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• W2136558272 abstract "Pulmonary surfactant proteins A (SP-A) and D (SP-D), members of the collectin family, play important roles in the innate immune system of the lung. Here, we show that SP-A but not SP-D augmented phagocytosis of Streptococcus pneumoniae by alveolar macrophages, independent of its binding to the bacteria. Analysis of the SP-A/SP-D chimeras, in which progressively longer carboxyl-terminal regions of SP-A were replaced with the corresponding SP-D regions, has revealed that the SP-D region Gly346-Phe355 can be substituted for the SP-A region Leu219-Phe228 without altering the SP-A activity of enhancing the phagocytosis and that the SP-A region Cys204-Cys218 is required for the SP-A-mediated phagocytosis. Acetylated low density lipoprotein significantly reduced the SP-A-stimulated uptake of the bacteria. SP-A failed to enhance the phagocytosis of S. pneumoniae by alveolar macrophages derived from scavenger receptor A (SR-A)-deficient mice, demonstrating that SP-A augments SRA-mediated phagocytosis. Preincubation of macrophages with SP-A at 37 °C but not at 4 °C stimulated the phagocytosis. The SP-A-mediated enhanced phagocytosis was not inhibited by the presence of cycloheximide. SP-A increased cell surface localization of SR-A that was inhibitable by apigenin, a casein kinase 2 (CK2) inhibitor. SP-A-treated macrophages exhibited significantly greater binding of acetylated low density lipoprotein than nontreated cells. The SP-A-stimulated phagocytosis was also abolished by apigenin. In addition, SP-A stimulated CK2 activity. These results demonstrate that SP-A enhances the phagocytosis of S. pneumoniae by alveolar macrophages through a CK2-dependent increase of cell surface SR-A localization. This study reveals a novel mechanism of bacterial clearance by alveolar macrophages. Pulmonary surfactant proteins A (SP-A) and D (SP-D), members of the collectin family, play important roles in the innate immune system of the lung. Here, we show that SP-A but not SP-D augmented phagocytosis of Streptococcus pneumoniae by alveolar macrophages, independent of its binding to the bacteria. Analysis of the SP-A/SP-D chimeras, in which progressively longer carboxyl-terminal regions of SP-A were replaced with the corresponding SP-D regions, has revealed that the SP-D region Gly346-Phe355 can be substituted for the SP-A region Leu219-Phe228 without altering the SP-A activity of enhancing the phagocytosis and that the SP-A region Cys204-Cys218 is required for the SP-A-mediated phagocytosis. Acetylated low density lipoprotein significantly reduced the SP-A-stimulated uptake of the bacteria. SP-A failed to enhance the phagocytosis of S. pneumoniae by alveolar macrophages derived from scavenger receptor A (SR-A)-deficient mice, demonstrating that SP-A augments SRA-mediated phagocytosis. Preincubation of macrophages with SP-A at 37 °C but not at 4 °C stimulated the phagocytosis. The SP-A-mediated enhanced phagocytosis was not inhibited by the presence of cycloheximide. SP-A increased cell surface localization of SR-A that was inhibitable by apigenin, a casein kinase 2 (CK2) inhibitor. SP-A-treated macrophages exhibited significantly greater binding of acetylated low density lipoprotein than nontreated cells. The SP-A-stimulated phagocytosis was also abolished by apigenin. In addition, SP-A stimulated CK2 activity. These results demonstrate that SP-A enhances the phagocytosis of S. pneumoniae by alveolar macrophages through a CK2-dependent increase of cell surface SR-A localization. This study reveals a novel mechanism of bacterial clearance by alveolar macrophages. Pulmonary surfactant is a mixture of lipids and proteins that function to keep alveoli from collapsing during expiration (1King R.J. Clements J.A. Am. J. Physiol. 