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The Journal of Immunology, 2005, 174: 7352-7358.
Copyright © 2005 by The American Association of Immunologists

Innate Immune Collectin Surfactant Protein D Enhances the Clearance of DNA by Macrophages and Minimizes Anti-DNA Antibody Generation1

Nades Palaniyar2,*,{dagger}, Howard Clark*, Jeya Nadesalingam*, Michael J. Shih*, Samuel Hawgood{ddagger} and Kenneth B. M. Reid*

* Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom; {dagger} Lung Biology Research Program, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada; and {ddagger} Department of Pediatrics and Cardiovascular Research Institute, University of California, San Francisco, CA 94143


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dying microbes and necrotic cells release highly viscous DNA that induces inflammation and septic shock, and apoptotic cells display DNA, a potential autoantigen, on their surfaces. However, innate immune proteins that mediate the clearance of free DNA and surface DNA-containing cells are not clearly established. Pulmonary surfactant proteins (SP-) A and D are innate immune pattern recognition collectins that contain fibrillar collagen-like regions and globular carbohydrate recognition domains (CRDs). We have recently shown that collectins SP-A, SP-D, and mannose binding lectin recognize DNA and RNA via their collagen-like regions and CRDs. Here we show that SP-D enhances the uptake of Cy3-labeled fragments of DNA and DNA-coated beads by U937 human monocytic cells, in vitro. Analysis of DNA uptake by freshly isolated mouse alveolar macrophages shows that SP-D, but not SP-A, deficiency results in reduced clearance of DNA, ex vivo. Analysis of bronchoalveolar lavage fluid shows that SP-D- but not SP-A-deficient mice are defective in clearing free DNA from the lung. Additionally, both SP-A- and SP-D-deficient mice accumulate anti-DNA Abs in sera in an age-dependent manner. Thus, we conclude that collectins such as SP-A and SP-D reduce the generation of anti-DNA autoantibody, which may be explained in part by the defective clearance of DNA from the lungs in the absence of these proteins. Our findings establish two new roles for these innate immune proteins and that SP-D enhances efficient pinocytosis and phagocytosis of DNA by macrophages and minimizes anti-DNA Ab generation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
On average, an adult human inhales ~10 liters of air every minute with ~107 microorganisms, which spread over the enormously large alveolar surface area (>100 m2) of the lung. To effectively remove these microbes and their debris (1, 2, 3), as well as dying epithelial cells and phagocytes (4), higher organisms use multiple pathways that include physical, chemical, and biological methods (5, 6, 7). The thin alveolar lining consists only of a single layer of epithelial cells and an overlay of pulmonary surfactant, an oily substance that contains surfactant proteins (SP-) 3 (10%, w/w) and lipids (90%, w/w) (6, 7). The innate immune collectins, SP-A (~90%, w/w of SPs) and SP-D (~3%, w/w of SPs), are the major hydrophilic proteins found in pulmonary surfactant and are primarily responsible for maintaining an infection- and inflammation-free lung (7, 8, 9, 10). During acute lung infections, these innate immune molecules opsonize and enhance the phagocytosis of microbes by freshly recruited polymorphonuclear leukocytes. The same collectins also bind to apoptotic polymorphonuclear leukocytes and alveolar macrophages (AMs), and enhance their clearance by healthy resident macrophages (11, 12). Ligands and regulatory proteins (13, 14, 15, 16) that are involved in the identification and clearance of apoptotic cells are not completely understood.

SP-A and SP-D are named surfactant proteins because of their discovery in pulmonary surfactant, but recent studies show that these collectins, SP-D in particular, are present in many exocrine secretions and mucus membranes (17). Mannose-binding lectin (MBL) is a liver-secreted acute phase collectin, primarily present in the serum and activates complement via the lectin pathway (18). A typical collectin is composed of polypeptide chains containing a short interchain disulfide bond-forming N-terminal domain, a collagen-like region with Gly-X-Y repeats (where X is any amino acid and Y is often hydroxyproline or hydroxylysine), an {alpha}-helical hydrophobic neck region and a C-terminal globular carbohydrate recognition domain (CRD) (19, 20, 21). Three of these polypeptides form a trimeric subunit, which assembles to form a higher order structure. The oligomeric assembly of SP-D (21, 22) resembles that of an "X" (4 subunits) or "asterisk" (>10 subunits) whereas the other collectins SP-A and MBL (2–6 subunits) and a noncollectin, the complement system protein C1q (6 subunits) appear as a "bouquet of flowers" (5, 22, 23, 24). Although the trimeric subunits of the collectins have limited affinity (µM) for several carbohydrate targets, their oligomeric assembly provides high avidity so that the proteins bind to ligands selectively and with high affinity (nM–pM).

