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* Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom; and
Department of Pediatrics and Cardiovascular Research Institute, University of California, San Francisco, CA 94143
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The alteration of surfactant lipid metabolism and turnover was an unexpected finding in SP-D-deficient mice because previous in vitro studies had not provided evidence of a role for SP-D in regulating surfactant metabolism or activity. However, there is increasing evidence that surfactant metabolism is influenced by the local immune environment, with altered levels of immunoregulatory molecules found in inflammation leading to perturbed surfactant homeostasis (7). For example, surfactant lipidosis and inflammation quite similar to the phenotype of SP-D-deficient mice have been described in transgenic mice overexpressing IL-4 (8). Mice deficient in GM-CSF also develop a surfactant lipidosis secondary to decreased surfactant clearance by alveolar macrophages (9, 10). The effects of SP-D gene ablation on surfactant turnover therefore might be secondary to inflammatory processes triggered by the absence of SP-D.
Rapid removal of apoptotic cells is recognized as a centrally important mechanism for maintenance of immune homeostasis and the resolution of inflammation (11, 12). As apoptosis progresses, the integrity of the plasma membrane is lost with consequent leakage of potentially toxic intracellular contents and triggering of an inflammatory response in bystander cells (13). If recognition and clearance of apoptotic cells by healthy macrophages is not efficient, tissue damage and prolonged low-grade inflammation could result. Interestingly, SP-A and SP-D promote clearance of apoptotic human neutrophils by alveolar macrophages in vitro (14). The mechanisms involved are uncertain but appear to involve preferential recognition of the apoptotic cell surface by the collectins (14). The structurally related proteins, MBL and C1q, bind apoptotic Jurkat T cells in vitro and enhance their clearance by monocyte-derived macrophages (15). These recent studies suggest the collectins might act as opsonins for apoptotic and/or necrotic cells.
We hypothesized that lung injury seemingly mediated by the presence of large numbers of alveolar macrophages in SP-D knockout mice may be due in part to impaired clearance of apoptotic alveolar macrophages. We report here that SP-D-deficient mice have an up to 10-fold increase in apoptotic and/or necrotic alveolar macrophages in the alveolar space compared with wild-type mice. It is known that overexpression of whole-length rat SP-D in the knockout background corrects the abnormal lung phenotype (16). Therefore we aimed to assess the therapeutic efficacy of administering a truncated trimeric fragment of human SP-D in correcting the phenotype of SP-D knockout mice. We report here that administering a 60-kDa recombinant fragment of human SP-D significantly reduced the numbers of apoptotic and/or necrotic alveolar macrophages and the surfactant lipidosis. Defective control of apoptotic macrophage numbers may be a factor that contributes to the dysregulated surfactant homeostasis observed in SP-D-deficient animals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported the generation of gene-targeted SP-D-deficient mice (4). SP-D-deficient mice backcrossed 10 generations into a C57BL/6 background were fed ad libitum and housed in isolators in a pathogen-free environment in the Biomedical Services Unit, Oxford University. Pathogen-free C57BL/6 wild-type mice for control experiments were obtained from Harlan-OLAC (Shaw’s Farm, Bicester, Oxfordshire, U.K.). All experimental protocols were approved by appropriate U.K. Home Office licensing authorities and by the University of Oxford Ethical Committee.
Generation of recombinant SP-A and SP-D
A recombinant homotrimer of SP-D (rfhSP-D), composed of eight
Gly-Xaa-Yaa repeats from the collagen region, the 
-helical
coiled-coil neck region, and the carbohydrate recognition domain (CRD)
of human SP-D, was expressed in Escherichia coli. After
solubilization and refolding of expressed protein from inclusion bodies
into a functional trimeric form, the rfhSP-D trimer was purified by ion
exchange, affinity, and gel filtration chromatography as described
previously (17). The recombinant preparation was judged to
be pure by using SDS-PAGE, immunoblotting, and amino-terminal
sequencing. The purified trimeric recombinant protein was assessed for
correct folding by disulfide mapping and by its crystallographic
structure complexed with maltose in the carbohydrate-binding pockets
(A. Shrive, H. Tharia, P. Strong, U. Kishore, I. Burns, P. Rizkallah,
K. Reid, and T. Greenhough, unpublished data). Before administration to
mice, rfhSP-D was passed over a column of polymyxin beads to remove
endotoxin. The final amount of endotoxin present in the rfhSP-D
preparations was <0.1 endotoxin units/µg protein. Whole-length
recombinant human SP-A (rhSP-A) expressed in a mammalian expression
system was obtained from Byk-Gulden Pharmaceuticals (Lake Constance,
Germany).