1972; 223: 707-714Crossref PubMed Scopus (146) Google Scholar). Surfactant protein A (SP-A) 1The abbreviations used are: SP-A, surfactant protein A; SP-D, surfactant protein D; SR-A, scavenger receptor A; CK2, casein kinase 2; LDL, low density lipoprotein; AcLDL, acetylated LDL; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; BSA, bovine serum albumin. is a major constituent of the surfactant (2Kuroki Y. Voelker D.R. J. Biol. Chem. 1994; 269: 25943-25946Abstract Full Text PDF PubMed Google Scholar). SP-A belongs to the collectin subgroup of the C-type lectin superfamily (3Day A.J. Biochem. Soc. Trans. 1994; 22: 83-88Crossref PubMed Scopus (150) Google Scholar) along with surfactant protein D (SP-D) and mannose-binding lectin. SP-A and SP-D are believed to play important roles in the innate immune system of the lung. SP-A-deficient mice exhibit reduced bacterial clearance and elevated pulmonary inflammation in response to microbial challenge (4Borron P. McIntosh J.C. Korfhagen T.R. Whitsett J.A. Taylor J. Wright J.R. Am. J. Physiol. 2000; 278: L840-L847Crossref PubMed Google Scholar, 5LeVine A.M. Kurak K.E. Bruno M.D. Stark J.M. Whitsett J.A. Korfhagen T.M. Am. J. Respir. Cell Mol. Biol. 1998; 19: 700-708Crossref PubMed Scopus (281) Google Scholar, 6LeVine A.M. Kurak K.E. Wright J.R. Watford W.T. Bruno M.D. Ross G.F. Whitsett J.A. Korfhagen T.R. Am. J. Respir. Cell Mol. Biol. 1999; 20: 279-286Crossref PubMed Scopus (170) Google Scholar). Recent studies from this and other laboratories have revealed that SP-A and SP-D modulate lung inflammation by interacting with cell surface receptors on macrophages including CD14, Toll-like receptor 2, signal-inhibitory regulatory protein α, and calreticulin/CD91 (7Gardai S.J. Xiao Y.-Q. Dickinson M. Nick J.A. Voelker D.R. Greene K.E. Henson P.H. Cell. 2003; 115: 13-23Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 8Murakami S. Iwaki D. Mitsuzawa H. Sano H. Takahashi H. Voelker D.R. Akino T. Kuroki Y. J. Biol. Chem. 2002; 277: 6830-6837Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 9Sano H. Sohma H. Muta T. Nomura S. Voelker D.R. Kuroki Y. J. Immunol. 1999; 163: 387-395PubMed Google Scholar, 10Sato M. Sano H. Iwaki D. Kudo K. Konishi M. Takahashi H. Takahashi T. Imaizumi H. Asai Y. Kuroki Y. J. Immunol. 2003; 171: 417-425Crossref PubMed Scopus (287) Google Scholar). SP-A binds to and enhances the phagocytosis of bacteria including Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, and Haemophilus influenza by immune cells (11Geertsma M.F. Nibbering P.H. Haagsman H.P. Daha M.R. Van Furth R. Am. J. Physiol. 1994; 267: L578-L584PubMed Google Scholar, 12Hartshorn K.L. Crouch E. White M.R. Colamussi M.L. Kakkanatt A. Tauber B. Shephard V. Sastry K.N. Am. J. Physiol. 1998; 274: L958-L969Crossref PubMed Google Scholar, 13Kabha K. Schmegner J. Keisari Y. Parolis H. Schlepper-Schaefer J. Ofek I. Am. J. Physiol. 1997; 272: L344-L352PubMed Google Scholar, 14Tino M.J. Wright J.R. Am. J. Physiol. 1996; 270: L677-L688PubMed Google Scholar). However, the relationship between the binding to the bacteria and the phagocytic effect and the mechanism of the SP-A-mediated uptake of the bacteria are not completely understood. Macrophage scavenger receptor A (SR-A) interacts with a number of ligands including the modified lipoproteins, lipopolysaccharides, lipoteichoic acid, and Gram-negative and -positive bacteria (15Dunne D.W. Resnick D. Greenberg J. Krieger M. Joiner K.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1863-1867Crossref PubMed Scopus (306) Google Scholar, 16Platt N. Gordon S. J. Clin. Invest. 2001; 108: 649-654Crossref PubMed Scopus (256) Google Scholar) and functions as a pattern recognition receptor as a mannose receptor (17Stahl P.D. Ezekowitz R.A.B. Curr. Opin. Immunol. 