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.

Here, we showed that SP-A but of Streptococcus pneumoniae by macrophages, independent of its binding the bacteria. Analysis of the SP-A/SP-D chimeras, in which progressively longer carboxy terminal of SP-A were replaced with the corresponding SP-D regions, has revealed that the SP-D region Gly 346 Phe 355 can be substituted for the SP-A region Leu 219 -Phe 228 without altering the SP-A’s activity of enhancing the phagocytosis, and that the SP-A region Cys 204 -Cys 218 is required for the SP-A-mediated phagocytosis. Acetylated low density lipoprotein (LDL) 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 SR-A-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 LDL than non-treated 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. increases cell surface localization of SR-A in a CK2-dependent manner. This study reveals a novel mechanism of bacterial clearance by alveolar macrophages. importance of augmentation the


INTRODUCTION
8 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 fluorescence microscope at x400 magnification. The results were expressed as 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, and 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 casein kinase 2 (CK2) assay.
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-9 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 (methanol-fixed samples) or a x20 (for paraformaldehyde-fixed samples) oil planapochromatic lens (NA1.4). 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 cenrifugation at 3,000 rpm for 10 min at 4°C. The cells were then suspended with HBSS and incubated onto lysine-coated coverslips.
Binding of 125 I-acetylated LDL to alveolar macrophages. Acetylated LDL (AcLDL) were labeled with 125 I using Bolton-Hunter reagent (Amersham Pharmacia Biotech) by the method described by Bolton and Hunter (22). The methods used in this study was adapted from those for binding of LDL to its receptor (25). Rat alveolar macrophages 11 were cultured at a density of 5 x 10 5 /well in 24-well plate in RPMI 1640 containing 10 % (v/v) FCS 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 125 I-AcLDL in 500 µl/well of ice-cold RPMI 1640 containing 10 mM Hepes (pH 7.4) and 10 % (v/v) FCS at 4°C for 4 h. After 4hincubation, the medium containing 125 I-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 12 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 125 I-labeled SP-A. 125 I-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 Ca 2+ -dependent.
Recombinant wt SP-A as well as native SP-A showed a concentration-dependent and saturable binding to S. pneumoniae (Fig. 1b). The mutant SP-A R197A, K201A, K203A exhibited a binding comparable to that of wt SP-A. Excess mannan, C1q or EDTA failed to alter the SP-Astimulated phagocytosis of S. pneumoniae (Fig. 2c). This suggests that mannose 13 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 the SP-A's 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 (PMB). Neither the heat-treated SP-A nor LPS 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.

SP-
We also examined whether another collectin SP-D, a structural homologue to SP-A,

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 acetylated LDL (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 the scavenger receptor A (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 (16), these results indicate that S. pneumoniae binds to SR-A. The effect of SP-A on the phagocytosis of FITC-labeled Staphylococcus aureus, an SR-A ligand (15), 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.

