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* Department of Biochemistry,
Department of Microbiology, and
Third Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan;
Department of Biosciences, School of Science, Kitasato University, Sagamihara, Japan;
¶ Japan Bacillus Calmette-Guérin laboratory, Tokyo, Japan;
|| Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206; and Octothorpe CREST, Japan Science and Technology Agency, Kawaguchi, Japan
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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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secretion by direct interaction with Toll-like receptor 2 on macrophages (21, 22). Lung collectins have also been shown to modulate lung inflammation by interacting with signal inhibitory regulatory protein and calreticulin/CD91 (23). These studies indicate that SP-A and SP-D play crucial roles in the regulation of pulmonary inflammation and host defense of the lung.
Mycobacterium avium is a member of group III nontuberculous mycobacteria and a facultative intracellular pathogen similar to Mycobacterium tuberculosis (M.tb). Attention has been focused on M. avium because the bacteria infects patients suffering from AIDS. The cell wall envelope of M. avium consists of large amounts of fatty acids (mycolic acids) which are covalently linked to an arabinogalactan-peptidoglycan cell wall core and a number of highly unusual lipids and glycolipids, including lipoarabinomannan (LAM), phenolic glycolipids, and glycopeptidolipids (24, 25, 26). During the early phase of the infection, M. avium stimulates secretion of cytokines including TNF-, IL-6, IL-12, and IL-10 (27) and subsequent IFN-
production by NK cells. Recent studies have demonstrated that Toll-like receptor 2 mRNA is induced following infection of murine macrophages with M. avium (28), and SP-A decreases M. avium-elicited NO production by IFN--primed murine alveolar macrophages (29). M. avium enters and can survive in macrophages, although the mechanism of entry into host cells is poorly understood.
M.tb is also a intracellular bacterial pathogen. Both SP-A and SP-D bind M.tb, but the effects of these proteins on the M.tb uptake are different (30, 31). SP-A directly interacts with macrophages and enhances phagocytosis of the virulent Erdman strain of M.tb by up-regulation of mannose receptor (MR) activity (30), and enhances MR expression on monocyte-derived macrophages (MDMs) (32). In contrast, SP-D exhibits reduced adherence of M.tb to MDMs (33, 34).
It is very difficult to manage M. avium infection because of the ineffective nature of the cell-mediated immune response by immunosuppressed hosts and the intrinsic drug resistance of the bacteria. Since the interaction of M. avium with the collectins and its uptake into macrophages are not well understood, we examined these processes. The purposes of this study were to determine 1) the direct binding of the collectins to M. avium, 2) the effect of these proteins on the phagocytosis of M.avium by mononuclear phagocytes, and 3) the role of the proteins in altering phagocytic receptor activity. Our findings demonstrate that SP-A, SP-D, and MBL bind to M. avium and that SP-A and SP-D but not MBL enhance the phagocytosis of M. avium by macrophages through up-regulating MR activity.
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Materials and Methods |
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Human SP-A and SP-D derived from bronchoalveolar lavage fluid of an alveolar proteinosis patient were purified by affinity chromatography on mannose-Sepharose 6B, followed by gel filtration as described previously (35, 36). Rat SP-A and SP-D were also purified from the lung lavage fluids of Sprague Dawley rats that had been given intratracheal instillation of silica (37) by the method described elsewhere (35, 38). Recombinant wild-type (wt) rat SP-A, a mutant SP-A (SP-AE195Q, R197D), and recombinant rat MBL (mannose-binding protein A) were expressed by the baculovirus-insect cell expression system and purified as described previously (39, 40).
Isolation of rat alveolar macrophages
Alveolar macrophages were isolated from bronchoalveolar lavage fluids of Sprague Dawley rats by lavaging the lungs with pyrogen-free saline (Otsuka Pharmaceutical, Tokyo Japan) and sedimenting the cells at 150 x g for 10 min.
Isolation of human macrophages
Human MDMs were isolated from whole blood buffy coats obtained from healthy volunteers (Hokkaido Red Cross Blood Center, Sapporo, Japan) according to the method of Ferguson et al. (33). Mononuclear cells were isolated from heparinized blood on Ficoll gradients and the MDM fraction was purified by adherence. The MDMs were cultured in Teflon wells (Savillex, Minnetonka, MN) for 5 days in the presence of 10% pooled AB+ human serum (Sigma-Aldrich, St. Louis, MO).
Growth and preparation of bacteria
M. avium was obtained from sputum of an infected patient. The bacteria were cultured for 14 days in Mycobroth (Kyokuto Pharmaceutical, Tokyo, Japan) and were then heat-killed at 90°C for 5 min. The bacterial suspension was centrifuged at 2500 x g and the pellet was resuspended in PBS. The concentration of bacteria was adjusted at 0.4–1.0 x 108 CFU/ml by measuring absorbance at 600 nm and the suspension was stored at –80°C until its use.