In the lung, SP-A and SP-D opsonize microbes, allergens, and other foreign bodies to varying degrees, and signal their clearance by resident AM and other leukocytes (7). These proteins also bind some of the well-known inflammation-causing ligands from bacterial cell walls, such as LPS, peptidoglycan, and lipoteichoic acid primarily via hexose sugars (5). We have recently shown that SP-D also binds to proteoglycans with long glycosaminoglycan chains such as decorin via both protein-protein and protein-carbohydrate interactions, and SP-D-decorin interaction may be important in limiting tissue inflammation (25).

Nucleic acid is a pentose-based anionic phosphate polymer, and DNA and RNA are usually present within organelles/nucleus and cytoplasm, respectively. Apoptotic cell death results in the fragmentation of DNA and its subsequent display on their surface as blebs (4). Inefficient removal of apoptotic cells leads to disintegration of its contents and the formation of necrotic cells (4). These leaky cells eventually release their intracellular components, and many of these components elicit tissue inflammation. Furthermore, certain pulmonary pathogens such as Pseudomonas aeruginosa actively secretes DNA on to the extracellular matrix to form active biofilm and subsequently establish chronic infection (3). Removal of dying cells and their components, including DNA, is therefore essential to maintain inflammation-free tissues and prevent autoimmune diseases (4). Although the multiple pathways and proteins involved in the apoptotic process have been studied in great detail, clearance of cellular debris and autoantigens is poorly understood. We have recently shown that free DNA and RNA as well as the DNA present on apoptotic cells are a novel class of ligands for collectins SP-A, SP-D, and MBL (26, 27, 28).

We hypothesized that innate immune collectins can bind free DNA and DNA present on the apoptotic and necrotic cells, enhance their phagocytosis, and minimize autoantibody generation. Here we show that collectins, SP-D in particular, effectively enhance the uptake of free DNA and DNA-containing beads by macrophages and reduce the generation of anti-DNA autoantibodies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Chemicals

All the chemicals were purchased from Sigma-Aldrich unless otherwise stated.

DNA and proteins

Plasmid DNAs were purified by midiprep procedures (Qiagen) whereas genomic DNA was isolated from mouse lung by DNeasy tissue kit (Qiagen). Genomic DNA (10–50 µg) was labeled with Cy3-dUTP (Amersham Biosciences) or biotinylated dUTP (Stratagene) by nick translation in the presence of 10 pg/ml DNase I and Klenow DNA polymerase (10 U) for 5–6 h at 15°C (AmershamBiosciences). Agarose gel electrophoresis showed that the average size of DNA after labeling was ~3 kb, with a range of 1–5 kb (data not shown). Labeled DNA was purified by phenol/chloroform extraction and ethanol precipitation or by DNeasy tissue kit (Qiagen). Nucleic acids in mice bronchoalveolar lavage (BAL) fluid were detected with OliGreen (ssDNA) and PicoGreen (dsDNA) quantitation kits (Molecular Probes) by the manufacturer’s instructions. Briefly, 100 µl of sample was added to 96-well MaxiSorp plates (Nunc). Fluorescent label was diluted (1:200) in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. A volume of 100 µl of label was added to the sample and allowed to incubate at 23°C for 5 min. Fluorescence was detected at 485 nm excitation, 530 nm emission, and 55–65 gain with a CytoFluor plate reader (Applied Biosystems). SP-A and SP-D were purified from therapeutic lung lavage obtained from alveolar proteinosis patients as described previously (25).

U937 monocytic cells

U937 cells were maintained in RPMI 1640 with 10% (v/v) FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a 5% CO2 environment. Actively growing cells from flasks (Nunc) were harvested by centrifugation at 1000 x g for 5 min and washed with PBS or HBSS (Flow Laboratories) before experiments.

