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* Division of Pulmonary Sciences and Critical Care Medicine, Departments of Medicine and
Pathology, University of Colorado Health Sciences Center, and
Program in Cell Biology, Departments of Pediatrics,
Immunology, and
¶ Medicine, National Jewish Medical and Research Center, Denver, CO 80262; and
|| Rheumatology Section, Imperial College School of Medicine, London, United Kingdom
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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 collectins, including SP-A, SP-D, mannose-binding lectin (MBL), and bovine conglutinin, are pattern-recognition proteins of the innate immune system known for their ability to facilitate the removal of microorganisms through opsonin-mediated phagocytosis. Collectins are composed of structurally similar oligomers that contain an N-terminal collagenous tail, and a C-terminal globular head group with lectin (carbohydrate-binding) activity (14). C1q, the first member of the classical complement cascade, is related to the collectins by structure and function, except that its globular head lacks lectin activity (15). The finding that C1q and MBL participate in apoptotic cell clearance, in vitro (5) and in vivo (9, 10), followed initial observations that C1q binds to blebs on the surface of apoptotic cells (16). Most recently, SP-A and SP-D have been shown to bind rat apoptotic neutrophils (PMNs) and drive their ingestion by rat AMs in vitro (13). These findings, and the abundance of SP-A and SP-D in the lung (17), suggest that collectins may play a significant role in pulmonary clearance of apoptotic cells, maintenance of homeostasis, and resolution of inflammation.
C1q and MBL have been suggested to drive apoptotic cell uptake through
engagement of cell surface calreticulin and CD91 (5).
Calreticulin is a multifunctional protein located in the endoplasmic
reticulum (18) and on the surface of many cell types,
including macrophages (5); it has been shown to
specifically bind to the collagenous tail of C1q and MBL (14, 19), and has been identified as the collectin receptor, cC1qR
(20). However, calreticulin lacks a transmembrane domain.
Therefore, for signal transduction, it is likely that calreticulin must
associate with a transmembrane receptor. CD91, also known as the
2 macroglobulin receptor, may be this
receptor, because CD91 complexes with calreticulin (21)
and participates in apoptotic cell phagocytosis mediated by C1q and MBL
(5). SP-A (22) and SP-D (P. Eggleton,
personal communication) also bind calreticulin, suggesting that
they may drive clearance of apoptotic cells through a similar
CD91-mediated mechanism (15).
Studies were designed to determine the relative in vivo and in vitro importance of C1q, SP-A, and SP-D in pulmonary clearance of apoptotic cells. Our findings indicate that SP-D appears to be particularly important in apoptotic cell removal from the naive murine lung, because genetic deletion or overexpression of SP-D, alone, alters apoptotic cell removal in vivo. In contrast, genetic deletion of SP-A or C1q has no effect on pulmonary apoptotic cell clearance. In vitro, SP-A, SP-D, and C1q bind to apoptotic cells and drive their ingestion by AMs through interaction with calreticulin and CD91. These results suggest that the entire extended family of collectins, including C1q, may contribute to apoptotic cell removal through a common receptor complex, but that in the naive murine lung SP-D appears to play a dominant role.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs obtained from BD PharMingen (San Diego, CA) included mouse
monoclonal anti-human Fc
RII (CD32) IgG, mouse monoclonal
anti-human CD45 IgG, and rat monoclonal anti-mouse FcRIII/II
(CD16/32) IgG. Mouse monoclonal anti-human CD91 IgG was obtained
from American Diagnostica (Greenwich, CT). Chicken polyclonal
anti-mouse calreticulin IgY was obtained from Affinity Bioreagents
(Golden, CO). Human C1q was obtained from Quidel (Santa Clara, CA). C1q
tails were prepared, as previously described (5). C1q was
confirmed to be endotoxin free (data not shown). Human
2 macroglobulin and bovine calreticulin were
obtained from Sigma-Aldrich (St. Louis, MO).