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 by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 16 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. 5 c). These results correlate well with those obtained from the phagocytosis assay ( Fig. 4b 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 constitutively expressed on cell surfaces of alveolar macrophages (Fig. 6a, gray line). After exposure of 20 µg/ml SP-A for 30 min, cell surface expression of SR-A on alveolar macrophages was enhanced (Fig. 6a, solid black line). When the mean fluorescence intensity (MFI) was calculated as a ratio to control, SP-A significantly increased the MFI ratio ( Fig. 6a, inset). These results support the conclusion obtained from confocal microscopy that SP-A increases cell surface SR-A localization on alveolar macrophages. However, the flow cytometric analysis of peritoneal macrophages revealed that SP-A failed to stimulate cell surface expression of SR-A (Fig. 6b). In addition, the MFI ratio of peritoneal macrophages without SP-A treatment was higher than that of untreated alveolar macrophages (Fig. 6ab, insets), indicating the cell surface expression of SR-A on untreated peritoneal macrophages is stronger than that on untreated alveolar macrophages, These results are consistent with those obtained from the phagocytsosis assay by peritoneal macrophages (Fig. 4c), showing that the percent phagocytosis without SP-A by peritoneal macrophages is greater than that by alveolar macrophages, and that SP-A did not stimulate the phagocytosis by peritoneal macrophages. In order to examine total SR-A expression in alveolar macrophages, the cells were treated with saponin after paraformaldehyde fixation and the flow cytometric analysis was performed (Fig. 6c). The fluorescence intensity in the saponin-treated cells was clearly increased when compared with that in the saponin-untreated cells, and total SR-A expression was not altered in the cells with or without SP-A exposure. In addition, immunoblotting analysis of the whole cell lysate of macrophages with SR-A antibody showed that total content of SR-A protein in the cells with SP-A exposure was not different from that in the cells without SP-A exposure (Fig. 6d). These results indicate that SP-A did not affect total SR-A expression in macrophages. Taken together, these results support the conclusion that SP-A increases cell surface expression of SR-A but not total SR-A expression in alveolar macrophages, and are consistent with the idea that new protein synthesis is not involved (see Fig. 4d and Fig. 5c).
The effects of protein kinase inhibitors were next examined to determine whether Likewise, apigenin also inhibited the SP-A-stimulated phagocytosis in a concentration-dependent fashion (Fig. 7c). SP-A still enhanced the phagocytosis in the presence of PD98059 and H89, although bisindolylmaleimide somewhat augmented the basal level of phagocytosis. Taken together, these results indicate that protein phosphorylation by CK2 is involved in the increased cell surface localization of SR-A and in the enhanced phagocytosis of S. pneumoniae that have been caused by SP-A. In addition, the results also suggest the idea that SP-A stimulates CK2 activity.

SP-A stimulates casein kinase 2 activity.
Thus, we examined whether SP-A stimulated CK2 activity. Immunoblotting analysis revealed that the cell extracts of alveolar macrophages under different conditions contained almost equal levels of CK2 protein (Fig. 8). Next, in vitro CK activity was examined by using immunoprecipitated CK2 and β-casein. β-Casein was phosphorylated only when macrophages were preincubated with SP-A. These results clearly demonstrate that SP-A stimulates CK2 activity. Incubation of macrophages with apigenin completely abolished the CK activity in the presence of SP-A. These results support the idea that the protein phosphorylation caused by SP-A-stimulated CK2 increases cell surface SR-A localization and S. pneumoniae phagocytosis.

DISCUSSION
The present study shows that SP-A augments the scavenger receptor A (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 manner dependent upon the CK2 activity. This study reveals a novel mechanism of bacterial clearance by alveolar macrophages.
A previous study (11) has shown that C1q inhibits the binding of SP-A to monocytes and the SP-A-enhanced uptake of Staphylococcus aureus by monocytes, indicating that the binding of SP-A to C1q receptor mediates phagocytosis of the bacteria by monocytes. However, another (14) and the present (see Fig. 2c) studies have revealed that C1q failed to attenuate the SP-A-stimulated phagocytosis by alveolar macrophages.
This may imply that the stimulatory effect of SP-A on phagocytosis was cell-type specific.
Although SP-A does not stimulate the uptake of S. pneumoniae by peritoneal macrophages, the level of phagocytosis without SP-A by peritoneal macrophages is significantly greater than that by alveolar macrophages (see Fig. 4c). This corresponds to the results obtained from flow cytometric analysis of cell surface SR-A expression (see Fig. 6ab, insets). Fluorescence intensity obtained for peritoneal macrophages without SP-A treatment is stronger than that for alveolar macrophages. In addition, the phagocytosis without SP-A by peritoneal macrophages is at the level almost equivalent to that with SP-A by alveolar macrophages (see Fig. 4c). Thus, it is possible to assume that the non-stimulatory effect of SP-A on the phagocytosis by preritoneal macrophages is due to the saturated cell surface expression of SR-A on peritoneal macrophages. completely block its binding (see Fig. 1a). Thus, there is a possibility that the failure of inhibiting the SP-A-stimulated phagocytosis by EDTA (see Fig. 2c) is because the weak binding of SP-A to the bacteria in the presence of EDTA still affects the phagocytosis.
However, the preincubation experiments (see Fig. 4ab) clearly demonstrate that the stimulatory effect of SP-A on the phagocytosis is independent of its binding to the bacteria. In addition, the mutant SP-A R197A, K201A, K203A that exhibits comparable binding to the bacteria fails to stimulate the phagocytosis by alveolar macrophages (see Fig. 1b and   2d). These results also supports the conclusion that the phagocytosis and the bacterial binding are independent processes. Thus, it is likely that SP-A does not serve as an