Labeling of M. avium with FITC
FITC- labeling of heat-treated M. avium was performed by a method based on that described by Tino and Wright (41). Briefly, a 1 ml-suspension of M. avium in 0.1 M sodium carbonate (pH 9.0) was mixed with 1 µl of FITC (10 mg/ml in DMSO; Molecular Probes, Eugene, OR) and incubated for 1 h in the dark, at room temperature, with gentle agitation. FITC-labeled bacteria were washed by centrifugation in PBS and diluted to 1.0 x 109 CFU/ml and stored at –80°C.
Extraction of crude LAM
Extraction of LAM from the M. avium-intracellular complex (MAC) was performed according to the method of Khoo et al. (42). Briefly, cells (10 g wet weight) were suspended in PBS containing 2% (v/v) Triton X-114 (PBS/Triton X-114) and were disrupted by probe sonication. The suspension was centrifuged at 27,000 x g for 1 h at 4°C. The cell pellet was suspended with PBS/Triton X-114 and centrifuged as described above. The combined supernatant was placed at 37°C to initiate biphase formation and centrifuged at 12,000 x g for 15 min at room temperature. The aqueous layer was re-extracted and the detergent layer was washed with PBS. The final detergent layers were precipitated with 9 volumes of acetone. The acetone precipitates were dried under a stream of nitrogen and partitioned between phenol and water. The aqueous layer containing LAM and lipomannan (LM) was freeze dried after dialysis against water. The final fraction is referred to as crude LAM.
Binding of collectins to M. avium
Rat SP-A was iodinated according to the method of Bolton and Hunter (43) using Bolton-Hunter reagent (Amersham Biosciences, Arlington Heights. IL). The specific radioactivity was typically 200–400 cpm/ng, and >85% of the radioactivity was precipitated by treatment with 10% (w/v) trichloroacetic acid. The binding of 125I-SP-A to M. avium was examined using microtiter wells. The bacterial suspensions (1.0 x 106 CFU in 40 µl of PBS/well) were put onto the wells, dried under a vacuum, and then fixed by incubating the wells with PBS containing 0.25% glutaraldehyde. After blocking with PBS (pH 7.4) containing 100 mM glycine, nonspecific binding to the wells was blocked with 20 mM Tris buffer (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl2, and 2% (w/v) BSA (blocking buffer). The indicated concentration of 125I-SP-A (50 µl/well) in blocking buffer was added to the wells with bacteria and incubated for 1 h at 37°C. The wells were washed three times with blocking buffer and the 125I-SP-A specifically bound to the bacteria was solubilized in 200 µl of 0.1 M NaOH. The radioactivity of 125I-SP-A recovered was finally determined by using a gamma counter.
Binding experiments were also performed using specific Abs to detect the recombinant SP-As, SP-D, and MBL. SP-A, SP-D, or MBL (50 µl/well) in blocking buffer was incubated with M. avium coated onto the microtiter wells. The wells were washed with PBS (pH 7.4) containing 0.1% (v/v) Triton X-100 (buffer A). Subsequently, either HRP-labeled anti-rat SP-A IgG (1/250), or anti-rat SP-D IgG (1/50) or anti-rat MBL IgG in PBS (pH 7.4) containing 0.1% (v/v) Triton X-100 and 3% (w/v) skim milk were added and incubated for 1 h at 37°C. After washing the wells with buffer A, the peroxidase reaction was performed using o-phenylenediamine as a substrate. The binding of the collectins to M. avium was quantified by measuring absorbance at 492 nm.
In some experiments, the wells were incubated with the indicated concentrations of the collectins in the absence or the presence of 5 mM EDTA to determine Ca2+-dependent binding. Where indicated, unlabeled rat SP-A (20–200 µg/ml), crude LAM (0.1–100 µg/ml) from MAC or 50 µg/ml mAbs or mouse control IgG were added as selective inhibitors of specific binding.
Phagocytosis of M. avium by macrophages
Rat alveolar macrophages (2.0 x 105) were incubated with FITC-labeled M. avium (1.0 x 107 CFU) in 200 µl of HBSS (Life Technologies, Grand Island, NY) in the absence or the presence of the indicated concentrations of the collectins for 30 min at 37°C. The assay was stopped by the addition of ice-cold PBS to the macrophage-bacteria suspension. The bacteria that had not been associated with the cells were separated from cell-associated bacteria by centrifugation at 150 x g. The cell pellet was washed three times with PBS. The cells were next suspended with 5 µl of 25 µg/ml ethidium bromide in PBS and pipetted onto a slide. The number of macrophages with or without intracellular (green fluorescent) bacteria were counted in at least 100 macrophages per slide for duplicate samples using fluorescence microscopy at x200 magnification. The results were expressed as the percentage of total macrophages that contained intracellular bacteria. In some experiments, the phagocytosis assay was performed in the absence or the presence of 2 mM EDTA, 4 mg/ml mannan, 50 µg/ml crude LAM, or 10–100 µg/ml zymosan. To determine whether new protein synthesis was involved in collectin-enhanced phagocytosis, 10 µg/ml cycloheximide (Sigma-Aldrich) was preincubated with the macrophages for 60 min at 37°C before the addition of the collectin and the bacteria.