Modification of rfhSP-D by FITC labeling
To assess in vitro binding to macrophages isolated by bronchoalveolar lavage (BAL) of mice, rfhSP-D was labeled with the amine reactive probe FITC (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. Briefly, 50 µg of FITC incubated with1 mg of rfhSP-D in 200 ml of 0.1 M sodium bicarbonate buffer (pH 9) at room temperature for 2 h. The labeled protein was then separated from free FITC using a G-25 column. FITC-rfhSP-D (20 µg/ml) was incubated with freshly isolated alveolar macrophages from wild-type and SP-D knockout mice in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2) and assessed for colabeling with propidium iodide (PI) or PE-labeled annexin V as outlined below by flow cytometry on a Consort 32 FACS system.
Administration of rfhSP-D, rhSP-A, or BSA
Six-week-old SP-D-deficient mice underwent multiple intranasal administrations of protein or PBS alone over a period of 3–6 wk using one of three protocols. For each protein administration, mice were lightly anesthetized with isofluorane before rfhSP-D or control protein (30 µg, 25 µg, or 10 µg of rfhSP-D, rhSP-A, or BSA in 50 µl of PBS) was applied to the nostrils using a sterile micropipette. Mice were held upright after each dose until all of the fluid was inhaled. In the first treatment protocol, 12 mice received 30-µg doses of rfhSP-D every 3 days from age 6 wk. Six mice were sacrificed after 3 wk for assay of alveolar macrophage number and alveolar phospholipid content. The remaining six mice in the treatment group completed 6 wk of treatment before sacrifice and assay. In the second treatment protocol, mice were treated from age 12 wk with 10-µg doses of rfhSP-D, rhSP-A, or BSA intranasally five times per week for 3 wk before sacrifice and assay. Additional controls were untreated mice and mice treated with PBS. To assess the effect of treatment from age 4 wk, before a significant increase in alveolar macrophage numbers occurs, six mice were treated in a third protocol for 2 wk with 30-µg doses of rfhSP-D. These mice were sacrificed at age 6 wk for assay of phospholipids and alveolar macrophage numbers and were compared with untreated age-matched controls.
BAL fluid, total phospholipid, and protein measurements
At the time points indicated in individual experiments, four to
six mice in each treatment group were sacrificed by asphyxiation with
carbon dioxide and underwent BAL with sterile PBS containing 0.25 mM
EDTA. The lungs were lavaged with 1 ml four times to yield a total
lavage volume of 
3 ml. The BAL fluid was centrifuged at 250 x
g for 5 min at room temperature. Total protein concentration
in the cell-free supernatant was determined using bicinchoninic acid as
a substrate. Cell-free BAL fluid was extracted into chloroform methanol
and the total phospholipid concentration was derived from the
phosphorous concentration.
rfhSP-D levels in mouse BAL fluid
Serial dilutions of cell-free BAL fluid from rfhSP-D-treated mice were analyzed for rfhSP-D content by standard sandwich ELISA methodology using biotinylated and nonbiotinylated, monospecific, polyclonal Abs raised against the recombinant fragment of human SP-D. This Ab showed no cross-reactivity with mouse SP-A or mouse SP-D. Standard curves using the recombinant fragment of human SP-D were used to calculate the absolute amounts of alveolar rfhSP-D recovered at specific time points after administration.
