Natural IgM and innate immune collectin SP-D bind to late apoptotic cells and enhance their clearance by alveolar macrophages in vivo

Molecular Immunology 48 (2010) 37–47

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Natural IgM and innate immune collectin SP-D bind to late apoptotic cells and enhance their clearance by alveolar macrophages in vivo Michael L. Litvack a,b,c , Pascal Djiadeu a,b , Sri Dushyaanthan Sri Renganathan d , Sarah Sy d , Martin Post b,c,d , Nades Palaniyar a,b,c,d,∗ a

Lung Innate Immunity Research Laboratory, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8 Physiology and Experimental Medicine, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8 Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada d Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada b c

a r t i c l e

i n f o

Article history: Received 15 March 2010 Received in revised form 16 September 2010 Accepted 22 September 2010 Available online 29 October 2010 Keywords: Innate immune collectin Surfactant protein D (SP-D) Immunoglobulin M (IgM) Late apoptotic cells Apoptotic cell clearance Alveolar macrophages

a b s t r a c t Innate immune collectin surfactant protein D (SP-D) and natural immunoglobulin M (IgM) are two soluble proteins. These opsonic proteins are good candidates for enhancing late apoptotic cell clearance. However, effects of these proteins on late apoptotic cell clearance in the lungs are not clearly established. We have recently shown that SP-D can bind several immunoglobulin isotypes, including IgM. Here we hypothesized that IgM and SP-D bind to late apoptotic cells and enhance their clearance from the lungs. We show that IgM and SP-D bind to late apoptotic secondary necrotic cells, and that IgM and SP-D either co-localize to the same regions or to different regions of late apoptotic Jurkat T cells. Mouse alveolar macrophages internalized late apoptotic cells, in vivo. We induced lung inflammation in mice using LPS and show that airway IgM and SP-D levels and the clearance of late apoptotic cells by alveolar macrophages increases under these conditions. We then coated late apoptotic cells with IgM, SP-D, or both and instilled them into the mouse airways. We found that alveolar macrophages internalize IgM- and SP-D-coated late apoptotic cells more effectively than uncoated late apoptotic cells, in vivo. None of these conditions cause inflammation in the naïve lungs. Therefore, these data suggest that both IgM and SP-D effectively opsonize late apoptotic cells and directly enhance their clearance by alveolar macrophages in the lungs. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Ineffective clearance of apoptotic cells can lead to the accumulation of late apoptotic cells or secondary necrotic cells in the lungs (Bianchi et al., 2008; Litvack and Palaniyar, 2010; Vandivier et al., 2006). When apoptotic cells are not cleared an inflammatory immune response may occur to reduce the accumulation of these dying cells (Clark et al., 2002; Palaniyar et al., 2005; Savill et al., 2002). To avoid this type of inflammation, apoptotic cells must be removed quickly and efficiently from tissues by phagocytic cells to escape immune response (Munoz et al., 2010). Macrophages are an important population of innate immune phagocytic cells that internalize and remove apoptotic cells. In this study we aimed to determine the effects of two opsonins – immunoglobulin IgM and

∗ Corresponding author at: Lung Innate Immunity Research Laboratory, Physiology and Experimental Medicine, 555 University Avenue, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8. Tel.: +1 416 813 7654x2328; fax: +1 416 813 5771. E-mail address: [email protected] (N. Palaniyar). 0161-5890/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2010.09.014

surfactant protein D (SP-D) – in apoptotic cell clearance by alveolar macrophages (AMs) in the lungs. The surfactant protein D is an innate immune pattern recognition protein originally isolated from lung tissue and samples (Persson et al., 1990, 1989). This protein is a member of the carbohydrate-binding collectin (collagenous lectin) group of innate immune proteins (Kingma and Whitsett, 2006; Litvack and Palaniyar, 2010; Palaniyar, 2010; Wright, 2005) and is characterized by its ability to increase and facilitate uptake of biological material by macrophages in vivo and in vitro including DNA (Palaniyar et al., 2005, 2004, 2003b), viruses (Gaiha et al., 2008; LeVine et al., 2004; Meschi et al., 2005), bacteria (Palaniyar et al., 2002; Wright, 2005) and apoptotic cells (Clark et al., 2002; Gardai et al., 2003; Palaniyar et al., 2003a; Vandivier et al., 2002). Recently, we have shown that SP-D can bind to adaptive immune proteins such as IgM, IgG, IgE, and secteroty IgA, but not IgA (Nadesalingam et al., 2005). This is of specific significance as natural IgM has been shown to bind to late apoptotic cells (Kim et al., 2002). Immunoglobulin M can enter the airways via epithelial cell transcytosis (Jaffar et al., 2009; Rojas and Apodaca, 2002); however, the role of IgM in apoptotic cell clearance in the lungs is unknown.

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In this study we sought to determine the binding pattern of SP-D and IgM to apoptotic cells and to further investigate their ability to influence late apoptotic cell clearance in the lungs. Here we show that SP-D and IgM can bind to each other and to late apoptotic cells in mutually distinct regions and co-localize on the cell surface. We illustrate that AMs internalize late apoptotic cells, in vivo. During LPS-induced lung inflammation, airway IgM and SP-D levels increase, as does the internalization potential of the AMs. Finally, we show that in naïve mice, SP-D and IgM facilitate the clearance of late apoptotic cells in the lungs by AMs. This study shows for the first time that IgM and SP-D can interact on late apoptotic cells and enhance the clearance of these cells in the lungs. These opsonins do not significantly inhibit each other’s ability to promote the clearance of late apoptotic Jurkat T cells by the alveolar macrophages, in vivo. 2. Materials and methods

Maxisorp polystyrine ELISA plates (Nunc) in sodium bicarbonate coating buffer (15 mM Na2 CO3 , 35 mM NaHCO3 , pH 9.5) overnight at 4 ◦ C. The wells were then washed three times with TSCT (20 mM Tris, 150 mM NaCl, 5 mM CaCl2 , 0.05% (v/v) Tween 20, pH 7.4) between each of the following steps and all incubations were completed at 37 ◦ C unless otherwise specified. All wells were blocked with 5% (w/v) BSA in TSCT for 30–45 min. Following blocking, wells were incubated with SP-D (1 ␮g/ml) for 2 h. Wells were then incubated with biotinylated anti-SP-D antibody (1 ␮g/ml) followed by a strepavidin-horseradish peroxidase conjugate (1:10,000). Wells were then washed four times and developed with 100 ␮l of TMB (BioRad) at room temperature. Reactions were stopped with 100 ␮l 0.5 M H2 SO4 . Murine lung IgM measurements were made using mouse-IgM ELISA quantitation kit (Catalog #E90-101, Bethyl Laboratories, Montgomery, TX, USA). The colour read-out was made by a spectrophotometer at 450 nm and analyzed using SoftMax Pro software and Microsoft Excel.