Knockout mice

SP-A (29) and SP-D (8) genes were ablated by gene-targeting of embryonic stem cells, backcrossed 10 generations into a C57BL/6 genetic background, and maintained at the animal house of the Department of Biochemistry, Oxford University, under barrier facilities (11). For each experiment, AMs from 3- to 12-mo-old SP-A(–/–), SP-D(–/–) and C57BL/6 wild-type mice (Harlan-OLAC) were isolated from the lung lavage by low speed centrifugation (500 x g) and PBS wash. The cell-free supernatant was used for DNA analysis. DNA from 1 ml of these samples was isolated by DNeasy kit (Qiagen) and analyzed in agarose gels (0.8%, w/v). The blood samples obtained from these mice were used for anti-nuclear Ab (ANA) or anti-dsDNA Ab determination.

ELISA for ANA and anti-dsDNA Ab determination

Anti-dsDNA Abs were detected by ELISA as previously described (30). Briefly, polystyrene 96-well plates (Greiner) were coated with 50 µg/ml polylysine in PBS for 1 h at 23°C, washed, and coated with 5 µg/ml plasmid DNA for 18 h. Wells were blocked with 3% (w/v) BSA/PBS, and replaced with diluted sera samples (1:200) in 1% (w/v) BSA, PBS, 0.05% (v/v) Tween 20 buffer, incubated for 3–4 h, washed once with PBS, 0.2% (v/v) Tween 20 buffer, and twice with PBS. The anti-DNA Abs were detected by anti-mouse Ab-HRP conjugate and (3,5,3',5')-tetramethylbenzidine substrate (Bio-Rad). As positive controls, sera samples from MRL/lpr mouse strain (Harlan-OLAC) were used. ANA concentrations were determined by a kit designed to detect these Ags in mouse serum, as instructed by the manufacturer (US Biological). In the ANA ELISA, any absorbance (450 nm) value <2-fold of the blank was considered not positive for the assay.

Pinocytosis of DNA

The Cy3-labeled double-stranded fragments of genomic DNA (2–5 µg) were mixed with different concentrations of proteins (0–25 µg/ml) in HBSS (Flow Laboratories) containing 5 mM CaCl2, and added to RPMI 1640 medium (Invitrogen Life Technologies). This mixture was added to U937 cells (105/well) grown in 24-well plates (Nunc) in RPMI 1640 medium or AMs obtained from the mouse lungs, incubated at 37°C for 0–60 min, washed twice with PBS, fixed in 0.5% (v/v) formaldehyde/PBS, and counted for the intensity of cell-Cy3 fluorescence in a FACS station (BD Biosciences).

Phagocytosis of DNA

To determine the phagocytosis of surface DNA-containing microspheres, 50 µl of 1.3 x 1010 1-µm diameter neutravidin FluoSpheres (Molecular Probes) were sonicated and mixed with PBS or ~3-kb-long fragments of genomic DNA (10 µg) that contained biotin-12 dUTP (nick translation kit; New England Biolabs). The volume of the mixture was brought to 1 ml with PBS and rotated overnight at 23°C. Free neutravidin sites were blocked with excess biotin (10 mM), and the beads were centrifuged at 10,000 x g for 5 min. The pellets were resuspended in 1 ml of PBS, washed three times in the same buffer, and stored at 4°C. Before each experiment the beads were sonicated, mixed with 0–5 µg/ml BSA or SP-D and incubated with 200 µl of 1 x 105 U937 cells or mouse AMs in PBS containing calcium and magnesium or HBSS (ICN Pharmaceuticals) with 2–5 mM CaCl2. Cell to beads ratio was maintained at ~1:50. Phagocytosis of the beads was monitored by FACS analysis after 15, 30, and 60 min sample incubation at 37°C. Both forward and side scatter values were set to 100 to exclude free beads and cell debris from the analyses. Under these settings, 10,000 cells were counted for each sample. In some studies, the cells that contained beads (FL-1 > ~102) were gated to determine the mean fluorescence intensity, and the gated regions are indicated in the figures.