Cytospin preparations of alveolar macrophages
Alveolar macrophages isolated by BAL using PBS with 0.25 mM EDTA and centrifugation at 250 x g were resuspended in 1 ml of PBS. Aliquots were taken for total cell counting by hemocytometer after staining with malachite green or crystal violet and preparation of cytospin slides using standard procedures. Differential cell counts on cytospin preparations after staining with DiffQuik (Scientific Products, McGaw Park, IL) confirmed that 98% of the cells isolated in this way were alveolar macrophages.
Flow cytometry and detection of apoptotic and necrotic cells
Cells isolated from BAL of SP-D-deficient mice with RPMI were resuspended in PBS and analyzed on a Consort 32 FACS system (BD Biosciences, San Jose, CA) to characterize the macrophage population by size and granularity. Cell preparations with obvious blood staining were discarded. Apoptotic cells and necrotic cells were detected by annexin V and PI staining using an Annexin-V-FLUOS staining kit (Roche Diagnostics, Mannheim, Germany). Apoptotic and necrotic cells expose phosphatidylserine (PS), which is normally present on the inner cell membrane leaflet to the outer leaflet, allowing annexin V to bind to PS at the cell surface (18). Cell aliquots were stained with fluorescent-labeled annexin V and counterstained with PI, which stains primary necrotic cells or cells becoming necrotic after apoptosis. In colabeling experiments, binding of FITC-labeled rfhSP-D to cells staining with PI or PE-labeled annexin V (BD PharMingen, San Diego, CA) was also assessed after compensation for overlap of fluorescent signals.
Confocal microscopy of TUNEL staining of alveolar macrophages from wild-type and SP-D knockout mice
Labeling of DNA breaks to detect apoptotic cells was conducted on macrophages isolated by BAL from wild-type and SP-D knockout mice using an APO-DIRECT single-step staining kit in accordance with the manufacturer’s instructions (BD PharMingen). Cytospins of cells stained with FITC-dUTP-detecting DNA breaks were examined using confocal microscopy.
Data analysis
Results are given as means ± SEM. Comparisons between groups of animals at individual time points were made with two-tailed t tests assuming unequal variance. Significance was accepted at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alveolar macrophage numbers were increased in SP-D-deficient
compared with wild-type mice as previously reported. The effect of
treatment with rfhSP-D on the number of alveolar macrophages isolated
from BAL fluid is shown in Fig. 1
A. By contrast with
treatment with rhSP-A, BSA, or PBS, the number of macrophages was
significantly decreased in rfhSP-D-treated SP-D-deficient mice at 9,
12, and 15 wk. Macrophage numbers in rfhSP-D-treated animals remained
higher than in wild-type mice at all ages.
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The effect of treatment on cell-free lavage total phospholipid
levels is shown in Fig. 1
B. As previously reported, there
was a marked increase in BAL phospholipid levels in SP-D-deficient mice
by 6 wk of age. There was no difference in phospholipid levels in mice
receiving 2 wk of treatment with rfhSP-D from age 4 wk compared with
untreated 6-wk-old mice. The same mice as shown in Fig. 1A,
which were treated from age 6 wk for 3 or 6 wk were also assayed for
cell-free BAL phospholipid. There was no difference in BAL
phospholipids after 3 wk of treatment (sacrifice age, 9 wk) compared
with untreated 9-wk-old controls. However, mice treated for 6 wk
(sacrificed for assay age 12 wk) showed a 50% reduction in excess
phospholipid compared with untreated 12-wk-old controls
(p < 0.05). There was no significant effect of
treatment on lavage total protein levels (data not shown) at any
age.
rfhSP-D levels in mouse BAL fluid
Fig. 2 shows the levels of rfhSP-D
at various time points after intranasal administration of a 10-µg
dose of rfhSP-D. A total of 35–40% of the administered dose was
recovered by BAL 1 h after intranasal administration. The small SE
on these measurements indicates that this is a consistent and reliable
method of administering rfhSP-D into the lungs of SP-D-deficient mice.
Using antisera specific for murine SP-D, the native recoverable pool of
SP-D in wild-type mice in cell-free supernatant is 3 µg (data not
shown). Intranasal administration of a 10-µg dose of recombinant
protein appeared sufficient to immediately replace endogenous pools.