The analysis of phagocytosis was also conducted using MDMs. MDMs were plated onto glass coverslips in 24-well plates (Falcon; Costar, Cambridge, MA) for 2 h at 37°C (2.0 x 105/coverslip). The wells were washed with RPMI 1640 and then incubated with FITC-labeled M. avium (1.0 x 107 CFU) in 200 µl of HBSS in the absence or presence of the indicated concentrations of human SP-A, rat SP-D, recombinant rat SP-A, or recombinant rat MBL for 1 h at 37°C in a tissue culture incubator. After the incubation, the cells were washed with PBS, stained with ethidium bromide, and M. avium phagocytosis was evaluated as described above.
Uptake of 125I-mannosylated BSA by rat alveolar macrophages
Mannosylated BSA was iodinated according to the method of Bolton and Hunter (43) using Bolton-Hunter reagent (Amersham Biosciences). The specific radioactivity ranged from 51 to 57 cpm/ng, and >96% of the radioactivity was precipitated by treatment with 10% (w/v) trichloroacetic acid. Rat alveolar macrophages (2 x 105/well) were seeded onto 48-well plates (Falcon) for 2 h in RPMI 1640 containing 10% (v/v) FCS (RPMI 1640/FCS). The medium was removed and each monolayer was washed with RPMI 1640/FCS. Next, rat SP-A (0–20 µg/ml) or SP-D (5 µg/ml) in RPMI 1640 containing 1 mg/ml BSA (RPMI 1640/BSA) was incubated with the cells for 1 h at 37°C. After the incubation with the collectins, the cells were washed with RPMI 1640/BSA. Subsequently, the indicated concentration of 125I-mannosylated BSA (200 µl/well) in RPMI 1640/BSA was added, followed by further incubation for 30 min at 37°C. The monolayers were then washed with 50 mM Tris (pH 7.4) containing 0.15 M NaCl and 2 mg/ml BSA. A final wash was performed with 50 mM Tris (pH 7.4) containing 0.15 M NaCl, and the macrophages were removed from the wells by dissolution in 0.1 M NaOH. The amount of 125I-mannosylated BSA associated with the cells was determined using a gamma counter.
Immunostaining of the MR and fluorescence microscopy
MDMs seeded onto glass coverslips were incubated with or without human SP-A (20 µg/ml) or human SP-D (5 µg/ml) for 1 h at 37°C. After the incubation, the cells were washed with PBS, and the cell monolayers were fixed with 4% (w/v) paraformaldehyde for 10 min at room temperature. The cells were then washed with 50 mM PIPES buffer (pH 7.2) containing 100 mM NaCl, 1 mM EGTA, 2 mM MgCl2, and 0.5% (w/v) BSA (buffer B) and were incubated with monoclonal anti-human MR Ab (clone 19.2, 1/100; BD PharMingen, San Diego, CA) in buffer B for 30 min at room temperature. The cells were then washed with buffer B and further incubated with Alexa 488-conjugated anti-mouse IgG (1/500; Molecular Probes) for 45 min at room temperature. The cells were finally washed with buffer B, sealed in the presence of Vectorshield Antifade (Vector Laboratories, Burlingame, CA), and examined using a laser microscope (LSM510; Zeiss, Tokyo, Japan) with a x63 oil planapochromatic lens (NA 1.4).
Binding of collectins to crude LAM isolated from MAC
Crude LAM was isolated from MAC as described above and was electrophoresed and transferred onto the nitrocellulose membrane. The membrane was overlayed and incubated with 5 µg/ml rat SP-A, SP-D, or BSA for 3 h at room temperature after blocking the membrane with buffer A containing 3% (w/v) skim milk. The protein binding to LAM and LM was detected by Ab to each protein followed by the incubation with HRP-labeled anti-rabbit IgG.
The binding of SP-D to crude LAM was also performed by using crude LAM (1 µg/well) coated onto microtiter wells. The wells were incubated with the indicated concentrations of rat SP-D in the presence of 5 mM CaCl2 or 5 mM EDTA for 3 h at 37°C. The SP-D binding to crude LAM was detected by anti-SP-D Ab.
Flow cytometry
MDMs (1.0 x 106) were incubated with or without 20 µg/ml human SP-A, human SP-D, or MBL. After washing the cells with PBS, they were fixed with 1% (w/v) paraformaldehyde. The cells were then washed and incubated on ice for 45 min with PE-conjugated mouse anti-human MR mAb (clone 19.2; BD PharMingen) or PE-conjugated mouse IgG1, 
monoclonal Ig isotype control (clone MOPC-31C; BD PharMingen) in PBS containing 2% FCS (v/v) and 0.1% (w/v) sodium azide. The cells were finally washed and analyzed by using FACSCalibur and CellQuest software (BD Biosciences).