Clearance of DNA from lungs

Plasmid DNA (pUC18) was isolated from 500 ml of Escherichia coli culture by Endofree Giga kit (Qiagen) according to manufacturer’s protocol. Samples were stored in pyrogen-free tubes at –80°C in 10 mM Tris (pH 8), 1 mM EDTA buffer. DNA (10 µg) was diluted in 50 µl of PBS and instilled intranasally to lungs of mice that were anesthetized by isoflurone/O2. After 30 min or 1 h in room air, the mice were sacrificed by CO2 asphyxiation, and their lungs were lavaged with 1 ml of PBS. Cells were separated by centrifugation at 500 x g, and the supernatant was used for the analysis of pUC18 DNA.

Semiquantitative PCR

Mouse cell-free BAL samples were brought to 23°C, vortexed, and recentrifuged for 1 min at 4000 x g. A volume of 25 µl of each sample was heated for 20 min at 95°C. A 10-µl aliquot was added on ice to a PCR mixture including 1x PCR buffer (Roche), 0.2 mM dNTPs (Roche), 0.5 µM M13/pUC sequencing primer (17-mer, 5'-d(GTA AAA CGA CGG CCA GT)-3'; New England Biolabs), 0.5 µM M13/pUC reverse sequencing primer (24-mer, 5'-d(AGC GGA TAA CAA TTT CAC ACA GGA)-3'; New England Biolabs), 1.25 U of TaqDNA polymerase (Roche), and brought to a final reaction volume of 50 µl with dH2O; one drop of mineral oil was then added to each sample. A 120-bp fragment was amplified by a 30 cycle PCR at 95°C, 1 min/53°C, 30 s/72°C, 1 min. PCR products were analyzed using electrophoresis in a 1.2% agarose gel with ethidium bromide stain.

Statistical analyses

Mean, SD, and SE were calculated by Excel software, and p values for the differences between means were determined by Student’s t test with equal variance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
SP-D enhances pinocytosis of DNA by U937 cells

To examine whether SP-D can enhance the uptake of DNA, we labeled mouse lung DNA with Cy3-dUTP by nick translation and incubated it with U937 monocytic cells in the presence of SP-D for different time periods. FACS analyses of Cy3 label distribution showed that SP-D enhanced the uptake of the DNA (Fig. 1). side scatter values indicated that neither SP-D nor Cy3-labeled DNA altered U937 cells during this experimental period (Fig. 1A). The highest amount of label in the cells was detectable after 20–30 min of incubation, at which point, the cell-associated fluorescence subsequently decreased (Fig. 2A). Unlabeled DNA competed with labeled ligand, and its effect was higher in the presence of SP-D (Fig. 2B), suggesting that SP-D facilitated the enhancement of DNA uptake of U937 cells.



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  		FIGURE 1. Effect of SP-D on the pinocytic uptake of DNA by U937 monocytic cells. A, FACS analysis of U937 cells that were incubated with Cy3-labeled mouse lung genomic DNA in the absence or presence of SP-D (5 µg/ml). Side scatter parameter indicates the nature of the cells under various experimental conditions. B, Histogram showing the effect of SP-D (5 µg/ml) on the pinocytosis of Cy3-labeld DNA by U937 cells. Cells in the absence (black line) or presence (red line) of Cy3-labeled DNA are shown. This data set represents one of three experiments.

 


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  		FIGURE 2. Time course of SP-D-mediated pinocytosis of Cy3-labeled DNA. A, Effect of SP-D concentrations ({blacksquare}, 0; {blacktriangleup}, 1; •, 5 µg/ml) on the kinetics of Cy3 label uptake by U937 cells. *, a significant difference between SP-D-treated and nonprotein-treated cells at each time point (p < 0.05). B, Effect of unlabeled DNA in the clearance of Cy3-labeled DNA. *, a significant difference between SP-D treatments in the presence or absence of unlabeled DNA at each time point (p < 0.05, n = 3). This data set represents one of two experiments.