1998; 10: 50-55Crossref PubMed Scopus (545) Google Scholar). These receptors are responsible for the phagocytosis of various bacteria and are essential components in the innate immune system. The purpose of this study was to investigate the mechanism of the SP-A-mediated phagocytosis of S. pneumoniae by alveolar macrophages. In this report, we show that SP-A augments the SR-A-mediated phagocytosis of S. pneumoniae by alveolar macrophages, independent of its binding to the bacteria, and that SP-A increases cell surface localization of SR-A in a CK2-dependent manner. This study reveals a novel mechanism of bacterial clearance by alveolar macrophages. Reagents—Fluorescein isothiocyanate (FITC) and Alexa 594-donkey anti-goat IgG were obtained from Molecular Probes. Mannan, C1q, cycloheximide, apigenin, PD98059, bisindolylmaleimide, and β-casein were purchased from Sigma. H89, acetylated low density lipoprotein (AcLDL) and [γ-32P]ATP were obtained from Seikagaku Corp., Biomedical Technologies Inc., and Amersham Biosciences, respectively. Goat anti-scavenger receptor A (anti-SR-A) antibody and rabbit anti-casein kinase 2 (anti-CK2)-α chain antiserum were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Surfactant Proteins and Recombinant Proteins—SP-A and SP-D were purified from rat bronchoalveolar lavage fluids as previously described (18Sano H. Chiba H. Iwaki D. Sohma H. Voelker D.R. Kuroki Y. J. Biol. Chem. 2000; 275: 22442-22451Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Recombinant wild type SP-A, mutant SPAR197A,K201A,K203A, SP-D, and SP-A/SP-D chimeras were expressed in a baculovirus-insect cell expression system and were purified as previously described (19Saitoh M. Sano H. Chiba H. Murakami S. Iwaki D. Sohma H. Voelker D.R. Akino T. Kuroki Y. Biochemistry. 2000; 39: 1059-1066Crossref PubMed Scopus (18) Google Scholar, 20Sano H. Kuroki Y. Honma T. Ogasawara Y. Sohma H. Voelker D.R. Akino T. J. Biol. Chem. 1998; 273: 4783-4789Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21Tsunezawa W. Sano H. Sohma H. McCormack F.X. Voelker D.R. Kuroki Y. Biochim. Biophys. Acta. 1998; 1387: 433-446Crossref PubMed Scopus (15) Google Scholar). Binding of SP-A to S. pneumoniae—S. pneumoniae was heat-killed (95 °C for 10 min) and used for the experiments. The binding study was performed using S. pneumoniae coated onto microtiter wells. S. pneumoniae (106 colony-forming units) in 40 μl of PBS was added onto the wells, and the wells were dried under vacuum, and then the bacteria was fixed in 100 μl/well of 0.25% (v/v) glutaraldehyde in PBS for 10 min at room temperature followed by incubation with 0.1 m glycine in PBS for 30 min. Nonspecific binding was blocked by incubating the wells with 20 mm Tris buffer (pH 7.4) containing 0.15 m NaCl, 5 mm CaCl2 and 0.1% (w/v) BSA (buffer A). The wells were then incubated with the indicated concentrations of SP-A or the recombinant proteins in the buffer A for 1 h at 37 °C. Next, horseradish peroxidase-labeled anti-SP-A antibody was added and incubated after the wells had been washed with PBS containing 3% (w/v) skim milk and 0.1% (v/v) Triton X-100. The amount of the proteins binding to S. pneumoniae was determined by measuring the absorbance at 492 nm using o-phenylenediamine as a substrate for the peroxidase reaction. In some experiments, 5 mm EDTA was used instead of CaCl2. 125I-SP-A labeled by Bolton-Hunter reagent (22Bolton A.E. Hunter W.M. Biochem. J. 1973; 133: 529-539Crossref PubMed Scopus (2398) Google Scholar) was also used for the binding study to S. pneumoniae coated onto microtiter wells, and the amount of 125ISP-A binding to the bacteria was determined by measuring the radioactivities using a γ-counter. Phagocytosis Assay of S. pneumoniae by Macrophages—FITC labeling of heat-treated S. pneumoniae was performed by a method based on that described by Tino and Wright (14Tino M.J. Wright J.R. Am. J. Physiol. 