MR, as demonstrated by both flow cytometry and confocal microscopy. The newly expressed MR was functional, as determined by mannan-inhibitable pinocytosis of the ligand mannose-BSA and phagocytosis of M.tb LAM-coated particles after SP-A treatment. It is unlikely that the increased uptake of ligands was due to an SP-A-induced change in affinity of the receptor for the ligand because there are no reports of affinity modification of MR as described for CR3 (43).
The effect of SP-A on MR expression was selective for cell type and
receptor type. In contrast to its effect on macrophages, short-term
(60-min) incubation with SP-A did not induce MR expression on
monocytes. MR expression becomes apparent upon monocyte maturation over
3–4 days in culture (3). Thus, expression is
differentiation dependent. The mechanisms for regulation of MR
expression have not been clearly elucidated, but potentially include
transcriptional regulation via the PU.1 site in the MR gene promoter
(44, 45, 46). The effect of SP-A is receptor specific under
the conditions of our assays. SP-A did not affect expression of CRs on
macrophages in our present study or Fc
R function in a previous study
(12). In contrast with our results, Tenner et al.
(18) reported that SP-A increased phagocytosis of both
E-IgG by FcR on monocytes and complement-coated erythrocytes by CR1 on
macrophages. SP-A up-regulation of cell surface expression of FcR or
CR1 was not addressed in their study (18). Additionally,
Kremlev et al. (19) reported that SP-A treatment of the
human monocytic cell line THP-1 significantly increased the levels of
expression of CD14, ICAM-1, and CD11b as measured by flow cytometry.
Possible explanations for these apparent discordant data include the
assay conditions such as the ligand density on the phagocytic particles
or, alternatively, the cell types used. Another difference between the
current study and that by Kremlev et al. (19) is the
method used to purify SP-A. Kremlev et al. (19) used an
isoelectric focusing method after solubilization of APP-SP-A.
MR is synthesized as a 154-kDa precursor, which is processed to its
mature form of 162 kDa in 
90 min. (47, 48).
Permeabilization studies indicate that in differentiated macrophages,
80% of the MR is localized intracellularly in vesicles (49, 50). The intracellular pool may include newly synthesized
receptor en route to the endosomal apparatus and receptors moving from
one pool to another. Newly synthesized MR has a half-life of 33 h
as determined by pulse-chase studies. This indicates that on the
average each molecule recycles between the cell surface and endosomes
hundreds of time before degradation (47).
Viewed as a whole, our data favor the idea that short-term incubation of SP-A increases trafficking of preformed MR to the cell surface. First, increased surface expression of MR in response to SP-A can be seen as early as 1 h. Second, uptake of 125I-labeled mannose-BSA at 37°C, a temperature at which receptor recycling occurs, increased in the presence of SP-A. Third, the addition of CHX before SP-A did not significantly reduce the up-regulation of MR, indicating that new protein synthesis was not necessary for the SP-A induction of MR. Phagocytosis of LAM microspheres is rapid and MR specific. Both surface-expressed MR and preformed intracellular MRs that are recycled to the surface are involved in phagocytosis of LAM microspheres. Thus, the fact that CHX had only a small effect on both MR expression and the phagocytosis of microspheres (40) supports the notion that SP-A primarily affects recycling of preformed MR in macrophages rather than induction of newly synthesized MR. They do not, however, rule out that SP-A could also affect transcription and/or posttranscriptional modification of the MR during longer periods of incubation. MR expression and function is highly regulated (4).
The magnitude of the SP-A-induced MR expression was dependent on the type of SP-A used: APP-SP-A consistently induced a larger increase in expression than did native human or native rat SP-A and a significantly larger increase in expression was induced than did recombinant rat SP-A. This is consistent with our previous finding that APP-SP-A was more effective than native human or recombinant rat SP-A in enhancing phagocytosis of M.tb by MDMs (12). These differences may reflect the degree of oligomerization and/or self-association of SP-A, or the preferential interactions of human cells with human SP-A species. Thus, both the form of SP-A and the nature of the host cell appear to influence the magnitude of MR response to SP-A.
The present study, combined with our previous work (12), shows that the magnitude of MR expression is also dependent on whether SP-A is presented in solution or on a matrix. We speculate that macrophages will encounter SP-A in different contexts within the alveoli, i.e., immigrating as a monocyte from the microvasculature into the alveoli and as a resident macrophage in the alveolus. These functional differences may give clues as to how blood monocytes uniquely differentiate to the AM.
Our data using macrophages from SPA-/- mice support our hypothesis that continuous interactions between alveolar surface lining constituents, particularly SP-A, and AMs affect the phenotype and function of these cells. AMs from the SP-A-/- mice express less MR than do those from control mice, and they phenotypically resemble peritoneal macrophages. In contrast, no differences in MR expression were found in peritoneal macrophages between SP-A-/- and SP-A+/+ mice. The observed differences in MR expression can be attributed to differences in SP-A levels because surfactant isolated from SP-A-deficient mice appears to function normally, and lungs of these mice appear normal histologically and ultrastructurally, except for the absence of tubular myelin (30).