 
SP-D enhances phagocytosis of DNA by U937 cells

To study the uptake of DNA present on the surface of a particle by U937 cells, we coated 1-µm FluoSpheres with mouse genomic DNA fragments and mixed them with the phagocytes. FACS analysis showed no significant differences in the uptake of blank (FL-1 gated mean, 609 ± 105) or DNA (FL-1 gated mean, 698 ± 185)-coated fluorescence beads (p < 0.12). Addition of purified SP-D increased the uptake of DNA-coated beads (FL-1 gated mean, 1037 ± 54) to a greater extent (p < 0.05) than that seen with blank beads (FL-1 gated mean, 770 ± 65) by U937 cells (Fig. 3). Increasing amounts of SP-D increased both fluorescent intensity and the number of cells involved in the phagocytosis (data not shown). Because SP-D but not BSA enhanced the uptake of DNA-containing beads, this collectin specifically enhances the uptake of DNA-containing particles.



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  		FIGURE 3. Effect of SP-D on the phagocytic uptake of DNA-coated beads by U937 monocytic cells. FACS analysis of blank beads or DNA-coated beads by U937 cells in the presence of 5 µg/ml BSA or SP-D. Bead uptake (FL-1 mean) is significantly higher in the presence of SP-D than BSA (p < 0.05). This data set represents one of three experiments.

 
Macrophages of SP-D(–/–) mice are defective in clearing DNA by pinocytosis

To investigate the pinocytic ability of mice in clearing DNA from the lung, we isolated AMs, incubated them with Cy3-labeled mouse lung DNA and analyzed the label present in the phagocytes by FACS. We found that the AMs isolated from the SP-D(–/–) mice were 2–5-fold less efficient in the uptake of DNA compared with those of SP-A(–/–) and wild-type mice (Fig. 4). These results suggest that AMs of SP-D(–/–) mice are defective in clearing DNA.



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  		FIGURE 4. Uptake of Cy3-labeled DNA by alveolar macrophages. AMs isolated from 8-mo-old wild-type (A), SP-A(–/–) (B), and SP-D(–/–) (C) mice were incubated with Cy3-labeled genomic DNA for 20 min, and the cell-associated Cy3 label (FL-2-H) was determined by FACS. The mean Cy3 intensities in wild-type, SP-A(–/–), and SP-D(–/–) mice are 147, 115, and 60 arbitrary units, respectively. The data represent one of three experiments. Tracings indicate FL2-H signal of AMs in the presence (red) or absence (black) of Cy3-labeled DNA.

 
Macrophages of SP-D(–/–) mice are defective in clearing DNA by phagocytosis

To determine whether the AMs of SP-D(–/–) mice have a general defect in phagocytic clearance of particles, we incubated them with inert FluoSpheres. FACS analysis suggested that the label uptake by AMs of SP-D(–/–) mice was ~30–40% less effective than that of the wild-type or SP-A(–/–) mice (Fig. 5A). Although AMs of SP-D(–/–) mice phagocytosed both blank and DNA-containing beads approximately to a similar extent, AMs of wild-type mice showed enhanced uptake of DNA-containing beads (p < 0.05) (Fig. 5A). To further confirm that the defect is related to SP-D, we added purified SP-D to the beads and repeated the experiment. Addition of SP-D specifically and significantly (p < 0.05) enhanced the uptake of DNA-containing beads by AMs of SP-D(–/–) mice (Fig. 5, B and C). These results show that although the AMs of SP-D(–/–) mice are slightly less effective in the phagocytosis of particles, clearance of DNA-containing beads is significantly enhanced by the presence of SP-D. Hence the defect seen in SP-D(–/–) mice is specific to the clearance of DNA.



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  		FIGURE 5. FACS analyses of the uptake of DNA-coated FITC-labeled beads by alveolar macrophages. A, AMs isolated from 4-mo-old mice (n = 4/genotype) were incubated with blank beads or DNA-coated beads, in the presence of 5 µg/ml BSA or SP-D for 30 min. Specific defect in the clearance of DNA beads by SP-D(–/–) mice macrophage population was determined by FACS analysis. Gated mean ± SD values of a typical experiment are indicated in each panel (A). Comparisons between FL-1 means of the bead (B) or DNA bead (C) uptake by AMs from different mice are also shown. *, a significant difference between the indicated data points (p < 0.05). {square}, wild type; {boxtimes}, SP-A(–/–); {blacksquare}, SP-D(–/–). The data (B, C) represent average values of three experiments.