1996; 270: L677-L688PubMed Google Scholar). A 1-ml suspension of S. pneumoniae in 0.1 m sodium carbonate (pH 9.0) was mixed with 1 μl of FITC (Molecular Probes; 10 mg/ml in Me2SO) and stirred at room temperature for 1 h in the dark. FITC-labeled bacteria was washed four times with PBS by centrifugation at 5000 rpm for 5 min. The final pellet of FITC-bacteria obtained was suspended in PBS and stored in 100-μl aliquots at -80 °C. The phagocytosis assay was carried out using macrophages in suspension by a modification of the method described by Geertsma et al. (11Geertsma M.F. Nibbering P.H. Haagsman H.P. Daha M.R. Van Furth R. Am. J. Physiol. 1994; 267: L578-L584PubMed Google Scholar). SR-A I/II knockout mice (SR-A-/-) are described in detail elsewhere (23Suzuki H. Kurihara Y. Takeya M. Kamada M. Kataoka M. Jishage K. Ueda O. Sakaguchi H. Higashi T. Suzuki T. Takashima Y. Kawabe Y. Cynshi O. Wada Y. Honda M. Kurihara H. Aburatani H. Doi T. Matsumoto A. Azuma S. Noda T. Toyoda Y. Itakura H. Yazaki Y. Horiuchi S. Takahashi K. Kruijt J.K. Van Berkel T.J.C. Steinbrechner U.P. Ishibashi S. Maeda N. Gordon S. Kodama T. Nature. 1997; 386: 292-296Crossref PubMed Scopus (1010) Google Scholar). Briefly, alveolar macrophages were first isolated from Sprague-Dawley rats or C57BL/6 mice by washing the lung with 0.9% NaCl containing 1 mm EDTA and centrifuging the lavage fluids at 1,000 rpm for 10 min at 4 °C. Alveolar macrophages (2 × 105 in 100 μl of Hanks' balanced salt solution) were mixed with FITC-labeled S. pneumoniae (2 × 106 colony-forming units in 100 μl of Hanks' balanced salt solution) and incubated for 30 min at 37 °C in the dark in the absence or the presence of the indicated concentrations of SP-A. 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 of the suspension at 2,500 rpm for 10 min at 4 °C, and the cell pellet was washed three times with ice-cold PBS. The cells were next suspended with 5 μl of 50 μg/ml ethidium bromide in PBS and pipetted to a microscopic slide glass. The number of macrophages with or without intracellular (green fluorescent) bacteria were counted for at least 100 macrophages in duplicate samples using a fluorescence microscope at × 400 magnification. The results were expressed as the percentage of macrophages that contained intracellular bacteria in total macrophages counted. Mouse-derived macrophages were used only for the phagocytosis experiments with SR-A-deficient cells. Other experiments were performed using rat macrophages. Casein Kinase 2 Assay—Alveolar macrophages isolated from rats were first incubated with or without 40 μm apigenin at 37 °C for 1 h, SP-A (20 μg/ml) was then added, and the cells were further incubated at 37 °C for 30 min. The cells were washed with PBS and processed for the CK2 assay. The CK2 assay was performed by the method described by Mead et al. (24Mead J.R. Hughes T.R. Irvine S.A. Sigh N.N. Ramji D.P. J. Biol. Chem. 2003; 278: 17741-17751Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Briefly, alveolar macrophages (1 × 106) were suspended in 500 μl of phosphatase-free cell extraction buffer consisting of 10 mm Tris buffer (pH 7.05), 50 mm NaCl, 50 mm NaF, 1% (v/v) Triton X-100, 30 mm sodium pyrophosphate. 