2.1. Reagents

2.4. Ligand blots

All reagents were purchased from Sigma–Aldrich Canada Ltd. (Oakville, Ontario) unless otherwise stated. Additional commercial human IgM from pooled human serum was purchased from Calbiochem (cat. #40 1799) and Oxford Biotechnology (OBT1524). Custom antibodies against SP-D were generated in rabbits using SP-D[G-x-y]8 (n/CRD) as the antigen by Cocalico Biologicals Inc. (Reamstown, PA). Polyclonal rabbit IgG was purified from rabbit serum with sodium sulphate precipitation. This was followed by sodium phosphate elution using protein G affinity chromatography as described by manufacturers recommendation (GE Healthcare) followed by elution with glycine buffer. Antibodies were further purified by Superose 6 gel filtration column chromatography in PBS and frozen in aliquots at −20 ◦ C.

One microlitre of serially diluted IgM (from different sources) was placed on a nitrocellulose membrane, allowed to dry and incubated with 2% (w/v) BSA in TSC for 18 h at 4 ◦ C. The membranes were washed with TSCT, incubated with SP-D (0.25 ␮g/ml) for 2 h at 37 ◦ C, washed with TSCT and incubated with biotinylated-rabbit anti human SP-D (1 ␮g/ml). SP-D that bound to the membranes were detected by Steptavidin conjugated HRP (1:10,000, Sigma) and ECL reagents (Pierce).

2.2. Protein purification Human IgM was purified from normal human plasma (NHS) (HD Supplies, UK). Briefly, NHS was thawed on ice and buffer exchanged using a 13.5 ml PD-10 desalting column (GE Healthcare) to prepare the plasma for use with the HiTrap IgM Purification HP, 1 ml (GE Healthcare 17-5110-01) as per the manufacturers instructions in an AKTA purifier (GE Healthcare). IgM was eluted with 20 mM sodium phosphate (pH 7.5). Following this, the appropriate fractions were further purified using a Superose 6 gel filtration column in the AKTA to isolate IgM. Western blot and Coomassie blue staining confirmed successful IgM purification using this method. Human SP-D was purified from human bronchoalveolar lavage fluid as described previously (Nadesalingam et al., 2005; Palaniyar et al., 2005, 2004). Briefly, human bronchoalveolar lavage fluid was incubated with maltose–agarose beads overnight in the presence of 10 mM CaCl2 . The beads were poured into an empty column and the protein was purified by FPLC on an AKTA purifier and eluted with a Tris–MnCl2 buffer (20 mM Tris, 100 mM MnCl2 ). Following this, the fractions containing SP-D were concentrated and the combined concentrate was further purified using a Superose 6 gel filtration column (10 mm × 300 mm; GE Healthcare) to separate SP-D from other proteins. SP-D eluted at the void volume of the column away from other minor protein components and SP-A. There was no SP-D trimers or monomers present in these SP-D preparations. These results are consistent with the fact that our SP-D preparations contained multimers (Nadesalingam et al., 2003; Palaniyar et al., 2005, 2004). 2.3. Enzyme-linked immunosorbent assay (ELISA) IgM purified from NHS as described in Section 2.2 or as purchased from Sigma (product #I8260) was immobilized onto

2.5. Western blots Twenty microlitres of BAL from mice instilled with or without LPS was resolved on a gradient (4–20%, w/v) SDS-PAGE gel (BioRad) under reducing conditions. The gel was transferred to a nitrocellulose membrane that was probed for SP-D with anti-SP-D antibodies (described above) followed by chemiluminescent detection. 2.6. Labelling of Jurkat cells The human Jurkat T-cell was purchased from ATCC (clone E6-1 ATCC# TIB-152) and cultured in RMPI media (Wisent) supplemented with 10% (v/v) FBS (Gibco), 10 U/ml penicillin (Gibco) and 10 ␮g/ml streptomycin (Gibco) (complete culture media) at 37 ◦ C with 5% (v/v) CO2 . Cell membranes were labelled with the fluorescent dye PKH 67 as described by the manufacturer’s directions (Sigma, PKH67-GL) or with DiD (Molecular Probes, Invitrogen). For DiD labelling the cell concentration was adjusted to 1 × 106 cells/ml in serum free conditions and 1 ␮l of DiD dye was added to the cells for each millilitre of suspension. The cells were incubated at 37 ◦ C for 4–8 min and then washed 2–4 times in HBSS by centrifugation for 5 min each at 400 × g. 2.7. Induction of apoptosis Apoptosis of Jurkat cells was induced by 30 s of UV-irradiation (254 nm) with a Stratalinker 2400 crosslinker (Stratagene) using the default power settings (120,000 ␮J/cm2 ). Briefly, cells were washed and irradiated in serum-free conditions in HBSS and incubated for the desired time at 37 ◦ C with 5% (v/v) CO2 environment in a Tris buffer as previously described (10 mM Tris, 140 mM NaCl, 2 mM CaCl2 , 1 mM MgCl2 , 1% (w/v) BSA) (Kim et al., 2002). All experiments and washes involving apoptotic cells were performed using this buffer as the vehicle unless otherwise indicated. Late apoptosis was verified by DNA laddering, examination of nuclear morphology and Trypan Blue exclusion analysis. Briefly, DNA was purified