opsonin. In contrast, SP-A has been shown to bind Staphylococcus aureus and induce
C1q receptor-mediated phagocytosis by monocytes (11) (19) from this laboratory has shown that the chimera ad1 fails to enhance the uptake of liposome containing dipalmitoylphosphatidylcholine by alveolar type II cells. Thus, taken together with this study, structural requirements for interaction of SP-A with alveolar macrophages are different from those with alveolar type II cells. Since the stimulatory effect of SP-A on S. pneumoniae phagocytosis occurs through SP-A-cell interaction and is specific for alveolar macrophages, it is presumed that the SP-A CRD recognizes the putative SP-A receptor on alveolar macrophages.
One recent study (7) has shown that the collagenous tails of SP-A and the SP-A mutant lacking functional head groups enhance inflammatory cytokine production by binding to calreticulin /CD91, suggesting a proinflammatory function of SP-A. They propose that the binding of SP-A to foreign organism and cell debris via the globular heads of the protein and the presentation of the collagenous tails to calreticulin/CD91, stimulate phagocytosis and proinflammatory responses. The present study also supports the concept that SP-A exhibits proinflammatory functions. However, the mutant SP-A R197A, K201A, K203A with intact collagenous tails, in which the amino acid residues in the head group are mutated, fails to stimulate the bacterial phagocytosis by alveolar macrophages (see Fig. 2d). In addition, the SP-A/SP-D chimera ad2 containing the SP-A tails, in which the SP-D region Cys 331 -Phe 355 is substituted for the corresponding SP-A region Cys 204 -Phe 228 , also fails to augment the uptake of S. pneumoniae (see Fig. 2d).

22
These results indicate the importance of the SP-A head groups in the SP-A-stimulated phagocytosis of S. pneumoniae. Thus, it is unlikely that the presentation of the SP-A tails to calreticulin/CD91, as reported by Gardai et al. (7), causes the increased phagocytosis of S. pneumoniae by alveolar macrophages. These studies support the idea that SP-A exhibits proinflammatory functions by different mechanisms.

Macrophage scavenger receptor A (SR-A) (16,27) recognizes a number of ligands
including chemically modified lipoprotein such as acetylated LDL (AcLDL) and oxidized LDL. A study using SR-A-deficient mice (23) has revealed that SR-A contributes to the generation of atherosclerotic lesions in vivo and also that it is an element of the innate immune system. SR-A recognizes a variety of polyanions and bacterial cell wall components (15,28,29). In addition, a study with macrophages derived from SR-A -/mice has revealed that SR-A mediates opsonin-independent phagocytosis of gram-positive bacteria (30), indicating that SR-A serves as a phagocytic receptor. The present results (see Fig. 3) clearly demonstrate that SP-A increases the SR-A-mediated phagocytosis of S. pneumoniae. We then pursued the mechanism of SP-A-stimulated phagocytosis. Immunocytochemical observation of SP-A-treated macrophages by anti-SR-A antibody revealed increased cell surface localization of SR-A and this was confirmed by the binding study at 4°C with an SR-A ligand, AcLDL (see