5 μm ZnCl2, 100 μm sodium orthovanadate, 1 mm dithiothreitol, 2.8 μg/ml aprotinin, 2.5 μg/ml leupeptin, 0.5 mm benzamidine, and 0.5 mm phenylmethanesulfonyl fluoride. The cell suspension was vortexed for 45 s at 4 °C and centrifuged at 11,000 rpm for 10 min. The supernatant was collected as the whole cell extract and stored at -80 °C. The whole cell extracts were first precleared by the addition of protein A-agarose beads (20 μl) and the incubation at 4 °C for 1 h, followed by centrifugation at 14,000 rpm for 5 min to remove the beads. The precleared supernatant was mixed with anti-CK2-α chain antiserum (2 μg/ml) and incubated overnight at 4 °C. The CK2-antibody immune complex was isolated by the addition of protein A-agarose beads (20 μl) with gentle shaking for 2 h at 4 °C. The beads binding to the immune complex were collected by centrifugation at 14,000 rpm for 5 min at 4 °C and washed once with the phosphatase-free cell extraction buffer. The pellet obtained was resuspended in 25 μl of kinase buffer consisting of 100 mm Tris buffer (pH 8.0), 100 μm sodium orthovanadate, 100 mm NaCl, 20 mm MgCl2, 50 mm KCl, 5 μCi of [γ-32P]ATP, and 5 mg/ml β-casein. The mixture was then incubated for 15 min at 37 °C, and the reaction was stopped by the addition of 10 μl of reducing solubilizing buffer (50 mm Tris buffer (pH 6.8) containing 100 mm dithiothreitol, 2% (w/v) SDS, 0.1% (w/v) bromphenol blue, and 10% (v/v) glycerol). Samples were subjected to SDS-PAGE after boiling for 10 min. After electrophoresis, the gel was fixed for 20 min in a solution containing 40% (v/v) methanol and 10% (v/v) acetic acid and dried after washing once with distilled water. The phosphorylated β-casein was finally visualized by autoradiography. Immunoblot Analysis—Immunoblotting analysis was performed to examine whether the whole cell extracts under different conditions contained equal amounts of CK2. The proteins were precipitated from 10 μg of the whole cell extracts using 4 volumes of ice-cold acetone. The precipitated proteins were subjected to SDS-PAGE, and the proteins on the gel were transferred onto polyvinylidene difluoride membrane. Nonspecific binding to the polyvinylidene difluoride membrane was blocked by the incubation with TBS (20 mm Tris buffer (pH 7.4) and 150 mm NaCl) containing 5% (w/v) skim milk and 0.05% (v/v) Tween 20. The membrane was then incubated with anti-CK2 antiserum, followed by incubation with horseradish peroxidase-labeled goat anti-rabbit antibody. After washing the membrane with TBS containing 0.05% (v/v) Tween 20, the protein bands were visualized using an enhanced chemiluminescence detection kit (Amersham Biosciences). Confocal Microscope—Alveolar macrophages (5 × 105) were suspended in Hanks' balanced salt solution and incubated with or without protein kinase inhibitors at 37 °C for 1 h and further incubated at 37 °C for 30 min on polylysine-coated coverslips in the absence or the presence of 20 μg/ml SP-A. The cells on coverslips were washed with PBS and fixed in 4% (w/v) paraformaldehyde in PBS or in methanol at -20 °C for 10 min. The cells were washed with PBS containing 2% (w/v) BSA (BSA/PBS) and incubated with BSA/PBS for 30 min and then incubated with anti-SR-A antibody (2 μg/ml) in BSA/PBS at room temperature for 60 min. The cells were then washed three times with BSA/PBS and incubated with Alexa 594-anti-goat IgG (1:500) in BSA/PBS at room temperature for 45 min in the dark. The cells were finally washed three times with BSA/PBS and with PBS, sealed in the presence of Vectashield Antifade (Vector Laboratories), and examined using a laser microscope (LSM510; Carl Zeiss, Tokyo, Japan) with a ×63 (methanol-fixed samples) or a ×20 (for paraformaldehyde-fixed samples) oil planapochromatic lens (numerical aperture 1.4). Digital images were acquired and processed using Adobe Photoshop, version 5.0 (Mountain View, CA) and CorelDRAW software (Corel Corp.). In some experiments, alveolar macrophages were first incubated with 20 μg/ml SP-A at 4 °C or at 37 °C for 30 min and washed with ice-cold PBS by centrifugation at 3,000 rpm for 10 min at 4 °C. The cells were then suspended with Hanks' balanced salt solution and