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from 5 × 106 apoptotic Jurkat T-cells using the GenEluteTM Mammalian Genomic DNA Miniprep Kit according to the manufacturers instructions (Sigma, G1N70) and eluted in a volume of 200 ␮l. Ten microlitres of the purified DNA sample was resolved on a 1% (w/v) agarose gel. Apoptotic cells were also stained with Trypan blue to assess membrane integrity and cells were counted for incorporation of the dye, which indicates permeability and secondary necrosis or late apoptosis. Cells were examined under a microscope to establish nuclear morphology. Cells allowed to undergo apoptosis overnight for a period of 24 h were considered ‘late apoptotic cells’ and are detailed herein as such unless otherwise indicated. 2.8. Flow cytometry Jurkat cells were cultured as described above and 2 × 106 cells were acquired from live culture in a T75 culture flask (BD Bioscience). The cells were UV-irradiated in HBSS as described above and centrifuged at 400 × g for 5 min. Irradiated cells were then resuspended in the indicated Tris buffer containing 1% (w/v) BSA to a concentration of 1 × 106 cells/ml. The cells were allowed to undergo apoptosis for the desired period. Following this, cells were centrifuged for 5 min at 400 × g and then resuspended in 20% (v/v) normal human plasma (NHS), 10 ␮g/ml of IgM (Sigma), or in the Tris buffer with BSA only, and incubated for 30 min at 37 ◦ C. Cells were then washed and centrifuged twice at 400 × g for 5 min. Cells were then stained with FITC-labelled anti-human IgM antibody (5–20 ␮g/ml) by incubating on ice in the dark for 30 min. These labelled cells were then washed once again with Tris buffer and centrifuged at 400 × g for 5 min. Labelled cell pellets were resuspended in 2% (w/v) paraformaldehyde (PFA) fixative and allowed to fix for 30–60 min at 4 ◦ C in the dark. Fixed cells were then centrifuged at 1000 × g for 15 min and resuspended in PBS until they were analyzed by flow cytometry using a FACScan flow cytometer (Becton Dickinson, Mississauga, ON). Secondary antibody controls were used to calibrate the flow cytometer and correct for non-specific binding and auto-fluorescence. For flow cytometry experiments involving IgM, SP-D and propidium idodide, incubation with both IgM and SP-D was preformed as described in Section 2.9. Briefly, apoptotic cells were incubated with IgM for the indicated time of apoptosis, followed by SP-D incubation for 2 h. Following this, cells in suspension were incubated with propidium idodide and were probed for IgM and SP-D using a FITC-labelled anti-␮ antibody and an ALEXA-647 tagged anti-SP-D antibody, respectively. In all cases, cells were analyzed by flow cytometry using both Flow Jo and FCS Express software packages. 2.9. In vitro cell culture for IgM and SP-D cell binding Apoptotic Jurkat T-cells were UV-irradiated as previously described in 24- or 48-well cell culture plates on autoclaved glass cover slips, or glass chamber slides. To establish the appropriate IgM concentration detectable by microscopy, several IgM concentrations were tested from low (10 ␮g/ml) to high (1 mg/ml). Visual examination and flow cytometry indicated that IgM binding saturated approximately at 10 ␮g/ml of IgM. Cells were therefore incubated with human IgM (10 ␮g/ml) in HBSS with 3–5 mM CaCl2 for 24 h at 37 ◦ C, followed by a 2 h incubation with human SP-D at the physiologically relevant concentration of 5 ␮g/ml (Palaniyar et al., 2008; Wright, 2005) at room temperature. Cells were fixed with 2% (w/v) PFA. Fixed cells were blocked with 5% (w/v) BSA for 30 min then IgM was probed with FITC conjugated anti-human IgM antibody (10–20 ␮g/ml) (green) and SP-D was probed with Alexa-555 conjugated anti-human SP-D (10 ␮g/ml) (red). Finally, cover slips were incubated with mounting media containing 4 ,6-

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diamidino-2-phenylindole (DAPI) and slides were adhered to a glass microscope slide and examined with a fluorescence microscope (Leica, Bannockburn, IL, USA). Images were created using the OpenLab (Improvision, UK). For images presented by confocal microscopy, SP-D was probed using an ALEXA-647 tagged antihuman SP-D antibody and presented in the corresponding ‘red’ false colour. Confocal images were analyzed using the Volocity software package (Improvision, UK). 2.10. In vivo mouse experiments Animal utilization protocols necessary for this study were approved by the Research Ethics Board of the Hospital for Sick Children. Labelled late apoptotic cells were adjusted to a concentration of 1 × 106 cells/ml. At the desired time, 1 ml of apoptotic cell suspension containing apoptotic cells (∼106 ) and apoptotic material for each condition was centrifuged for 10 min at 400 × g. This pellet displayed a notable blue hue from the DiD dye and the supernatant was removed and centrifuged at a high speed of 25,000 × g for 20 min to pellet all remaining apoptotic material. This high-speed pellet also displayed a blue hue from the DiD dye, albeit a much smaller sized pellet, and was treated in the same manner as the low speed pellet. Pellets were incubated with IgM (10 ␮g/ml) and/or SPD (5 ␮g/ml) proteins or Tris buffer only in a volume of 100 ␮l for 30 min at 37 ◦ C, followed by a 20–30 incubation on ice. The pellets were then washed one time in Tris buffer to a total volume of 800 ␮l and centrifuged at their respective speeds. The resulting pellets within an experimental condition from each centrifugation speed were combined and resuspended to a volume of 50 ␮l and stored on ice for 30 min. Six to ten week old CD1 mice (Charles River Canada, Saint-Constant, Québec) were gently anesthetized with isoflurane, and these cells (∼106 cells/mouse) were intranasally instilled into the lungs. One day prior to the late apoptotic cell instillation, these mice were intranasally instilled with PKH 67 green fluorescent dye in the phagocytic Diluent B (Sigma) to label alveolar macrophages. Bronchoalveolar lavages were performed on mice intraperitoneally euthanized with Euthanyl (CDMV Laboratories, St. Hyacinthe, Québec). Briefly, thoracic and abdominal cavities of the mice were opened, and exsanguinated, revealing the lungs. The lungs were inflated and deflated 3 times with 1 ml of DPBS (Gibco) using a 1 ml syringe and a BD Angiocath (BD Bioscience). This was repeated 5 times for a total lavage fluid volume of 5 ml. The BAL was kept on ice for the duration of the lavage. Pooled BAL fluid was centrifuged for 20 min at 400 × g at 4 ◦ C and cell pellets were treated with sterile water for 10 s to lyse residual erythrocytes followed by a 1:9 dilution of the water with complete RMPI cell culture media. The cells were plated in chambers of 8-well chamber slides (BD Falcon) and macrophages were allowed to adhere to the glass slides for 30 min at 37 ◦ C. In some experiments a portion of the cells were removed prior to incubation for a cytospin followed by a differential cell staining. After 30 min, cells that did not adhere to the chamber slide were removed and fixed with 2% (w/v) PFA for 1–18 h at 4 ◦ C. The remaining adherent cells were washed 1–2 times with buffer or media and fixed with 4% (w/v) PFA. Fixed cells were stained with DAPI and slides were mounted using Dako Fluorescent Mounting Media (DakoCytomation). Slides were left to dry for at least 24 h and were examined using Z-stack imaging from a spinning disc confocal microscope (Carl Zeiss Canada, Toronto) and analyzed in two and three dimensions for multiple fluorescent channels with Volocity (Improvision, UK). 2.11. Calculation of attachment and phagocytic indices Phagocytic indices were calculated by counting the number of macrophages in a field, which contained material from apoptotic cells and dividing that value by the total number of