 
DNA accumulates in SP-D(–/–) mice lungs

To determine DNA-related pathophysiological consequences of collectin gene deficiency, we obtained cell-free BAL fluid from SP-A(–/–), SP-D(–/–) and wild-type mice at different ages and measured their absorbance at 260 nm (A260). The BAL of SP-D(–/–) mice had higher absorbance readings, which increased with age, compared with those of SP-A(–/–) and wild-type mice (Fig. 6A). This analysis suggested that SP-D(–/–) mice might contain more free DNA in the lung. Direct analysis of DNA by OliGreen (Fig. 6B) and PicoGreen (Fig. 6C) label fluorometry and agarose gels with ethidium bromide staining (Fig. 6D) confirmed that SP-D(–/–) mice had more DNA in the lung washings than those of the other mice at different ages. A high SD of the data reflected the differences among individual SP-D(–/–) mice (Fig. 6), and it was consistent with the patchy and highly variable lung phenotype known for SP-D(–/–) mice (8, 9). These results suggest that SP-D deficiency leads to age-dependent accumulation of free DNA in the lung.



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  		FIGURE 6. Accumulation of free DNA in the lung. A, A260 of BAL fluid obtained from mice (n = 3–6/group). {diamondsuit}, wild type; {blacksquare}, SP-A(–/–); {blacktriangleup}, SP-D(–/–). Concentrations of ssDNA (B) and dsDNA (C) present in the cell-free BAL supernatant were determined by OliGreen and PicoGreen fluorescence, respectively. *, a significant difference between DNA in SP-D(–/–) BAL with those of SP-A(–/–) and wild-type mice (p < 0.05; n = 5–13/genotype/age group). D, Agarose gel (1%, w/v) showing the DNA present in the cell-free supernatant of BAL fluid at different ages. M, month, Wt, wild type; A, SP-A(–/–); D, SP-D(–/–).

 
SP-D(–/–) mice clear plasmid DNA less efficiently

To directly determine the effectiveness of DNA clearance by SP-A and SP-D, we instilled 10 µg of pUC18 DNA into the lungs of SP-A(–/–), SP-D(–/–), and wild-type mice. The mice were sacrificed after 0.5 or 1 h, and BAL samples were collected. Amount of DNA present in cell-free supernatant was determined by semiquantitative PCR. After 0.5 and 1 h of DNA instillation, only a small amount of DNA was detected in most of the wild-type and SP-A(–/–) mice (Fig. 7A). However, many SP-D(–/–) mice contained a large proportion of the administered plasmid DNA in the BAL fluid. To determine the relative clearance of DNA from the lung, intensity of each DNA band amplified by PCR was quantified and compared with the positive control value. The 100% positive control value was obtained from the PCR amplification of an equivalent amount of DNA that was administered to each mouse. Comparison of the percentages of DNA present in BAL fluid showed that SP-D(–/–) mice cleared bacterial plasmid DNA less effectively than SP-A(–/–) and wild-type controls (Fig. 7B; p < 0.05).



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  		FIGURE 7. Clearance of pUC18 plasmid DNA by mouse lungs. A, PCR results showing the amount of pUC18 plasmid present in the cell-free lung washings of wild type (Wt), SP-A(–/–), and SP-D(–/–) mice, 0.5 and 1 h after DNA instillation. B, Semiquantitative analysis of PCR is shown for each genotype at both time points. The data represent one of three experiments. (0.5 h, open symbols; 1 h, solid symbols).

 
SP-A(–/–)- and SP-D(–/–)-deficient mice generate anti-DNA antibodies

To determine the pathophysiological effect of the increased concentrations of DNA in the lungs, we obtained sera from these mice and analyzed the concentrations of anti-dsDNA and ANAs. The mean (±SE) ELISA readings for both SP-A(–/–) (0.26 ± 0.04) and SP-D(–/–) (0.27 ± 0.03) mice were higher than that of the wild-type mice (0.17 ± 0.02) after 9 mo of age (p < 0.05, n = 32/genotype) (Fig. 8). The analyses showed that a proportion of SP-A(–/–) and SP-D(–/–) mice generated anti-dsDNA Abs above the wild-type background level. The MRL/lpr mouse strain, which is known to contain high serum concentrations of these Abs and systemic lupus erythematosus (SLE)-like disease (31), had 3- to 5-fold higher titer than those of SP-A(–/–) and SP-D(–/–) mice.