Phagocytosis of zymosan
Rat alveolar macrophages (2.0 x 105) were incubated at 37°C for 30 min with or without 20 µg/ml rat SP-A or 5 µg/ml rat SP-D in the absence or presence of 2 mM EDTA or 4 mg/ml mannan. The cell suspension was further incubated with FITC-conjugated zymosan A (Molecular Probes) at 37°C for 30 min. The cells were then washed with PBS, stained with ethidium bromide, and the phagocytosis was evaluated as described above.
Uptake of FITC-conjugated mannosylated BSA by rat alveolar macrophages
Rat alveolar macrophages (1.0 x 105/well) were seeded onto 96-well plates (Matrix Technologies, Hudson, NH) and incubated for 3 h in RPMI 1640 containing 10% (v/v) FCS. After the incubation, the cells were washed with the medium and were incubated at 37°C for 30 min with the indicated concentrations of FITC-conjugated mannosylated BSA (Sigma-Aldrich) in the absence or presence of rat SP-A (20 µg/ml) or rat SP-D (5 µg/ml) in HBSS containing 1 mg/ml BSA. In some experiments, 2 mM EDTA was included in the incubation buffer. The cells were then washed three times with 50 mM Tris buffer (pH 7.4) containing 0.15 M NaCl and 2 mg/ml BSA and the final wash was performed by using Tris buffer without BSA. The fluorescence intensities of FITC-mannosylated BSA associated with the cells were measured at 485 nm (excitation) and 528 nm (emission) using Synergy HT and KC4 software (Bio-Tek Instruments, Watford Herts, U.K.). Specific uptake of mannosylated BSA was calculated by subtracting the fluorescence intensity of the FITC-labeled protein binding to the wells without cells from total fluorescence intensity.
Binding of FITC-conjugated mannosylated BSA and FITC-labeled M. avium to rat alveolar macrophages
Rat alveolar macrophages (5.0 x 105) were incubated with or without rat SP-A (20 µg/ml) at 4°C for 4 h in HBSS containing 1 mg/ml BSA. The cell suspension was washed three times with PBS and incubated at 4°C for 1 h with FITC-mannosylated BSA (5 µg/ml) or FITC-labeled M. avium (5.0 x 106 CFU) in HBSS containing 1 mg/ml BSA. After washing the cells with PBS, the cell suspension was resuspended with 200 µl of PBS containing 1% (w/v) paraformaldehyde and put onto 96-well plates (Matrix Technologies). The fluorescence intensities of FITC-mannosylated BSA or FITC-labeled M. avium binding to the cells were finally determined using Synergy HT and KC4 software (Bio-Tek Instruments) as described above.
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Results |
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We first examined the binding of SP-A to M. avium coated onto microtiter wells by using 125I-labeled rat SP-A.125I-SP-A bound to M. avium in a concentration-dependent manner (Fig. 1A). The Scatchard plot analysis (Fig. 1A, inset) gives an approximate dissociation constant of 32.6 ± 2.34 nM (n = 3, mean ± SE), assuming that the molecular mass of SP-A is 0.64 MDa (2). Eighty-six nanograms of SP-A was capable of binding to 106 CFU of bacteria. Inclusion of EDTA in the binding buffer almost completely abolished the binding of SP-A to M. avium (Fig. 1B). Unlabeled SP-A competed well with 125I-labeled SP-A for the binding to M. avium (Fig. 1C), indicating the specific binding of SP-A to M. avium. One hundred-fold excess unlabeled SP-A inhibited 125I-SP-A binding by 86.81 ± 2.52% (n = 3, mean ± SE). To further characterize the SP-A binding to M. avium, we examined the effect of crude LAM containing LAM and LM upon the interaction. LAM and LM are major cell wall-associated lipoglycans, which were isolated from MAC. Excess LAM failed to attenuate the binding of SP-A to M. avium (Fig. 1D), indicating that LAM is not a ligand for SP-A on M. avium. This was consistent with the result obtained from a ligand blot showing that no SP-A protein was detected on LAM and LM that were transferred onto the nitrocellulose membrane (Fig. 2C). The effect of anti-rat SP-A mAbs on the rat SP-A binding to the bacteria was also examined (Fig. 1E). Ab 1D6 completely blocked the SP-A binding to M. avium although Ab 6E3 decreased its binding to a significant extent. In contrast, control mouse IgG or anti-human SP-A mAb, PE10, did not affect the binding of rat SP-A to M. avium. Since the epitopes for Abs 1D6 and 6E3 are localized at the carbohydrate recognition domain (CRD) and the neck domain of rat SP-A, respectively (44), these data suggest that SP-A binds to M. avium through its CRD. This result is also consistent with the Ca2+ dependence of binding since most CRD interactions require Ca2+.