However, the administered protein is virtually undetectable in
cell-free lavage 21 h after administration, indicating that it is
rapidly cleared from the alveolar space or becomes inaccessible to
lavage.
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DiffQuick staining confirmed that the lavage cell pellets
consisted of 98% macrophages. Fig. 3
shows representative photomicrographs of cytospin preparations from the
various treatment groups. As previously reported, cells from
SP-D-deficient mice were large, foamy, and often multinucleated. In
contrast from BSA or SP-A-treated mice, alveolar macrophages from
rfhSP-D-treated mice were more frequently normal in appearance, with
fewer enlarged and foamy cells. This effect was quantified by forward
and side scatter flow cytometry.
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Fig. 4 shows a representative result
of forward scatter (cell size) and side scatter (granularity) flow
cytometry in SP-D-deficient mice compared with wild-type mice. Five
thousand cells were counted in each mouse from each group
(n = 6 per group). The histograms show a population of
larger and more granular cells in SP-D-deficient mice consistent with
the cytospin appearances of enlarged foamy macrophages in
SP-D-deficient mice compared with those isolated from wild-type mice.
The effect of rfhSP-D treatment on the forward and side scatter of the
cell population is shown using representative histogram overlays and
indicates a cell population with fewer abnormally large and granular
alveolar macrophages, consistent with the cytospin appearances.
Treatment with rhSP-A, PBS, or BSA had no effect on the shape of
forward and side scatter histograms (data not shown).
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Fig. 5 shows end labeling of DNA
breaks in macrophages from SP-D knockout mice by green FITC-dUTP
incorporation, but not in macrophages from wild-type mice. Cells
isolated from SP-D knockout mice showed a much higher degree of
staining for DNA breaks, consistent with increased numbers of
macrophages undergoing apoptosis in the SP-D knockout mice.
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Fig. 6 shows the typical patterns of
annexin V and PI staining of macrophages from SP-D-deficient mice
compared with wild-type mice. Consistently, the number of macrophages,
staining with annexin V, was significantly higher in SP-D-deficient
mice compared with wild type, and there was a 3- to 4-fold increase in
the number of cells that stained with both annexin V and PI in
SP-D-deficient mice. The arbitrary cutoff shown in the figure, dividing
the cell population into quadrants, allowed calculation of the
percentages of cells staining with PI or annexin V alone in each case.
The effect of treatment for 3 weeks with rfhSP-D on the percentages of
cells staining with either PI or annexin V is shown in Fig. 6c. There
was no significant effect of treatment with rhSP-A, BSA, or PBS on the
percentage of cells staining with annexin V and/or PI. By contrast,
rfhSP-D treatment resulted in a significant reduction in the number of
annexin V- and/or PI-positive cells in the BAL of SP-D-deficient mice,
though the percentage of annexin V- and/or PI-positive cells was still
significantly higher than wild type.
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The extent of colabeling of FITC-labeled rfhSP-D with annexin V-
and/or PI-positive cells is shown in Fig. 7. Results are representative of three
experiments. The dot plots show preferential binding of FITC-rfhSP-D to
early apoptotic (annexin V-positive) and necrotic (PI-positive) cells.
Overall, a higher proportion of cells isolated from knockout mice were
bound by FITC-rfhSP-D, compared with wild-type (45 ± 6 vs 15
± 1.8%). Of the small number of annexin V-positive cells in wild-type
mice, 80 ± 6% were bound by FITC-rfhSP-D, compared with only
1 ± 0.5% of annexin V-negative cells. In knockout mice, 55
± 4% of annexin V-positive alveolar macrophage-bound FITC- rfhSP-D,
compared with only 3.5 ± 1.7% of annexin V-negative cells. All
cells which stained with PI from wild-type or knockout mice were bound
by FITC-rfhSP-D. Thus, there was a low level of binding of FITC-rfhSP-D
to healthy cells (PI negative, annexin V negative) from wild-type mice
and knockout mice compared with the binding to annexin V- or
PI-positive cells. Coincubation of freshly isolated cells with
FITC-annexin V and unlabeled rfhSP-D did not significantly affect
annexin V binding (data not shown), indicating that there was no direct
interaction between annexin V and rfhSP-D, nor any competition by
rfhSP-D of annexin V binding to its PS ligand.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The increased number of apoptotic and/or necrotic cells may reflect an increased rate of apoptosis, decreased clearance, or some combination of the two. Alveolar macrophages from SP-D-deficient mice produce and release large quantities of ROS (6). It is not yet known whether it is the annexin V-positive or -negative population that is responsible for ROS production, but enhanced ROS production or other proapoptotic molecules in SP-D-deficient mice could drive increased apoptosis and/or necrosis of cells in the alveolar space. The recent report ascribing direct anti-oxidant activity to the CRD of SP-D in vitro provides a potential mechanism for the effect of exogenous truncated SP-D on the number of apoptotic cells (21).