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macrophages counted within the field. For our assays, this represents the percentage of alveolar macrophages involved in late apoptotic cell uptake. Values obtained from multiple image fields from 1 chamber in each experiment were combined to generate phagocytic indices for each mouse separately. Experiments were repeated 4 or more times and data are presented as the mean ± SEM. 2.12. Protein measurements Bronchoalveolar lavage fluid obtained from mice as described above was measured for TNF␣ concentrations using the mouse TNF␣ DuoSet ELISA Development kit (DY410) from R&D Systems, Inc. (Minneapolis, MN, USA) and for total protein concentration with the BCA® Protein Assay kit from Pierce (Rockford, IL, USA) both by following the package insert instructions. 2.13. Tissue sectioning Mice intranasally instilled with late apoptotic cells were sacrificed and their lungs were extracted, preserved, sectioned, stained and examined as described previously (Cao et al., 2009). Briefly, excised lungs were fixed overnight with 4% (v/v) PFA in PBS at 4 ◦ C and then dehydrated using a series of ethanol/xylene incubations, followed by embedding in paraffin wax. Lung tissue was sectioned in 5–10 ␮m slices onto a microscope slide and stained with Haemotoxyline and Eosin and air dried. Mounted slides were examined and photographed using a Leica digital imaging system at 40× magnification. 2.14. Statistical analysis Where applicable multiple means comparisons were completed using an analysis of variance (ANOVA) with a Tukey–Kramer HSD post hoc analysis using JMP software. A Student’s t-test was used to compare means from 2 samples using Microsoft Excel. Means were considered statistically significant with a p-value less than or equal to 0.05, unless otherwise indicated. 3. Results 3.1. SP-D binds to IgM Our previous studies show that SP-D interacts with several classes of antibodies including IgM (Nadesalingam et al., 2005). To verify the binding of SP-D to various preparations of IgM we performed a series of ligand blots and ELISA-style binding experiments using different preparations of purified IgM (Sigma, Calbiochem and Oxford) as targets for SP-D. We also purified IgM from normal human plasma using the IgM HiTrap HP binding column (GE Healthcare) and gel filtration chromatography and used these preparations in these assays. We show using ligand blots that the degree of SP-D:IgM interactions varies to some extent depending on the commercial IgM preparations (Fig. 1a). We also confirmed by ELISA that SP-D could bind to the commercial IgM in a concentration-dependent manner (Fig. 1b) and to the IgM we purified (Fig. 1c). These data confirmed that SP-D interacts with IgM purified from different lots of plasma. 3.2. IgM and SP-D bind to late apoptotic cells A previous study suggested that IgM present in NHS would bind late apoptotic cells (Kim et al., 2002). To establish the appropriate apoptotic stages for our studies, we rendered human Jurkat T-cells apoptotic by ultraviolet light as described in materials and methods. We verified the progression of apoptosis by DNA

Fig. 1. SP-D binds to IgM. (a) Ligand blots showing the interactions between SP-D and immobilized IgM [Sigma (S), Calbiochem (C) and Oxford (O)]. Positive control is SP-D and negative control is gelatin. (b) ELISA of serial dilutions of IgM as a target for SP-D binding. (c) ELISA showing that SP-D binds to IgM purified in our lab from normal human plasma (* p < 0.05).

laddering (Fig. 2a), Trypan blue staining (Fig. 2b), and nuclear morphology (Fig. 2c). At 24 h post UV irradiation, most of the cells (>70%) were non-viable and reached late apoptotic or secondary necrotic cell state. To determine the interaction between IgM and apoptotic cells, we used IgM from two different sources (normal human plasma or purified IgM). Using flow cytometry we found that IgM present within normal human plasma (Fig. 2d) and purified IgM obtained from Sigma (Fig. 2e) both sufficiently bound to late apoptotic cells. A significant degree of IgM binding occurred when the cells were undergoing apoptosis for 6–24 h. We then used propidium iodide (PI) staining to better define the nature of the apoptotic cells to which IgM binds and to determine if SP-D binds to the same cells. First we show that at 6 h (Fig. 3a, left) following UV-irradiation nearly half of the cells are late apoptotic or secondary necrotic cells (48.4%), whereas at 24 h (Fig. 3a, right) more than 60% (63.8%) of the cells are late apoptotic or secondary necrotic cells. We examined the degree of IgM staining for these cells at both 6 and 24 h in the absence (Fig. 3b) or presence of SP-D (Fig. 3c). In both cases at 6 h, more than 20% of the cells stained positive for IgM, but in the presence of SP-D this population was increased in proportion by nearly 10% (without SP-D, 24.1%; with SP-D, 33.46%). This 10% increase in IgM-positive cells was similarly observed in the 24 h cells (without SP-D, 62.1%; with SP-D, 73.3%). In separate sets of experiments, we evaluated the proportion of cells to which IgM and SP-D both bind (Fig. 3d). At 6 h, these IgM:SP-D double positive cells represented only 7.9% of cells analyzed. In contrast, at 24 h, this IgM:SP-D double positive cell population represented 31.9% of cells analyzed. Almost all of these SP-D binding cells were also IgM binding cells (d, 24 h; upper right quadrant). This constitutes a nearly 4-fold increase in