Human and animal experimentation
This study was approved by, and performed in accordance with the ethical standards of, the National Jewish Medical and Research Center Institutional Review Board on Human Experimentation. Written informed consent was obtained from each subject.
Mice were housed and studied under Institutional Animal Care and Use Committee-approved protocols in the animal facility of National Jewish Medical and Research Center. Experiments were performed with 8- to 12-wk-old mice matched for age.
Animals
SP-D-deficient mice (SP-D knockout (KO)) were prepared as previously described (23) and were outbred five generations with National Institutes of Health Swiss Black mice. SP-D KO mice had no detectable SP-D protein in bronchoalveolar lavage fluid. SP-A was decreased by 25%, and saturated phosphatidylcholine was increased 3-fold (23).
SP-D-overexpressing (SP-D OE) mice were prepared as previously
described (24) and were outbred five generations with
National Institutes of Health Swiss Black mice. SP-D OE mice had
5–10 times more SP-D protein in lung homogenates, 10–20 times
higher extracellular concentrations of SP-D protein in bronchoalveolar
lavage fluid, but no change in SP-A concentrations (24).
Rat SP-D expressed in these mice has been shown to be biologically
active by virtue of its ability to reverse the SP-D KO phenotype (i.e.,
emphysema and altered phospholipid homeostasis) in crossbreeding
experiments (24).
SP-D control mice (SP-D wild type (WT)) prepared in parallel with SP-D KO and SP-D OE mice were combined in all experiments. These mice had been outbred with National Institutes of Health Swiss Black mice for five generations.
SP-A-deficient mice (SP-A KO) mice were prepared as previously described (25) and were outbred seven generations with National Institutes of Health Swiss Black mice. SP-A KO mice had no detectable SP-A in lung homogenate or in alveolar type II cells, and SP-D was not altered (25). Control mice (SP-A WT) were prepared in the same fashion as the SP-A KO mice (25) and were outbred seven generations into National Institutes of Health Swiss Black mice.
C1q-deficient mice (C1q KO) were prepared as described (10) and were outbred eight generations with C57BL/6 mice. C1q KO mice had no detectable circulating C1q protein and no detectable functional classical pathway hemolytic activity (10). C1q KO mice exhibit an autoimmune phenotype including the development of nephritis, anti-nuclear, and anti-histone Abs (10). There was no alteration in the number or morphology of AMs in the C1q KO mice (data not shown). Control mice (C1q WT) were bred in the same manner and were outbred eight generations with C57BL/6 mice.
In vivo clearance of apoptotic cells
Mice were anesthetized with Avertin (Milwaukee, WI), and apoptotic or IgG-opsonized, human PMNs (10 x 106), suspended in 50 µl PBS, were instilled intratracheally through a modified animal feeding needle (Fisher, Pittsburgh, PA). Whole lung lavage was performed 30 min following cell instillation with a total of 5 ml ice-cold PBS. Total cell counts were made for each mouse. A cytospin slide of lung lavage was stained with modified Wright’s Giemsa (Leukostat; Fisher), and evaluated for macrophage phagocytosis of apoptotic cells (see In vitro phagocytosis assays). An additional cytospin slide was fixed in 10% neutral buffered Formalin (Fisher) for 10 min, washed with PBS, and stained for myeloperoxidase activity with dianisidine (Sigma-Aldrich), as described previously (26). This method stains human PMNs deep brown, but does not stain mouse PMNs.
Isolation and purification of human SP-A and SP-D
SP-A was isolated from whole lung lavage fluid taken from patients with pulmonary alveolar proteinosis, as previously described (27). SP-D was isolated from transfected Chinese hamster ovary cells, as previously described (28). LPS was stripped from these proteins using polymixin.
Cell isolation and culture
Human monocyte-derived macrophages (HMDMs) and human PMNs were isolated using Percoll gradient centrifugation, as previously described (29). Monocytes were plated in 48-well tissue culture plates (BD Biosciences, Franklin Lakes, NJ), and matured into HMDMs by culturing for 6–8 days (30). Approximately 5 x 105 HMDMs were present in each well.