Recent in vitro data support a potential role for the collectins in clearance of apoptotic cells. Normally macrophages are very efficient at recognizing and clearing immune and other cells in the early phases of apoptosis without stimulating a proinflammatory response. In fact, normal clearance of apoptotic cells may be associated with an anti-inflammatory response by the engulfing macrophages (22). The mechanisms of apoptotic cell recognition and clearance are complex and still incompletely understood (12). The PS exposed on the surface of apoptotic cells may be an important ligand for specific receptor-mediated phagocytosis (23), but several other changes on the surface of apoptotic cells including changes in cell surface carbohydrates with increased mannose expression may participate (24). Two recent reports support an opsonic role for the collectins in the clearance of apoptotic cells by macrophages. In the first, Schagat et al. (14) and colleagues reported that SP-A preferentially binds apoptotic neutrophils over viable neutrophils and enhances the alveolar macrophage uptake of apoptotic neutrophils almost 3-fold. The ligand for SP-A on the surface of the apoptotic neutrophils is unknown. SP-D also enhanced the phagocytosis of apoptotic neutrophils in this study but to a significantly lesser extent (14). In the second, Ogden et al. reported that the structurally related protein, MBL, also preferentially binds apoptotic Jurkat T cells, using the CRD domain of MBL (15). The binding of MBL to apoptotic cells enhances macrophage micropinocytosis and uptake of the apoptotic cells.
Consistent with these in vitro studies, we found the exogenous administration of a truncated fragment of human SP-D, lacking most of the collagenous domain, significantly reduced the total number, size, and granularity of alveolar macrophages in SP-D-deficient mice and the percentage of these cells that were apoptotic or necrotic. We do not have direct evidence that the truncated form of SP-D used in our experiments enhances clearance of apoptotic cells by macrophages in vivo, though we have demonstrated that the recombinant fragment preferentially binds to apoptotic and necrotic macrophages in vitro. Studies with MBL and the structurally related protein C1q in vitro suggest the collagenous tails of these two proteins mediate the interaction with the macrophage by binding to the multifunctional cell surface protein, calreticulin (15). If this result is applicable to other collectins, it would suggest both major domains of the collectins, the collagenous, and carbohydrate recognition domains, would be required for productive apoptotic cell clearance. Consistent with this observation, the SP-D-deficient phenotype is rescued by lung-specific transgenic over-expression of full-length SP-D (16), but not by a mutant SP-D that lacks N-terminal cysteines and fails to form high order oligomers characteristic of the collectin family (26). The truncated fragment of SP-D however does bind preferentially the apoptotic and/or necrotic BAL cells in vitro. This finding is most consistent with an opsonic function of truncated SP-D in facilitating clearance of apoptotic cells in vivo despite the lack of the collagenous domain. Whether the rate of macrophage apoptosis in SP-D-deficient mice reflects normal senescence or is enhanced by ROS or other factors remains to be determined.
SP-D-deficient mice have been shown to have decreased levels of SP-A in
the cell-free BAL fluid (4, 27), but measurements of SP-A
content on whole lavage without removal of cells showed an overall
2-fold increase in SP-A levels (4). It is possible that
endogenous SP-A may be bound to the apoptotic cells. However, despite
the fact that SP-A is effective in enhancing the alveolar macrophage
engulfment of apoptotic neutrophils in vitro (14), we saw
no detectable effect of exogenous SP-A on the number of apoptotic
and/or necrotic cells or other aspects of the SP-D-deficient phenotype
in vivo. This may be due to a preferential recognition of apoptotic
macrophages rather than neutrophils by SP-D or other aspects of the two
experimental designs, including specifics of the protein preparations.