The primary structure of an SP-A subunit is composed of several discrete domains including the following: 1) a short N-terminal segment, 2) a collagen-like sequence of Gly-X-Y repeats, where X is any amino acid and Y is often proline or hydroyproline, 3) a hydrophobic neck domain, and 4) a CRD. This monomer trimerizes by the folding of the collagen-like domains into triple helices (8, 51). Six SP-A trimers form an octadecamer through covalent and noncovalent interactions between the N-terminal segment and the first half of the collagen-like domain (8, 51). Although the structures of APP and native and recombinant SP-A are very similar, subtle differences have been noted that affect the level of protein multimerization. Differences in multimerization may potentially affect receptor binding, signaling, and consequently up-regulation of MR expression. APP-SP-A octadecamers can self-associate to form multimolecular complexes not found in SP-A obtained from healthy volunteers (10, 11). In contrast, recombinant SP-A synthesized in invertebrate cells is deficient in hydroxyproline and does not oligomerize to the same extent as native SP-A. Mutants lacking the collagen region can only form trimers and hexamers, whereas mutants lacking both the N-terminal and collagen regions can form only trimers (8). Neither of these variants was capable of stimulating an optimal increase in MR expression, but the experiments do not discriminate between the direct effects of deletional mutations on SP-A/SP-A receptor interactions (8, 52, 53) and indirect effects due to altered oligomerization (52, 53).
Our studies demonstrate that both the sugars and collagen-like domain of the protein play a role in SP-A induction of MR. SP-A contains two sugars at Asn1 in the N terminus region and Asn187 in the CRD region (8). The carbohydrate moieties of SP-A have been reported to be important for binding to macrophages (12) and herpes simplex virus type 1-infected cells (54), but not to type II epithelial cells (8). Our results indicate that the carbohydrates play a role in SP-A up-regulation of MR expression (12). Although it is true that the insect expression system modifies proteins in a simple, mannose-rich manner, which is different from native SP-A (23), this knowledge does not alter our interpretation of the data. TM2, which contains high mannose oligosaccharides, remains inactive. Collectively, our data argue most strongly for the three-dimensional oligomeric context of the oligosaccharides being important for full SP-A function.
The precise domains of SP-A involved in receptor binding are not well characterized. This may be due to the existence of more than one SP-A receptor, that a receptor complex is involved in binding, and/or that receptor expression varies with cell type (55). Thus, different cell types may vary in their host cell response to SP-A. Several proteins have been identified as putative receptors for SP-A, some of which are present on macrophages (24, 38, 56, 57, 58). Our studies suggest that SP-A binds to its receptor(s) on macrophages and induces the activation of a signal transduction pathway(s) leading to increased MR trafficking to the plasma membrane. The signal transduction pathways involved in this process are the focus of our current studies.
In conclusion, SP-A increases the surface expression of functional MR on macrophages. Up-regulation of the MR by SP-A provides a mechanism for enhanced phagocytosis of invading infectious organisms. Our data indicate that the majority of newly expressed MR comes from the preformed intracellular receptor pool and is induced to traffic to the cell surface by SP-A. The source of SP-A influences the magnitude of MR expressed, and an intact glycoprotein is necessary for optimal SP-A induction of MR expression. In most scenarios, up-regulation of MR expression on a professional phagocyte such as a macrophage would be beneficial for the host by initiating phagocytosis and killing of nonopsonized microbes, thereby participating in first-line host defense. However, intracellular pathogens such as M.tb may use increased levels of MR to reach their intracellular niche, the AM. The current study supports the idea that the alveolar lung constituent, SP-A, contributes to the outcome of lung invasion by inhaled particles and bacteria by modifying the biological properties of AM.
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2 Address correspondence and reprint requests to Dr. Larry S. Schlesinger, Department of Internal Medicine, University of Iowa, 200 Hawkins Drive SE-318 GH, Iowa City, IA 52242. E-mail address: larry-schlesinger{at}uiowa.edu
3 Abbreviations used in this paper: AM, alveolar macrophage; PRR, pattern recognition receptor; MR, mannose receptor; MDM, monocyte-derived macrophage; SP-A, surfactant protein A; CRD, carbohydrate recognition domain; APP, alveolar proteinosis; M.tb, Mycobacterium tuberculosis; CR, complement receptor; HSA, human serum albumin; PMB, polymyxin B; LAM, lipoarabinomannan; NRS, normal rabbit serum; CHX, cycloheximide; MFI, mean fluorescence intensity.
Received for publication April 17, 2002. Accepted for publication July 3, 2002.
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