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SP-A and SP-D enhance the phagocytosis of M. avium by macrophages
We next examined the effects of SP-A and SP-D on the phagocytosis of M. avium by macrophages. Rat alveolar macrophages (2 x 105) were incubated with FITC-labeled M. avium for 30 min in the absence or presence of rat SP-A or SP-D (Fig. 3, A and B). Both SP-A and SP-D enhanced the phagocytosis of M. avium by rat alveolar macrophages in a concentration-dependent manner. SP-A at 20 µg/ml increased the number of cells phagocytosing the bacteria from 13 to 32% of the population. SP-D at 5 µg/ml also increased the uptake of M. avium from 9 to 16% of the population. When 200 ng/ml LPS was incubated with alveolar macrophages in the presence of FITC-labeled M. avium, the uptake of M. avium was not augmented. In addition, the endotoxin-free SP-A, that had been treated with polymyxin B in the presence of octyl-
-D-glucoside (20) augmented M. avium phagocytosis at a level comparable to nontreated SP-A, 32.2 ± 5.6% (mean ± SE, n = 3) and 32.2 ± 2.7% for polymyxin-treated SP-A and nontreated SP-A, respectively. These results suggest that the effects of SP-A and SP-D are not due to the endotoxin contamination in the protein preparations. We also examined the effects of lung collectins on M. avium phagocytosis by human MDMs. Coincubation of human SP-A or rat SP-D with MDM monolayers and FITC-labeled M. avium exhibited a concentration-dependent stimulation of M. avium phagocytosis (Fig. 3, C and D). Human SP-A at 20 µg/ml increased the uptake of M. avium from the background level of 8% to the stimulated level of 18%. Rat SP-D at 5 µg/ml augmented the phagocytosis from 10 to 25%.
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Another collectin homologue, MBL, was also examined for M. avium binding and macrophage uptake of the bacteria. Recombinant rat MBL exhibited a concentration-dependent binding to M. avium (Fig. 4A). Its binding was not inhibitable by the presence of EDTA, indicating the Ca2+-independent binding of MBL to the bacteria. Excess crude LAM did not significantly inhibit the MBL binding to M. avium (Fig. 4B). When MDMs were incubated with FITC-labeled M. avium in the presence of 20 µg/ml MBL, the uptake of the bacteria was not significantly increased (Fig. 4C), whereas the presence of the pulmonary collectins in the same experiment significantly increased phagocytosis of the bacteria.
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To characterize SP-A- or SP-D-stimulated phagocytosis of M. avium, the phagocytosis assays were conducted under various conditions. Inclusion of 2 mM EDTA instead of CaCl2 did not block the SP-A-enhanced phagocytosis of M. avium by rat alveolar macrophages (Fig. 5A). Since the binding of SP-A to M. avium is blocked in the presence of EDTA (Fig. 1B), the results indicate that the SP-A-stimulated phagocytosis of M. avium can occur independently of its binding to the bacteria. EDTA also failed to block the SP-D-stimulated uptake of M. avium (Fig. 5D).
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Thus, we determined whether the preincubation of macrophages with the lung collectins stimulated M. avium phagocytosis. Native and wt rat SP-As and SP-AE195Q, R197D augmented the phagocytosis of M. avium by alveolar macrophages (Fig. 7A), even after the cells were washed to remove the proteins. Preincubation of SP-D with alveolar macrophages also stimulated M. avium uptake to a level comparable to that obtained by coincubation of SP-D and the bacteria with the phagocytes (Fig. 7B). These results indicate that interaction of the collectins with macrophages elicits the stimulation of M. avium phagocytosis. In addition, Ab experiments suggest that the CRDs or the neck domain of SP-A may not be involved in the SP-A-macrophage interaction. The effect of cycloheximide on the phagocytosis was also examined. SP-A and SP-D retained the activity of stimulating phagocytosis of M. avium even in the presence of 10 µg/ml cycloheximide (Fig. 7C), suggesting that new protein synthesis is not involved in the stimulatory effects of lung collectins on M. avium phagocytosis.
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SP-A and SP-D enhance macrophage MR activity
Since SP-A and SP-D enhance MR-mediated phagocytosis of M. avium by direct interaction with macrophages as shown above, we investigated whether these proteins alter MR activity by using 125I-mannosylated BSA as a ligand for MR. Rat alveolar macrophages were preincubated in the absence or presence of 20 µg/ml rat SP-A or 5 µg/ml rat SP-D for 1 h. After washing, the cells were further incubated with the indicated concentrations of 125I-mannosylated BSA for 30 min. The uptake of mannosylated BSA by rat alveolar macrophages was concentration dependent (Fig. 8A). Both SP-A and SP-D increased the uptake of mannosylated BSA when compared with that without collectin preincubation. SP-A enhanced mannosylated BSA uptake by 94 ± 28% (mean ± SE, n = 3, p < 0.02) at 10 µg/ml 125I-mannosylated BSA, when compared with the control. SP-D had an almost identical effect. When rat alveolar macrophages were incubated with FITC-labeled mannosylated BSA in the presence of collectins and EDTA, the fluorescence intensities of the proteins associated with the cells were almost completely diminished (Fig. 8B). These results are consistent with the characteristics of MR sharing homology with C-type lectins (49). Taken together, these data demonstrate that SP-A and SP-D directly interact with macrophages and up-regulate MR activity.