incubated onto lysine-coated coverslips. Binding of 125I-Acetylated LDL to Alveolar Macrophages—AcLDLs were labeled with 125I using Bolton-Hunter reagent (Amersham Biosciences) by the method described by Bolton and Hunter (22Bolton A.E. Hunter W.M. Biochem. J. 1973; 133: 529-539Crossref PubMed Scopus (2398) Google Scholar). The methods used in this study were adapted from those for binding of LDL to its receptor (25Goldstein J.L. Brown M.S. J. Biol. Chem. 1974; 249: 5153-5162Abstract Full Text PDF PubMed Google Scholar). Rat alveolar macrophages were cultured at a density of 5 × 105/well in a 24-well plate in RPMI 1640 containing 10% (v/v) fetal calf serum for 2 h. The cells were then incubated with or without SP-A (20 μg/ml) at 37 °C for 1 h and washed three times with 500 μl of RPMI 1640 containing 1 mg/ml BSA. The 24-well plate was then put on ice, and the cells were incubated with the indicated concentrations of 125I-AcLDL in 500 μl/well of ice-cold RPMI 1640 containing 10 mm Hepes (pH 7.4) and 10% (v/v) fetal calf serum at 4 °C for 4 h. After a 4-h incubation, the medium containing 125I-AcLDL was aspirated, and the cells were washed rapidly with 500 μl of ice-cold buffer B (50 mm Tris buffer (pH 7.4), 0.15 m NaCl, and 2 mg/ml BSA). The cell monolayer was then incubated on ice for 10 min with 500 μl of the ice-cold buffer B. The medium was removed, and these washing steps were repeated. Next, the cell monolayer was rapidly washed with 500 μl of ice-cold buffer containing 50 mm Tris (pH 7.4) and 0.15 m NaCl. The cells were removed from the wells by dissolution in 500 μl of NaOH, and the amount of 125I-AcLDL bound to the cells was determined with a γ-counter. Flow Cytometric Analysis—Alveolar macrophages and peritoneal macrophages isolated from rats (1 × 106 cells) were incubated with or without 20 μg/ml SP-A at 37 °C for 30 min. The cells were washed with PBS and fixed in 4% paraformaldehyde at room temperature for 10 min. After fixation, the macrophages were collected by centrifugation at 150 × g for 10 min and washed once with PBS. The fixed macrophages were incubated with anti-SR-A antibody (Santa Cruz Biotechnology) or control goat IgG in PBS containing 5 mg/ml BSA and 10 mm sodium azide, followed by the incubation with FITC-conjugated anti-goat IgG. After another washing, the labeled macrophages were analyzed by using FACSCalibur and CellQuest software (BD Biosciences). In some experiments, rat alveolar macrophages were treated with 0.1% saponin at 4 °C for 30 min after paraformaldehyde fixation, and total SR-A expression was analyzed as described above. Binding of SP-A to S. pneumoniae—We first examined whether SP-A bound to S. pneumoniae using 125I-labeled SP-A. 125I-SP-A exhibited a concentration-dependent binding to S. pneumoniae (Fig. 1a). Inclusion of 5 mm EDTA in the binding buffer inhibited the binding of SP-A to the bacteria, indicating that the binding of SP-A to S. pneumoniae is Ca2+-dependent. Recombinant wild type SP-A as well as native SP-A showed a concentration-dependent and saturable binding to S. pneumoniae (Fig. 1b). The mutant SP-AR197A,K201A,K203A exhibited a binding comparable with that of wild type SP-A. SP-A Enhances the Phagocytosis of S. pneumoniae by Alveolar Macrophages—The effect of SP-A on the phagocytosis of S. pneumoniae by alveolar macrophages was studied. The presence of SP-A significantly increased the phagocytosis of the bacteria in a manner dependent upon SP-A concentrations (Fig. 2a). SP-A augmented the phagocytosis in a time-dependent fashion (Fig. 2b). Only 5-min coincubation of macrophages with SP-A significantly increased the uptake of the bacteria. At the end of the phagocytosis assay, the viabilities of alveolar macrophages were determined. No differences in the viabilities