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Fig. 3. Flow cytometry showing that IgM and SP-D bind to late apoptotic (secondary necrotic) cells. (a–c) Propidium iodide (PI) staining of apoptotic cells at 6 h (left panels) or 24 h (right panel). (a) PI staining of the apoptotic cells in the absence (dotted line) or presence of SP-D (5 ␮g/ml; solid line). At 6 and 24 h, 47.2% (46.0–48.4%) and 62.9% (61.9–63.8%) of the cells are secondary necrotic, respectively. (b and c) IgM (10 ␮g/ml) binding to the cells in the absence (b) or presence of SP-D (c; 5 ␮g/ml). IgM binds to 24.1% and 62.1% of the cells at 6 and 24 h, respectively (left quadrants). In the presence of SP-D, 9.4–11.3% more cells are interacting with IgM (top 2 quadrants). (d) Separate sets of experiments showing that 7.85% and 31.88% of the cells are binding both IgM and SP-D at 6 h and 24 h, representing 20.1% and 44.3% of the IgM-positive population, respectively. PI (Texas Red channel); IgM, FITC (Alexa488 channel); SP-D, Alexa647 (APC channel).

Fig. 2. IgM binds to late apoptotic cells. (a) Agarose gel showing laddering of DNA isolated from Jurkat cells exposed to ultraviolet light—a hallmark of apoptosis. (b) Trypan blue exclusion vitality assay. Cells were counted for incorporation of Trypan blue indicating cell permeability and late apoptotic cells. (c) Nuclei of apoptotic cells stained with DAPI. Arrows indicate nuclei displaying typical blebbing of apoptotic cell nuclei. (d) Flow cytometry showing that IgM in normal human plasma (NHS) preferentially binds to a subpopulation of late apoptotic cells (6–24 h). (e) Flow cytometry showing that purified IgM purchased commercially (Sigma) also sufficiently binds to these apoptotic cells (6–24 h). In both (d) and (e) cells were probed with FITC-labelled anti-human IgM antibody.

double-positive cells at 24 h post UV-irradiation. The double staining population at 6 h was 20.9% of the IgM binding cells (d, top 2 quadrants, 37.6%). At 24 h, this double staining population was 44.3% of the IgM binding cells (d, top 2 quadrants; 71.9%). Therefore, at 24 h, 2-fold more IgM binding cells also bound SP-D. Taken together, these data illustrate that IgM binds to a population of late apoptotic secondary necrotic cells that stain positive for PI, and cells undergoing apoptosis for 24 h effectively bind both IgM and SP-D. SP-D primarily binds to the cell population to which IgM binds.

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(secondary necrotic) cells. Therefore, to better characterize the three-way interaction and distribution amongst IgM, SP-D and apoptotic cells we next co-incubated late apoptotic Jurkat T cells with IgM and SP-D, and then probed for these two immune proteins using fluorescent labels. We examined the distribution of IgM and SP-D binding on these 24 h late apoptotic cells (Fig. 4a, S1). Using fluorescence microscopy we found that some cells bound neither IgM nor SP-D, while others bound IgM in a diffuse or punctate pattern. Many cells bound both IgM and SP-D in a punctate pattern while exhibiting similar regional binding. This type of information cannot be obtained by flow cytometry. Therefore, we manually counted the number of different colour dots present on each cell (Fig. 4b). These analyses showed that many cells bound SP-D (red), IgM (green), and both SP-D and IgM (yellow). To determine whether SP-D and IgM truly co-localize with each other, we examined these cells using multi-layered Z-stack confocal microscopy with a layer depth of 0.25 ␮m. These data illustrate that IgM and SP-D bind to late apoptotic cells in mutually distinct regions while also exhibiting co-localization with each other (Fig. 4c). 3.4. Primary alveolar macrophages engulf late apoptotic cells, in vivo To determine whether late apoptotic cells can be internalized by AMs in vivo, we instilled late apoptotic cells into mouse lungs. Using distinct fluorescent labels to denote AMs (PKH 67, green)

Fig. 4. IgM and SP-D bind to late apoptotic cells in mutually distinct regions and co-localize on the cell surface. Late apoptotic cells were incubated with IgM and SP-D, then probed with fluorescent antibodies as described in materials and methods. Cell nuclei are stained blue with DAPI. Fluorescence microscopy identified a spectrum of four distinct populations of late apoptotic cells to which SP-D (red) and IgM (green) bind. (a) Cells are organized in 3 rows and 4 columns. Cell 1,1 represents a cell that binds neither SP-D nor IgM whereas cells 1,2 through 1,4 represent cells predominantly binding SP-D. Cells in row 2 display binding of SP-D with an increased frequency of IgM binding moving across the row from left to right. Row 3 exemplifies cells that predominately bind IgM, as in cell 3,1 on the left, to cells decorated with both IgM and SP-D, as in cells 3,3 and 3,4 (scale bar indicates 5 ␮m). (b) To better determine the relative proportions of these 4 cell populations, we analyzed the protein localization on late apoptotic cells by the number of IgM, SP-D, or dual colour (yellow) spots on the cells. The x-axis represents an arbitrary cell reference number while the y-axis represents the absolute number of coloured spots identified on each cell. Cells are ranked by increased number of red (SP-D) dots identified. (c) Confocal microscopy image of cells exemplifying the mutually distinct and co-localized regions of IgM and SP-D binding to the cell surface. Arrows identify regions of co-localization of IgM and SP-D on the cell surface—scale bar represents 5 ␮m (cells are shown without DAPI to present a clearer image).

3.3. IgM and SP-D co-localize on the surface of late apoptotic cells It has been shown previously that SP-D binds to apoptotic cells (Clark et al., 2002; Jakel et al., 2010; Palaniyar et al., 2003a; Vandivier et al., 2002); however, as we have established in Section 3.2, SP-D and IgM bind preferentially to the late apoptotic

Fig. 5. Alveolar macrophages internalize late apoptotic cells in vivo. (a) Bright field confocal image of AMs obtained from mice instilled intranasally with late apoptotic cells. (b) Deconvolved fluorescence confocal image of single plane from a Z-stack (same field as in ‘a’). The ‘+’ indicates the focal object of the field shown on the top (x) and the side (y). (c) Three dimensional structure of the cells present in the same field as in a and b. The image panel was rotated 100 degrees counterclockwise to provide a clear view of different cells. Colour threshold values were lowered to show the internal details of the cells. Cytoplasmic areas of AMs are green (PKH 67), late apoptotic cells and material from these cells are blue (DiD), and nucleus is purple (DAPI). These data indicate that alveolar macrophages internalize apoptotic cellular materials, in vivo.