Mouse AMs were isolated from 8- to 12-wk-old C57BL/6 mice (Harlan Sprague-Dawley, Indianapolis, IN). Unstimulated mouse lungs were lavaged with 5 ml ice-cold PBS. Mouse AMs were pelleted, resuspended in X-vivo medium (BioWhittaker, Walkersville, MD), plated in 96-well tissue culture plates at 1 x 105 AMs/well, and incubated at 37°C in 5% CO2. AMs were allowed to adhere for 30 min, washed with X-vivo medium, and incubated for an additional 2 h before experimentation.
Human AMs were isolated by bronchoalveolar lavage from normal volunteers. The right middle lobe was lavaged with 240 ml 0.9% saline solution at room temperature. Human AMs were plated in 96-well tissue culture plates (BD Biosciences) at 1 x 105 AMs/well, and were treated in the same manner as described above for mouse AMs.
Jurkat cells, a human T cell leukemia line, were obtained from American Type Culture Collection (Manassas, VA) and were cultured as previously described (30).
Induction of apoptosis
Jurkat cells or human PMNs were exposed to UV irradiation at 254
nm for 10 min. Jurkat cells were cultured in RPMI 1640 with 10% FCS
(Gemini Bio-Products, Woodland, CA) for 3.5 h at 37°C in 5%
CO2. Human PMNs were cultured in RPMI 1640 with
1%, endotoxin-free, fraction V BSA (Sigma-Aldrich) for 2.5 h at
37°C in 5% CO2. Jurkat cells and human PMNs
were 70% apoptotic by nuclear morphology, and have been shown to
maintain intact cell membranes that exclude trypan blue
(30)
Opsonization of PMNs
Freshly isolated human PMNs were resuspended in DMEM at 5 x 106 cells/ml. Mouse monoclonal anti-human CD45 IgG was added at 1 µg/1 x 106 PMNs, and PMNs were incubated with Ab for 30 min at 4°C while rotating. Chilled, opsonized PMNs were washed and used immediately.
In vitro phagocytosis assays
Six- to 8-day-old HMDMs, or freshly isolated AMs, were cocultured with apoptotic Jurkat cells or human PMNs in X-vivo medium (BioWhittaker) at 37°C in 5% CO2 in the absence of human serum for 1 h at a 5:1 ratio (apoptotic cell:macrophage). Macrophages were gently washed to remove uningested apoptotic cells, fixed, and stained with a modified Wright’s Giemsa stain (Fisher). Phagocytosis was determined by visual inspection of samples and was expressed as a phagocytic index, as previously described (31).
Single-ligand particles (Ebab): construction and ingestion assay
Human erythrocytes were obtained from normal donors and washed in PBS, pH 8.0. Thirty million erythrocytes were resuspended in 800 µl PBS (pH 8.0) and incubated with EZ-Link sulfo-NHS-LC-biotin (Pierce, Rockford, IL), according to manufacturer’s instructions. These biotinylated erythrocytes were washed twice, resuspended in PBS (pH 8), and incubated with 120 µg streptavidin (Sigma-Aldrich) at room temperature for 30 min. Erythrocytes were washed twice, resuspended in PBS (pH 7.4) and incubated with 5–10 µg biotinylated Ab or protein. Proper construction of Ebab ligand was confirmed by flow cytometry using the appropriate fluorochrome-conjugated Ab.
Target Ebab were added to HMDMs in duplicate wells at a 15:1 ratio and allowed to incubate at 37°C for 30 min. Unbound Ebab were washed away with HBSS. Uningested Ebab were lysed by adding deionized H2O for 10 s in one of the two duplicate wells. Cells were fixed with 0.75% glutaraldehyde, stained with dianisidine and H2O2 (Sigma-Aldrich), and counterstained with eosin. Two hundred cells were scored using light microscopy to quantify binding (before distilled water lysis) and engulfment of Ebab (after lysis).