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Alveolar macrophages were first preincubated with 20 µg/ml SP-A and were then washed as described in Fig. 8A legend. In our previous report (20), only 0.5 ng/ml unbound SP-A was found to be detectable in the medium after the cells were incubated with 20 µg/ml 125I-SP-A, washed, and replaced with fresh medium, indicating that most of the collectin had been removed by washing.
In addition, the binding experiments with FITC-labeled mannosylated BSA and FITC-labeled M. avium to rat alveolar macrophages were performed at 4°C after the cells were preincubated with 20 µg/ml rat SP-A at 4°C, washed, and replaced with the medium containing mannosylated BSA or M. avium. The fluorescence intensity of mannosylated BSA that bound to the cell surface with or without SP-A treatment was 1.91 ± 0.07 (mean ± SE, n = 3) or 1.19 ± 0.24, respectively. The fluorescence intensity of M. avium that bound to the cell surface with or without SP-A treatment was 11.43 ± 1.69 (mean ± SE, n = 3) or 12.26 ± 3.06, respectively. These results indicate that the amounts of mannosylated BSA or M. avium binding to the SP-A-treated cells are not different from those binding to the untreated cells when the cells were preincubated with SP-A at 4°C. Thus, it is unlikely that the increased association of mannosylated BSA shown in Fig. 8A is due to increased binding of the mannosylated BSA to the collectins that bound to the cell surface of macrophages.
SP-A and SP-D increase cell surface localization of MR on MDMs
We next examined whether SP-A and SP-D enhance MR expression on the MDM cell surface. Human MDMs were first incubated in the absence or presence of 20 µg/ml human SP-A or 5 µg/ml human SP-D for 1 h. After washing, the cells were fixed in paraformaldehyde and immunostained with anti-MR mAb and then analyzed using a confocal microscope. Both SP-A and SP-D up-regulated expression of the MR on MDMs when compared with control (Fig. 9), demonstrating that SP-A and SP-D up-regulate MR expression on MDMs. We further assessed the cell surface expression of MR on MDMs by flow cytometry. MR was constitutively expressed on cell surfaces of MDMs (Fig. 10, gray line). After exposure of SP-A and SP-D, cell surface expression of MR on MDMs was enhanced (Fig. 10, B and C, solid black line). These results are consistent with those obtained from the uptake experiments with 125I-mannosylated BSA. The data confirm a previous study (32) indicating the MR up-regulation by SP-A and demonstrate for the first time that SP-D increases the MR activity. MBL failed to increase the MR expression on MDMs (Fig. 10A), consistent with the finding that this collectin did not stimulate M. avium phagocytosis (Fig. 4). Taken together, these results support the idea that lung collectins enhance M. avium phagocytosis by increased MR activity on macrophages.
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Discussion |
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Competition experiments reveal that LAM derived from MAC fails to compete for SP-A binding to M. avium but blocks M. avium phagocytosis. Inclusion of EDTA, which abolishes the binding of SP-A to M. avium, does not affect the stimulatory effect of this collectin on M. avium phagocytosis. Recombinant rat SP-AE195Q, R197D, which exhibits the reduced binding to M. avium, still potently enhances the phagocytosis. In addition, anti-rat SP-A mAb 1D6, which completely blocks the SP-A binding to M. avium, fails to inhibit SP-A-stimulated phagocytosis. Collectively, these findings demonstrate that SP-A binding to M. avium does not parallel the collectin-mediated stimulation of M. avium phagocytosis. This conclusion is consistent with the results obtained from experiments, in which preincubation of lung collectins with macrophages enhances M. avium phagocytosis. These results support the idea that direct interaction of SP-A and SP-D with the macrophages induces the stimulated phagocytosis.
In this study, both SP-A and SP-D enhance the phagocytosis of M. avium by alveolar macrophages and MDMs. The phagocytosis of mycobacteria has been shown to be mediated by a variety of phagocytic receptors including complement receptor (CR) 1, CR3, CR4, MR, and scavenger receptors (46, 50, 51, 52). Because the inclusion of mannan, zymosan, or M. avium LAM blocks the stimulatory effects of the collectins on M. avium uptake, the proteins have been proposed to stimulate MR-mediated phagocytosis. This is consistent with the results that lung collectins up-regulate the MR activity. In addition to the previous study describing the up-regulation of MR by SP-A (32), we now show that SP-D also induces cell surface expression of MR on macrophages. The up-regulation of MR activity has been confirmed by uptake experiments with mannosylated BSA using the collectin-treated macrophages. Since the presence of cycloheximide did not inhibit the collectin-stimulated phagocytosis of M. avium, new protein synthesis is not involved in the effects by SP-A and SP-D. In addition, the stimulatory effects of M. avium uptake are specific for the lung collectins because MBL failed to stimulate M. avium phagocytosis.