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and late apoptotic cells (DiD, blue), we examined AMs obtained from bronchoalveolar lavages using confocal microscopy. To verify that apoptotic cells or materials derived from these cells were truly internalized by AMs, we performed 2-dimensional multiple Z-stack imaging (Fig. 5a and b). These images clearly show the presence of apoptotic cellular materials inside these alveolar macrophages (Fig. 5b). The 3D reconstruction shows the location of the internalized apoptotic material in the AMs (Fig. 5c). Unlike regular fluorescence microscopy, this method generated high quality images with precise phagocytic indices. Although laborious, these experimental and imaging procedures that we use are effective tools to assess the clearance of materials by AMs in vivo. These data indicate that late apoptotic cells are internalized well by AMs in vivo.

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3.5. Late apoptotic cells do not cause lung inflammation Lung tissues can be sensitive to inflammation, which can influence apoptotic cell clearance. To determine whether or not late apoptotic cells cause inflammation on their own we analyzed an array of inflammatory markers from mice instilled with late apoptotic cells. We performed differential cell counts of cells obtained from the BAL to identify macrophages and neutrophils. In all of the experimental conditions, mice instilled with late apoptotic cells for 4 h or 1 day, more than 85–95% of cells counted were macrophages (Fig. 6a). We performed a comparative ELISA to measure the TNF␣ levels in the BAL fluid of mice instilled for 4 h or 1 day with or without late apoptotic cells coated with IgM, SP-D, both, or neither (Fig. 6b). No measurable difference from the buffer control

Fig. 6. Late apoptotic cells do not cause inflammation in the mouse lung. (a) Differential cell counts for neutrophils and macrophages show that macrophages represent the predominant population of cells in the BAL of mice instilled with late apoptotic cells. (b) Mice instilled with late apoptotic cells for 4 h or 1 day show no difference in TNF-␣ levels in comparison to buffer-instilled mice for the same time points. (c and d) Protein concentrations of mouse BAL is similar amongst different coating conditions, including controls, of late apoptotic cells (c) 4 h after instillation or (d) 1 day after instillation. (e and f) Lung tissue sections from mice instilled with late apoptotic cells did not show lung inflammation (e) 4 h after late apoptotic cells instillation nor (f) 1 day after instillation.

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was identified amongst the conditions. To further validate these findings, we measured the total protein content of the BAL fluid obtained from mice instilled for 4 h (Fig. 6c) or 1 day (Fig. 6d) with or without late apoptotic cells coated with IgM, SP-D, both, or neither. Protein levels remained similar amongst all experimental and control conditions. Finally, we performed lung tissue sectioning for different treatment conditions to determine if there were any changes in tissue pathologies. No indications of inflammation were observed in the lung tissues of mice from either time point (Fig. 6e, 4 h; Fig. 6f, 1 day). Taken together these data indicate that late apoptotic cells do not cause detectable lung inflammation. 3.6. IgM and SP-D levels in the lungs and apoptotic cell clearance increases during lung inflammation Inflammation is known to alter the functional properties of macrophages (Chen et al., 2004; Medzhitov, 2008). We have established that late apoptotic cells do not cause inflammation by themselves; however, whether or not inflammation will alter the uptake of late apoptotic cells is not clear. To test this, we induced lung inflammation in mice using LPS (5 ␮g/ml; 250 ng/25 g mouse) and instilled late apoptotic cells intranasally into the airways of mice (co-administration of late apoptotic cells and LPS). Bronchoalveolar lavages were performed on the mice 4 h after instillation to assess the immediate effects of inflammation and clearance, and 1 day after instillation to assess the later clearance activities. Differential cell counting showed the presence of neutrophilic lung inflammation in these mice at both of the time points (data not shown). In addition to evaluating the differential cell populations in the BAL of LPS-instilled mice, we performed Western blot analysis to assess SP-D levels. Western blot analysis indicated that SP-D levels do not significantly change in mice instilled with LPS within 4 h; however, a significant difference between LPS-instilled and control mice occurred after 1 day (Fig. 7a). We have shown in Sections 3.1 and 3.3 that IgM can bind preferentially to late apoptotic cells and thus, we examined and compared the IgM concentration in BAL fluid obtained from mice instilled with apoptotic cells in the presence or absence of LPS. We found that over the course of 1 day the IgM concentration in the lungs increased significantly by nearly 3-fold (Fig. 7b). This data highlighted the relevance of IgM and SP-D in our studies. Alveolar macrophages are known to exist in two different forms (adherent and non-adherent cells (Spiteri et al., 1992; Watford et al., 2002)). We assessed the phagocytic index (PI) of both catagories of AMs to provide more complete information on the uptake of late apoptotic cells, in vivo. Prior selection of these two cell populations is not feasible in these in vivo experiments; hence, we have separated the two AM populations at the end of the experiment and determined their phagocytic indices, separately. The phagocytic index is indicative of the ability of the lungs to clear apoptotic material, and adherent AMs (Fig. 8a) obtained 4 h after instillation displayed phagocytic indices of 0.19 ± 0.02 without LPS and 0.31 ± 0.03 with LPS (p < 0.05). Cells obtained 1 day after instillation displayed phagocytic indices of 0.39 ± 0.08 without LPS and 0.62 ± 0.03 with LPS (p < 0.05). The non-adherent macrophages displayed a similar pattern of clearance potential. Non-adherent AMs (Fig. 8b) obtained 4 h after instillation displayed phagocytic indices of 0.12 ± 0.05 without LPS and 0.32 ± 0.03 with LPS (p < 0.05). Cells obtained 1 day after instillation displayed phagocytic indices of 0.10 ± 0.02 without LPS and 0.42 ± 0.05 with LPS. These data indicated that both adherent and non-adherent AMs from mice instilled with LPS and late apoptotic cells significantly increased clearance of the late apoptotic cells both within 4 h and over 1 day compared to control mice receiving instillations of late apoptotic cells without LPS. Taken together these data indicate that late apoptotic