were found in the absence or the presence of SP-A (90.3 ± 1.2 versus 89.8 ± 0.4% (means ± S.E.), n = 3). This result rules out a possibility that the stimulation of the phagocytosis by SP-A is due to the effect of SP-A on the macrophage viability. Excess mannan, C1q, or EDTA failed to alter the SP-A-stimulated phagocytosis of S. pneumoniae (Fig. 2c). This suggests that mannose receptor or C1q receptor is not involved in the SP-A-mediated enhanced phagocytosis of S. pneumoniae. The results also indicate that the augmentation of the phagocytosis by SP-A is independent of the binding of SP-A to S. pneumoniae, since the addition of EDTA that inhibited SP-A binding to S. pneumoniae showed no effect on the uptake enhanced by SP-A. In addition, SP-A retained the activity of enhancing the phagocytosis in the presence of polymyxin B. Neither the heat-treated SP-A nor lipopolysaccharide stimulated the phagocytosis (Fig. 2c). These results rule out the possibility that the enhanced phagocytosis is due to the endotoxin contamination in the SP-A preparation. We also examined whether another collectin SP-D, a structural homologue to SP-A, enhanced the uptake of S. pneumoniae by alveolar macrophages. Unlike SP-A, SP-D failed to augment the phagocytosis of the bacteria (Fig. 2d). Thus, the effect of SP-A/SP-D chimeras, in which progressively longer carboxyl-terminal regions of SP-A were replaced with the corresponding SP-D regions, was examined to identify the SP-A region required for augmentation of the phagocytosis (Fig. 2d). Only the chimera ad1, containing the extreme carboxyl terminus of SP-D, and not the chimeras ad2-ad5 exhibited the enhanced phagocytosis, indicating the importance of the carbohydrate recognition domain in the SP-A-stimulated phagocytosis of S. pneumoniae. The data clearly show that the SP-D region Gly346-Phe355 can be substituted for the SP-A region Leu219-Phe228 without altering the SP-A activity of enhancing the phagocytosis. The results also suggest that the SP-A region Cys204-Cys218 is required for the SP-A-mediated phagocytosis, since the chimera ad2 failed to augment the uptake of the bacteria. In addition, SP-AR197A,K201A,K203A did not enhance the phagocytosis of S. pneumoniae (Fig. 2d). Because this mutant avidly bound to S. pneumoniae at a level comparable with that of wild type SP-A (Fig. 1a), taken together with the results obtained from the experiments with EDTA, these results demonstrate that SP-A augments the phagocytosis of S. pneumoniae by alveolar macrophages, independent of its binding to the bacteria. SP-A Augments Scavenger Receptor A-mediated Phagocytosis of S. pneumoniae—When the phagocytosis assay was performed in the presence of 0-100 μg/ml AcLDL, the increasing concentrations of AcLDL significantly reduced the uptake of S. pneumoniae by alveolar macrophages (Fig. 3a). SP-A exhibited almost no stimulatory effect on the phagocytosis in the presence of 20 and 100 μg/ml AcLDL, suggesting that SP-A enhances SR-A-mediated phagocytosis. Consistent with these results, SP-A failed to stimulate the phagocytosis of S. pneumoniae by alveolar macrophages derived from SR-A-/- mice (Fig. 3b). These results demonstrate that SP-A stimulates SR-A-mediated phagocytosis of S. pneumoniae. When FITC-labeled S. pneumoniae was incubated with rat alveolar macrophages at 4 °C in the absence or the presence of fucoidan and mannan, fucoidan but not mannan significantly decreased the binding of the bacteria to macrophages (Fig. 3c). Since fucoidan is a ligand for SR-A (16Platt N. Gordon S. J. Clin. Invest. 2001; 108: 649-654Crossref PubMed Scopus (256) Google Scholar), these results indicate that S. pneumoniae binds to SR-A. The effects of SP-A on the phagocytosis of FITC-labeled Staphylococcus aureus, an SR-A ligand (15Dunne D.W. Resnick D. Greenberg J. Krieger M. Joiner K.