Analysis by confocal microscopy and flow cytometry has revealed that lung collectins enhance cell surface expression of MR (Figs. 9 and 10). These proteins also stimulate the uptake of mannosylated BSA by alveolar macrophages, which is completely diminished in the presence of EDTA (Fig. 8, A and B). These results are consistent with the idea that lung collectins enhance MR activity, since EDTA is a potent inhibitor of MR (49). In contrast, the uptake of zymosan is partially EDTA-resistant although some of the uptake is attenuated in the presence of EDTA (Fig. 8C). Likewise, the collectin-stimulated phagocytosis of M. avium is attenuated but is not completely blocked by EDTA (Fig. 5), suggesting the additional mechanisms may be involved in the phagocytosis of zymosan and mycobacterium in addition to the MR-involved mechanism. Since a variety of phagocytic receptors including MR, CRs, scavenger receptors, glucan receptors, and dectin-1 (50, 51, 52, 53, 54, 55, 56) mediate the phagocytosis of mycobacterium and zymosan, it is possible to assume that lung collectin may be involved in other mechanisms in addition to MR-mediated phagocytosis.
The mutant collectin, SP-AE195Q, R197D exhibits decreased binding to M. avium but stimulated phagocytosis to a level comparable to wt SP-A (Fig. 6). In addition, enhanced phagocytosis by macrophages persists even after preincubation and removal of lung collectins (Fig. 7). Thus, it is likely that lung collectin does not serve as an opsonin for the phagocytosis of M. avium. In contrast, opsonic functions of lung collectins in phagocytosis of HSV, H. influenzae, and Klebsiella pneumoniae have been shown (11, 12, 13). Taken together, these studies support the idea that lung collectins may stimulate the phagocytosis by two mechanisms, one of which by activating macrophages and the other by serving as an opsonin.
Ferguson et al. (33) have reported that coincubation of M.tb with SP-D reduces adherence of the bacteria to macrophages, whereas preincubation of SP-D with the macrophages does not affect bacterial adherence. They have also shown that SP-D binds to LAM on the surface of Erdman M.tb via the mannose cap on Erdman LAM and conclude that SP-D-M.tb interaction reduces adherence of M.tb to monocytes. In this study, both coincubation and preincubation of SP-D with M. avium provide consistent results of increased phagocytosis, which is consistent with the result that SP-D-treated macrophages exhibit higher expression of MR. One possible explanation of the difference between these earlier studies and this work is that different binding mechanisms of SP-D between M.tb and M. avium, due to the structural difference of LAM between these mycobacterium (42), may result in the distinct effects on macrophage interaction. Another possibility may be because of the different assay systems. We have discriminated between the intracellular bacteria and the cell surface-associated bacteria by quenching FITC-labeled M. avium with ethidium bromide.
SP-AE195Q, R197D has been shown to be inactive in the regulation of surfactant secretion and lipid uptake by alveolar type II cells and receptor binding on these cells (39). In addition, anti-SP-A Ab 1D6, whose epitope is located at the CRD (44), has previously been reported to block the inhibitory effect of SP-A on lipid secretion from type II cells, the receptor-binding activity of SP-A on type II cells, and the SP-A-stimulated lipid uptake by type II cells (44, 45, 57). These previous studies indicate that the CRD of SP-A is responsible for the SP-A action on alveolar type II cells. In contrast, SP-AE195Q, R197D stimulated M. avium phagocytosis by macrophages at a level comparable to that of wt SP-A, although this mutant exhibits significant decreased binding to M. avium. Ab 1D6 failed to inhibit the SP-A-enhanced phagocytosis of M. avium by alveolar macrophages as shown in this study. Thus, the structural requirement for SP-A-macrophage interaction in M. avium uptake is different from that for SP-A-type II cell interaction in regulating lipid secretion and uptake and receptor binding.
One recent study (32) has shown that SP-A up-regulates MR activity on MDMs and that alveolar macrophages from SP-A–/– mice have reduced MR expression relative to SP-A+/+. In this study, confocal microscopy has revealed that SP-D as well as SP-A enhances MR activity on MDMs. This finding is also confirmed by the collectin-induced increase in uptake of mannosylated BSA by alveolar macrophages. The up-regulation of MR by lung collectins provides a mechanism for enhanced phagocytosis of M. avium since the collectin-stimulated phagocytosis of the bacteria is abolished in the presence of LAM, mannan, or zymosan, which are all ligands for the MR (46, 47, 48). Eighty percent of the MR is localized in intracellular vesicles and the protein recycles between the intracellular pools and the cell surface (58, 59). Enhanced expression of MR on MDMs and increased uptake of mannosylated BSA by alveolar macrophages have been observed after 1-h incubation of the collectin with the cells. In addition, the presence of cycloheximide does not affect the SP-A- or SP-D-stimulated M. avium phagocytosis. Thus, it is likely that the lung collectins may stimulate the receptor recycling en route to the plasma membrane rather than new receptor synthesis at either the transcriptional or translational level. The precise mechanism by which SP-A and SP-D up-regulate the MR activity remains to be elucidated.