Fig. 7. SP-D and IgM levels increase in the BAL of mice during LPS-induced inflammation. (a) Western blot analysis (top) for SP-D in BAL fluid from mice instilled with or without LPS for 4 h or 1 day (top). Densitometry of the blot (bottom) reflecting the differences between mice instilled with or without LPS for 4 h or 1 day. Densitometry values were compared to the mean control value; n = 3 mice, * p < 0.05. (b) ELISA measuring the IgM concentration of BAL fluid from mice intranasally instilled with LPS and apoptotic cells for a duration of 4 h or 1 day (data presented as mean ± SEM, n > 4 for each condition; p < 0.01).

cells and material derived from these cells can be internalized by AMs in vivo and that late apoptotic (secondary necrotic) cells do not cause notable lung inflammation. Furthermore, during lung inflammation, IgM and SP-D levels in the lungs increased 2–3fold, and clearance of late apoptotic cells by AMs was increased significantly. 3.7. IgM and SP-D enhance uptake of late apoptotic cells in the absence of lung inflammation, in vivo After determining that IgM and SP-D levels significantly increase in the lungs during LPS-induced inflammation, we sought to determine if the interactions we describe between IgM, SP-D and late apoptotic cells could generate a functional effect within the lungs. To assess this functional effect we conducted exper-

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Fig. 8. Lung inflammation enhances the clearance of late apoptotic cells from the mouse lungs. Phagocytic indices obtained from (a) adherent and (b) nonadherent AMs 4 h or 1 day following LPS and late apoptotic cell instillation. Alveolar macrophages of both types engulf apoptotic material at a significantly higher rate during lung inflammation (data presented as mean ± SEM, n > 4 for each condition; some of these controls are included in Fig. 9; p < 0.05).

iments where there were no confounding factors using healthy naïve mice. We coated late apoptotic cells with IgM (10 ␮g/ml) and/or SP-D (5 ␮g/ml), or buffer and instilled them intranasally into the mouse airways. We obtained AMs by BAL 4 h or 1-day post instillation. The phagocytic indices of adherent (Fig. 9a) and nonadherent (Fig. 9b) AMs were calculated for both time points and for all four protein-coat conditions. During these non-inflammatory conditions, adherent AMs successfully performed uptake of late apoptotic cells and material within 4 h. No significant difference in mean phagocytic index (PI) was observed at this time point between the different protein coating conditions (Fig. 9a). However, the adherent AMs obtained from mice lavaged 1 day after instillation displayed higher mean phagocytic indices than those obtained from the lavages 4 h after instillation (Fig. 9a). Alveolar macrophages from mice instilled with late apoptotic cells coated with IgM or SP-D showed a significantly higher mean phagocytic index (0.70 ± 0.05, IgM) or (0.47 ± 0.06, SP-D) compared to the phagocytic index of the AMs from non-coated control late apoptotic cells (0.34 ± 0.04) (p < 0.05). Moreover, the combination of coating apoptotic cells with IgM + SP-D also significantly increased

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Fig. 9. IgM and SP-D enhance the uptake of late apoptotic cells by AMs in naïve mice. (a) Phagocytic indices of adherent AMs obtained from mouse lungs 4 h or 1 day after mice were intranasally instilled with late apoptotic cells coated with or without IgM and SP-D. Late apoptotic cells coated with IgM or SP-D were engulfed at a significantly higher rate compared to control (non-coated) cells 1 day after instillation. (b) Phagocytic indices of non-adherent AMs from mouse lungs 4 h or 1 day after mice were intranasally instilled with late apoptotic cells coated with or without IgM and SP-D. Late apoptotic cells coated with either IgM or SP-D or both were internalized at a significantly higher rate compared to control cells while cells coated with only SP-D were internalized more effectively than any of the other conditions after 1 day. Data presented as mean ± SEM, n ≥ 4 for each condition; * denotes that the values are significantly different from control cells or ** all other conditions; p < 0.05).

the phagocytic index of AMs (0.61 ± 0.08, IgM + SP-D) to controlled conditions. Non-adherent AMs obtained from lavages 4 h after instillation did not show significant differences in uptake of protein coated late apoptotic cells as measured by phagocytic index (Fig. 9b). Conversely, the non-adherent AMs that were obtained 1 day after instillation displayed significant phagocytic indices for late apoptotic cells that were coated with any combination of IgM or SP-D compared to the control (non-coated cells). Non-adherent AMs from mice receiving late apoptotic cells coated with SP-D displayed the greatest enhancement of phagocytic index compared to control mice (receiving non-coated late apoptotic cells) while late apoptotic cells coated with either IgM or the combination of IgM + SP-D also displayed a significant enhancement (SP-D, 0.65 ± 0.05; IgM, 0.40 ± 0.03; IgM+SP-D, 0.29 ± 0.03; no protein, 0.10 ± 0.03; Fig. 9b; (p < 0.05). The clearance enhancement observed in the SP-D-coating condition is significantly greater than that observed in the IgM- and IgM + SP-D coating condition; however, in the presence of IgM + SP-D, the effect attributed to SP-D is reduced and these AMs exhibit increased phagocytic indices that significantly differ from the control (non-coated cells) only. Overall, these data indicate that the natural IgM and the innate immune pattern recognition protein SP-D enhance the uptake of late apoptotic cells to different extent by AMs in the mouse lungs, in vivo.