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  		FIGURE 8. Age-dependent generation of anti-dsDNA antibody. ELISA results (450 nm) showing anti-dsDNA Ab concentrations in the sera of 6-mo-old (n = 10/group) and 9- to 12-mo-old (n = 32/group) mice.

 
ANAs represent the Abs generated against several intracellular constituents of cells. Although a suggestive trend existed toward an increased ANA concentration in the SP-A(–/–)- and SP-D(–/–)-deficient mice compared with that of the wild-type control mice (n = 10/genotype/age group), these ELISA reading differences did not fully satisfy the positive score criteria defined by the assay kit (data not shown). All of the MRL/lpr mice sera had >10-fold of the absorbance measured for the collectin-deficient mice, and scored positive for the test (n = 5). Hence, although a suggestive trend may exist, collectin-deficient mice did not generate ANA titers that we considered sufficient to classify them as SLE mice. Overall, these results indicate that collectin gene deficiency, particularly that of SP-D, leads to the accumulation of DNA in the lung and anti-DNA Abs in the serum.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Palaniyar et al. (26, 27, 28) have recently shown that three innate immune collectins, SP-A, SP-D, and MBL, bind DNA and RNA to varying degrees. Here we show that collectins, particularly SP-D, enhances pinocytic and phagocytic DNA clearance by monocytic cells, in vitro (Figs. 1–3). In addition, AMs of the SP-D(–/–) mice are less effective in clearing free DNA (Fig. 4) and DNA-containing beads ex vivo (Fig. 5), and the lungs of these knockout mice accumulate greater amounts of free DNA compared with SP-A(–/–) and wild-type mice (Figs. 6 and 7). We also found that collectin-deficient mice generate anti-DNA Abs in an age-dependent manner (Fig. 8). These results suggest that collectins bind DNA and enhance its clearance, both in vitro and in vivo. Our investigation therefore establishes two new roles for collectins—enhancing DNA clearance and minimizing autoantibody generation.

Innate immune collectins such as SP-A (12, 32), SP-D (12, 32), and MBL (33) have been shown to bind apoptotic cells; however, the ligands that they bind were not clearly identified. In our previous experiments, Clark et al. (11) showed that apoptotic/necrotic macrophages accumulate in the lungs of SP-D(–/–) mice, and this defect could be corrected by treating these animals with recombinant SP-D(n/CRD). Others have shown that only SP-D gene deficiency, but not deficiency of SP-A or C1q, leads to defective clearance of apoptotic cells from the lung (12). Hence, binding of SP-D with cell surface DNA could be a major signal for the phagocytosis of apoptotic cells in the lungs

Although the complement protein C1q binds DNA and activates the complement cascade (34), and its genetic deficiency leads to defective clearance of immune complexes and SLE (35), this protein may not play an essential role in clearing DNA from the lung (12). Recent studies have established that defects in degradation of DNA (36), apoptosis, and clearance of cell corpses (13) accelerate an autoimmune response (4), and serum proteins such as protein S bind apoptotic cell surface phosphatidylserine to stimulate the phagocytosis of apoptotic cells (14). Serum amyloid component P is also known to bind DNA; however, mouse models of serum amyloid component P genetic deficiency suggest that this acute phase protein selectively solubilizes chromatin from apoptotic cells and enhances its clearance by phagocytes (37). Whether the C-reactive protein, another pentraxin that binds DNA, plays any role in clearing this ligand in vivo is, at present, unknown. A putative DNA receptor, the macrophage scavenger receptor, binds CpG DNA (38), but whether it enhances the clearance of DNA in vivo is uncertain (39). Whether the other putative receptors bind apoptotic cells, or collectins, and enhance their engulfment is also not clearly established.