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1863-1867Crossref PubMed Scopus (306) Google Scholar), and of FITC-labeled latex beads were also examined. SP-A significantly increased the uptake of S. aureus as well as S. pneumoniae by rat alveolar macrophages (Fig. 3d). However, SP-A did not enhance the phagocytosis of latex beads. Taken together, these results are consistent with the conclusion that SP-A augments SR-A-mediated phagocytosis. It is unlikely that SP-A stimulates the general phagocytosis phenomenon. Requirement of SP-A-Macrophage Interaction—Since the SPA-mediated phagocytosis of S. pneumoniae is independent of its bacterial binding, alveolar macrophages were first preincubated with SP-A at 37 °C for various lengths of time, the medium containing SP-A was removed, and the cells were washed. The cells were then incubated with FITC-labeled S. pneumoniae. The alveolar macrophages preincubated with SP-A still exhibited the enhanced phagocytosis even after the cells had been washed (Fig. 4a). The longer the preincubation with SP-A, the greater the uptake of S. pneumoniae. However, preincubation at 4 °C failed to stimulate the uptake in the presence of SP-A (Fig. 4b). The basal uptake of the bacteria by peritoneal macrophages in the absence of SP-A is significantly greater than that by alveolar macrophages (Fig. 4c). Peritoneal macrophages did not exhibit stimulated phagocytosis in response to SP-A. The effect of cycloheximide on the phagocytosis was also examined. SP-A still enhanced the uptake of the bacteria by alveolar macrophages in the presence of 10 μg/ml cycloheximide (Fig. 4d), suggesting that new protein synthesis is not involved in the SP-A effect. SP-A Increases Cell Surface Localization of Scavenger Receptor A on Alveolar Macrophages—The effect of SP-A on cell surface expression of SR-A was next examined. Alveolar macrophages were preincubated with SP-A, and the cells were processed for immunochemistry with anti-SR-A antibody after fixation in paraformaldehyde. Incubation of SP-A with the macrophages clearly increased the expression of SR-A (Fig. 5a). Observation of the immunostained cells that had been fixed in methanol revealed that SP-A induced translocation of SR-A from cytoplasmic vesicles to plasma membrane. Consistent with these results, the binding of acetylated LDL, an SR-A ligand, to alveolar macrophages that had been preincubated with SP-A was significantly increased (Fig. 5b). These data support the concept that SP-A increases cell surface localization of SR-A. SP-A still increased cell surface localization of SR-A even in the presence of cycloheximide (Fig. 5c). In addition, preincubation of the cells with SP-A at 4 °C failed to enhance the immunostaining of SR-A (Fig. 5c). These results correlate well with those obtained from the phagocytosis assay (Fig. 4, b and d) and suggest that SP-A stimulates the phagocytosis and the cell surface SR-A localization by the mechanism that is temperature-dependent but is not involved in the new protein synthesis. We further assessed the cell surface expression of SR-A on alveolar and peritoneal macrophages by flow cytometry. SR-A was constitu" @default.
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• W2136558272 date "2004-05-01" @default.
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• W2136558272 title "Pulmonary Surfactant Protein A Augments the Phagocytosis of Streptococcus pneumoniae by Alveolar Macrophages through a Casein Kinase 2-dependent Increase of Cell Surface Localization of Scavenger Receptor A" @default.
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• W2136558272 doi "https://doi.org/10.1074/jbc.m312490200" @default.
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