Interestingly, the partial reduction in BAL surfactant lipidosis
mediated by exogenous rfhSP-D treatment in our study was delayed
relative to the reduction in macrophage number, possibly suggesting the
lipidosis is secondary to a product released by or stimulated by the
dying cells. Certainly, other proinflammatory mouse models have
resulted in a surfactant lipidosis of similar magnitude
(28). Replacement of full-length protein by transgenic
overexpression has been shown to completely reverse the abnormal
lipidosis in SP-D knockout mice (16). The fact that only
partial reversal of the lipidosis occurred in our experiments could be
the result of dosage effects, problems with distribution to the distal
lung, or due to the possibility that the truncated form of the protein
is less active than the full-length native protein in this regard.
We speculate that SP-D may have an important housekeeping role to play in minimizing inflammation in the lung by enhancing the clearance of dead and dying cells in the airspaces and/or reducing the basal rate of alveolar macrophage apoptosis. The clinical implications of this hypothesis are intriguing. Several environmental toxins promote alveolar macrophage apoptosis, including cigarette smoke (29). Smokers have decreased SP-D levels in their BAL (30). It is at least plausible that delayed clearance of apoptotic macrophages in smokers secondary to low SP-D levels could contribute to the chronic inflammation seen in this population. Our results also suggest that a recombinant fragment of human SP-D may provide a novel therapeutic agent in treating chronic lung inflammation in patients predisposed by relative SP-D deficiency.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Howard Clark, Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
3 Abbreviations used in this paper: SP-D, surfactant protein D; SP-A, surfactant protein A; MBL, mannan-binding lectin; rfhSP-D, recombinant homotrimer of SP-D; CRD, carbohydrate recognition domain; rhSP-A, recombinant human SP-A; BAL, bronchoalveolar lavage; PI, propidium iodide; PS, phosphatidylserine; ROS, reactive oxygen species.
Received for publication March 25, 2002. Accepted for publication July 17, 2002.
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, PGE2, and PAF. J. Clin. Invest. 101:890.[Medline]
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N. Palaniyar, H. Clark, J. Nadesalingam, M. J. Shih, S. Hawgood, and K. B. M. Reid Innate Immune Collectin Surfactant Protein D Enhances the Clearance of DNA by Macrophages and Minimizes Anti-DNA Antibody Generation J. Immunol., June 1, 2005; 174(11): 7352 - 7358. [Abstract] [Full Text] [PDF]
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 T. Betsuyaku, Y. Kuroki, K. Nagai, Y. Nasuhara, and M. Nishimura Effects of ageing and smoking on SP-A and SP-D levels in bronchoalveolar lavage fluid Eur. Respir. J., December 1, 2004; 24(6): 964 - 970. [Abstract] [Full Text] [PDF]
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V. Dave, T. Childs, and J. A. Whitsett Nuclear Factor of Activated T Cells Regulates Transcription of the Surfactant Protein D Gene (Sftpd) via Direct Interaction with Thyroid Transcription Factor-1 in Lung Epithelial Cells J. Biol. Chem., August 13, 2004; 279(33): 34578 - 34588. [Abstract] [Full Text] [PDF]
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N. Palaniyar, J. Nadesalingam, H. Clark, M. J. Shih, A. W. Dodds, and K. B. M. Reid Nucleic Acid Is a Novel Ligand for Innate, Immune Pattern Recognition Collectins Surfactant Proteins A and D and Mannose-binding Lectin J. Biol. Chem., July 30, 2004; 279(31): 32728 - 32736. [Abstract] [Full Text] [PDF]
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 T. P. Hickling, H. Clark, R. Malhotra, and R. B. Sim Collectins and their role in lung immunity J. Leukoc. Biol., January 1, 2004; 75(1): 27 - 33. [Abstract] [Full Text] [PDF]
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