Here we show that SP-D deficiency results in a specific defect in clearing free DNA or surface DNA-containing microspheres (Figs. 1–5). A slightly lower number of AMs of SP-D(–/–) mice participate in the phagocytosis of beads compared with that of wild-type AMs, agreeing with the presence of more apoptotic AMs in the SP-D(–/–) mice (11). The defect in clearing DNA-containing microspheres could significantly be corrected by SP-D but not by nonrelated protein BSA. Our results therefore suggest that collectins, particularly SP-D, play an important role in clearing free DNA and surface DNA-containing particles (Figs. 1–5) and apoptotic cells from the lung (11) and possibly other tissues (17), in vivo.

Relatively higher amounts of DNA accumulate in the lungs of SP-D- but not SP-A-deficient mice (Fig. 6), suggesting that SP-D is important for the effective clearance of DNA. Experiments with pUC18 plasmid DNA show that SP-D(–/–) mice are defective in clearing DNA from the lungs, in vivo (Fig. 7). These results are consistent with the findings that SP-D strongly binds DNA (26, 28) and enhances its clearance (Fig. 2). Furthermore, because SP-A(–/–) mice express wild-type levels of SP-D in their lungs (29), DNA clearance-related phenotypes in the lungs of these mice may not be detectable. DNA autoantibody concentrations in SP-A(–/–) and SP-D(–/–) mice sera are significantly higher than that of wild-type mice in an age-dependent manner, but do not reach levels comparable to severe autoimmune disease states (Fig. 8). Because innate immune collectins SP-A, SP-D, and MBL all bind DNA, presence of more than one of these proteins in a given higher vertebrate may indicate the redundancy in DNA clearance pathways. Deficiency of these collectins may confer increased risk of developing autoimmune diseases, and studying genetic polymorphisms may help to understand their contributory roles in severe autoimmune diseases such as SLE.

Increased number of apoptotic cells and cellular debris, and decreased concentrations of functional surfactant proteins SP-A and SP-D are found in the airways of patients with inflammatory disease/conditions (40, 41, 42). In addition, certain pathogenic bacteria secrete DNA into the lungs, particularly in cystic fibrosis, to form a coat of biofilm to establish persistent infection (3). Lungs from these patients are known to contain large amounts of free DNA (1, 3) and low concentrations of SP-A and SP-D (43). Increased concentrations of free DNA in the lungs also cause inflammation and septic shock (1) during gene therapy experiments in addition to the disease conditions (39). Because our results show that innate immune collectins bind both DNA and enhance their clearance (Figs. 1–4), these new findings could have significant implications in understanding the pathology and treatment of these diseases.

It is interesting to note that most of the previously described proteins implicated in DNA clearance, contain either lectin/DNA-binding domains or collagen-like regions. However, collectins contain both of those domains in the same protein and are capable of enhancing phagocytosis (5, 6). This may represent an evolutionary process where an important biological function, the clearance of DNA, becomes mediated by two distinct types of protein structures that are combined within a single molecule. Thus collectins are particularly well suited to promoting the clearance of free DNA and apoptotic cells.


   Acknowledgments
 
We thank Jackie Shaw and Beryl Moffatt for maintaining the U937 cell line, animal house staffs, Denise and coworkers for maintaining mice in a healthy condition, and Dr. Cliff Morgan (Royal Brompton National Heart and Lung Hospital, London) for supplying alveolar proteinosis BAL samples.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was partially supported by the research grants from Medical Research Council, European Union Framework 5 QL Contract 00325 (J.N., K.B.M.R.), and National Institutes of Health Grants HL-24075 and HL-58047 (S.H.). P.N. is a recipient of the Wellcome Trust and Canadian Institutes of Health Research postdoctoral fellowships, and H.C. holds a Beit Memorial fellowship for medical research. Back

2 Address correspondence and reprint requests to Dr. Nades Palaniyar, Lung Biology Research Program, The Hospital for Sick Children Research Institute, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. E-mail address: nades.palaniyar{at}sickkids.ca Back

3 Abbreviations used in this paper: SP-, surfactant-associated protein; AM, alveolar macrophage; ANA, anti-nuclear antibody; BAL, bronchoalveolar lavage; C1q, complement component 1q; CRD, carbohydrate recognition domain; MBL, mannose-binding lectin; SLE, systemic lupus erythematosus. Back

Received for publication August 18, 2003. Accepted for publication April 1, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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