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4. Discussion We have previously shown that the innate immune protein SPD can interact with various adaptive immune immunoglobulins (Nadesalingam et al., 2005). Here we show that SP-D binds to immobilized IgM in a concentration dependent manner as is depicted by the ligand blot (Fig. 1a). This also indicates that three separate commercially available preparations of IgM provided substantial targets for SP-D to bind. We show by ELISA (Fig. 1b) that IgM purified in our lab could also act as a target for SP-D to bind to IgM. The source of all of these IgM preparations is pooled normal human serum. This shows that SP-D has the ability to bind to various preparations of IgM derived from normal pooled plasma from different sources. We then illustrate that IgM from purified preparations and from NHS can bind to late apoptotic (secondary necrotic) cells generated by ultraviolet-light exposure (Figs. 2 and 3) and that SP-D can bind to a population of these cells (Fig. 3). We show that IgM and SP-D bind to independent locations and also co-localize on late apoptotic cells (Fig. 4) and that late apoptotic cells are successfully internalized in vivo by AMs (Fig. 5) and do not cause lung inflammation (Fig. 6). We also show that during LPS-induced lung inflammation, airway levels of SP-D and IgM (Fig. 7a, b) and the ability of AMs to perform uptake of apoptotic cells in vivo increase (Fig. 8). Finally, we show that late apoptotic cells coated with IgM and SP-D are cleared by lung macrophages more efficiently than control buffer uncoated cells (Fig. 9). These results suggest a role for these two interacting proteins in clearing late apoptotic cells from the lungs. In a previous study it was shown that IgM, but not IgG, can bind to apoptotic cells that are in the later stages of apoptosis when the apoptotic cells are incubated with normal human serum (Kim et al., 2002). Additional studies indicate that IgM contained within serum plays a critical role in systemic apoptotic cell clearance through its interaction with other serum proteins (Ogden et al., 2005; Peng et al., 2005; Zwart et al., 2004). Other studies have illustrated that naturally occurring IgM and monoclonal IgM antibodies will bind to oxidation-specific epitopes on late apoptotic cells (Chou et al., 2009; Ciurana and Hack, 2006; Das et al., 2008). Here we show that purified IgM binds to these late apoptotic cells with a comparable efficiency to that of IgM contained within pooled normal human serum preparations (Fig. 2). This illustrates explicitly that IgM does not require other serum components to bind to late apoptotic cells. Although SP-D is known to bind apoptotic Jurkat T-cells (Jakel et al., 2010; Palaniyar et al., 2004), its binding pattern on these cells is unknown. Our data indicates that SP-D binds to regions where IgM is present and co-localizes with IgM, but that the SP-D and IgM can bind to mutually distinct regions of the late apoptotic cells (Fig. 4). It is worth noting that natural IgM has been shown to function in autoimmunity (Zhang and Carroll, 2007). Hence, natural human IgM may be considered as an innate immune opsonin of late apoptotic cells in this context. Under conditions of severe lung inflammation, other serum proteins may leak into the edema fluid of the airways (Sarma et al., 2006). Additionally, IgM can enter the lung via epithelial cell transcytosis (Jaffar et al., 2009; Rojas and Apodaca, 2002). Thus, our finding that SP-D binds to IgM on late apoptotic cells represents a condition for potential competition with other serum proteins that IgM may interact with on late apoptotic cells. Previously published studies have used cell lines and primary peritoneal macrophages as model phagocytes to study the clearance of early apoptotic cells (Fu et al., 2007; McDonald et al., 1999; Ogden et al., 2005; Peng et al., 2005). Therefore we initially used cell-lines as our model to examine late apoptotic cell clearance by macrophages in vitro. It became clear that macrophage cell-lines were not adequate to study the clearance of late apoptotic cells (data not shown). Based on ex vivo studies, AMs are considered to be poor eaters of apoptotic cells (Hu et al., 2000).

However, apoptotic cells are effectively cleared in the lung. Therefore we focused our studies in vivo. There are two major contrasts of our work with that of others. First, we study late apoptotic cells while others exclusively study early apoptotic cells (Fadok et al., 2001; Hoffmann et al., 2001; Hu et al., 2000; Janssen et al., 2008; Licht et al., 2004, 1999). The late apoptotic cells may represent a population that becomes deficient in ‘eat me signals’ (Grimsley and Ravichandran, 2003). Second, the common technologies used to identify phagocytic events include flow cytometry, differential staining for light microscopy or regular fluorescence microscopy. These microscopy technologies are usually limited to 2-dimensional descriptions of cell-cell interactions. Data presented here are based on cell counts obtained from confocal microscopy and 3-dimensional Z-stack imaging using automated imaging at 0.25 ␮m increments. This technique ensures precise identification that the material derived from apoptotic cells is truly internalized by macrophages, and our phagocytic indices represent the relative proportion of real phagocytic events as depicted in Fig. 5c. After establishing that AMs are active in internalizing late apoptotic cells in vivo we considered how inflammation would influence the uptake of late apoptotic cells. We found that during the 1day period of inflammation, but not the 4-h period, SP-D levels in the inflamed mouse lungs increased (Fig. 7a). We then show that BAL fluid obtained from mice lungs during inflammation showed significantly higher concentrations of IgM compared to control mice (Fig. 7b). Additionally, here we show that both adherent and non-adherent AMs show increased phagocytic potential for late apoptotic cells during lung inflammation (Fig. 8a and b). Taken together these results suggest that the increased levels of IgM and SP-D found in the lungs during inflammation could influence the clearance of late apoptotic cells. We then performed experiments in the absence of inflammation to eliminate any confounding effects and showed that late apoptotic cells pre-coated with IgM or SP-D were cleared by adherent and non-adherent macrophages at a significantly higher rate compared to control (non-coated) late apoptotic cells (Fig. 9). Overall, our data indicates that IgM enhances the clearance of late apoptotic cells in lungs, while other studies suggest that IgM can enhance the clearance of apoptotic cells in other tissues (Fu et al., 2007; Ogden et al., 2005; Peng et al., 2005). The tissue-specific late apoptotic cell clearance is likely to be different because each tissue contains different groups of immunomodulatory proteins. In summary, the results we show here illustrate that the innate and natural immune proteins SP-D and IgM can interact with each other on late apoptotic cells and that both IgM and SP-D increase the clearance of late apoptotic cells from lungs. Under the conditions tested, SP-D:IgM interactions occurring on late apoptotic cells appear not to interfere with the clearance of these cells by alveolar macrophages. These results may provide a strategy for removing the late apoptotic cells that remain in the lungs during chronic lung inflammation. Thus these results should help to clarify the regulation of apoptotic cell clearance, lung inflammation and the associated diseases. Acknowledgements We thank the Laboratory Animal Services (LAS) members at the Hospital for Sick Children, Toronto, Canada for their help in maintaining our animals. This study was funded by CIHR (MOP-84312; N.P.) and CIHR (MOP-134761; N.P.); M.L.L. is a recipient of Sickkids Restracomp/OSOTF and of the Peterborough K.M. Hunter Graduate Studentships. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molimm.2010.09.014.

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