HIV-infected individuals are at risk for opportunistic infections with M.tb, M. avium, or Pneumocystis carinii. Increased recovery of SP-A has been reported in bronchoalveolar lavage fluids from HIV-infected patients with P. carinii (60). The increased SP-A level in bronchoalveolar lavage fluids from HIV-infected individuals is closely associated with significant enhancement of M.tb attachment to alveolar macrophages and is correlated with the severity of HIV disease (61). The present and other studies (30) have shown that lung collectins mediate enhanced phagocytosis of M. avium and M.tb. Once internalized, mycobacterium resides in a membrane-bound vacuole that is resistant to lysosomal fusion (49). In addition, SP-A decreases NO production by macrophages infected with M. avium (29), which may be a pathway for this bacteria to escape from the bactericidal mechanisms of the host. Thus, lung collectin may play a role in the early phase of M. avium infection. Increased SP-A levels in HIV patients, the SP-A-enhanced phagocytosis of M. avium, and the SP-A-mediated suppression of NO may explain the increased incidence of mycobacterial infection in HIV-infected individuals.
This study clearly demonstrates that both SP-A and SP-D enhances the phagocytosis of M. avium by alveolar macrophages through stimulating the mannose receptor activity. Because M. avium can survive in macrophages, the collectin-mediated entry of the bacteria into the host cells may be an important pathway to escape bactericidal killing. In conclusion, this study demonstrates that the collectins bind to M. avium and that SP-A and SP-D but not MBL enhance the phagocytosis of M. avium by macrophages through up-regulation of MR activity. These findings provide a significant new mechanism by which lung collectins can regulate the host response to M. avium infection.
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2 Address correspondence and reprint requests to Dr. Yoshio Kuroki, Department of Biochemistry, Sapporo Medical University School of Medicine, Souh-1 West-17, Chuo-ku, Sapporo 060-8556, Japan. E-mail address: kurokiy{at}sapmed.ac.jp
3 Abbreviations used in this paper: SP-A, surfactant protein A; SP-D, surfactant protein D; MBL, mannose-binding lectin; wt, wild type; MDM, monocyte-derived macrophage; LAM, lipoarabinomannan; LM, lipomannan; MAC, M. avium-intracellular complex; M.tb, Mycobacterium tuberculosis; MR, mannose receptor; CRD, carbohydrate recognition domain; CR, complement receptor.
Received for publication January 22, 2004. Accepted for publication April 2, 2004.
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secretion in U937 cells and alveolar macrophages by direct interaction with Toll-like receptor 2. J. Biol. Chem. 277:6830.B activation and TNF- secretion are down-regulated by lung collectin surfactant protein A. J. Immunol. 171:417. or calreticulin/CD91, lung collectin act as dual function surveillance molecules to suppress or enhance inflammation. Cell 115:13.[Medline]
-dependent mechanism. Am. J. Respir. Cell Mol. Biol. 28:520.
). The radioactivity bound to the wells was determined using a gamma counter as described in Materials and Methods. The results represent the specific binding calculated by subtracting the amounts of the labeled protein binding to the wells without the bacteria from total binding. The data shown are the mean ± SE of three experiments. *, p < 0.01 when compared with the binding in the presence of EDTA. The inset indicates a Scatchard plot of the binding data. C and D, 125I-rat SP-A (2 µg/ml) was incubated at 37°C for 1 h with M. avium coated onto microtiter wells in the presence of the indicated concentrations of unlabeled SP-A (C) or crude LAM (D) isolated from MAC. The amount of 125I-SP-A binding to M. avium was determined as described above. The data shown are the mean + SE of three experiments. *, p < 0.01 and **, p < 0.05 when compared with the values obtained without unlabeled SP-A. E, 125I-rat SP-A (2 µg/ml) was incubated at 37°C for 1 h with M. avium coated onto microtiter wells in the absence or presence of 50 µg/ml control mouse IgG, anti-human SP-A mAb PE10, anti-rat SP-A mAbs 1D6, and 6E3. The amount of 125I-SP-A binding to M. avium was determined as described above. The data shown are the mean + SE of three experiments. *, p < 0.01 when compared with the binding in the presence of control mouse IgG.
) at 37°C for 1 h. The binding of SP-A to M. avium was detected using anti-SP-A IgG as described in Materials and Methods. The data shown are the mean ± SE of three experiments. *, p < 0.01 when compared with the binding of wt SP-A at each concentration. B, Rat alveolar macrophages (2 x 105) were incubated with FITC-labeled M. avium at 37°C for 30 min in the absence or presence of the indicated concentrations of rat native SP-A (native) or wt SP-A (wt) or SP-AE195Q, R197D (E195Q, R197D). The percent phagocytosis of M. avium was determined as described in Materials and Methods. The data shown are the mean + SE of three experiments. *, p < 0.01 and **, p < 0.05 when compared with the incubation without SP-As. C, Rat SP-A (20 µg/ml) was preincubated with 50 µg/ml anti-rat SP-A mAb 1D6, or 6E3, or control mAb PE10 at 37°C for 1 h, and the mixture was further incubated with rat alveolar macrophages and FITC-labeled M. avium at 37°C for 30 min. Phagocytosis of M. avium was evaluated as described in Materials and Methods. The data shown are the mean + SE of three experiments.