Surface modulation experiments
Tissue culture plates were precoated with specific proteins to test their ability to modulate HMDM phagocytosis of apoptotic cells. C1q tails were prepared from human C1q (Quidel), as described previously (32). Proteins were suspended in DMEM at 0.1 µg/ml; 100 µl was added to each well and incubated for 2 h at 4°C. HMDMs (5 x 105) were adhered to precoated plates for 30 min at 37°C, following which target Ebab were added to each well at a 15:1 ratio for 1 h. Binding and ingestion of erythrocytes were assessed as described above.
Flow cytometry and fluorescence microscopy
Viable and apoptotic human PMNs were stained with 10 µg/ml FITC-conjugated SP-A or SP-D and analyzed on a FACScan cytometer, using PCLysys software (BD Biosciences), as described previously (5). Viable and apoptotic human PMNs were stained with 10 µg/ml FITC-conjugated SP-A or SP-D and analyzed using fluorescent microscopy, as previously described (5).
Statistics
The means were analyzed using ANOVA for multiple comparisons; when ANOVA indicated significance, the Dunnetts method was used to compare groups with an internal control. All data were analyzed using JMP (version 3) Statistical Software for the Macintosh (SAS Institute, Cary, NC) and are presented ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using a murine model of apoptotic cell removal, experiments were
conducted to determine the relative importance of SP-D, SP-A, and C1q
in the clearance of apoptotic cells from the naive lung (Fig. 1
). In these experiments, apoptotic PMNs
were instilled into mice intratracheally. Thirty minutes later, the
lungs were lavaged, and clearance of apoptotic cells was assessed by 1)
quantitation of recovered apoptotic PMNs, and 2) measurement of AM
ingestion of apoptotic cells (phagocytic index). These data suggested
that clearance of exogenous, apoptotic PMNs from the naive mouse lung
was specifically affected by SP-D, because recovery of apoptotic PMNs
was increased in SP-D-deficient mice vs SP-D overexpressors
(Fig. 1A), and the AM phagocytic index was decreased in
SP-D-deficient mice vs WT or SP-D overexpressors (Fig. 1B).
When lung lavage was examined separately, we found that endogenous
apoptotic cells did not accumulate in naive SP-D-deficient mice
compared with WT controls (data not shown). In contrast, neither SP-A,
nor C1q deficiency altered clearance of exogenous, apoptotic PMNs (Fig. 1, C–F). Intratracheal instillation of exogenous apoptotic
PMNs did not induce an inflammatory response in any of the mice tested,
as assessed by influx of murine PMNs (data not shown).
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R, rather than receptors involved with clearance of
apoptotic cells. These experiments did not demonstrate a significant
clearance defect for IgG-opsonized PMNs by any of the groups tested
(data not shown), suggesting that removal of apoptotic cells from the
naive lung is specifically affected by the presence or absence of SP-D.
SP-A and C1q appear to play lesser roles, individually, in the
clearance of apoptotic cells from the unstimulated lung. Because SP-A
has been shown to be involved with AM clearance of apoptotic cells in
vitro (13), and because C1q has been shown to promote
clearance of apoptotic cells in vitro (5), in the kidney
(10), and in the peritoneum (9), experiments
were performed to confirm the activity of individual collectins in AM
phagocytosis of apoptotic cells in vitro. Human SP-A, SP-D, and C1q enhance phagocytosis of apoptotic cells by human and murine AMs in vitro, independent of the apoptotic cell type
Maintenance of homeostasis in the naive lung requires that
phagocytes recognize and remove a variety of apoptotic cells, including
lymphocytes and PMNs. The AM is the professional phagocyte in the lung
likely to be most involved in the removal of apoptotic cells
(33), although structural cells, such as airway epithelium
(34) and fibroblasts (35), almost surely
contribute to this process. Accordingly, in vitro experiments were
conducted to test whether pulmonary collectins (SP-A and SP-D) and the
nonpulmonary, collectin-like C1q enhance AM uptake of apoptotic cells.
These data demonstrated that human SP-A and SP-D increased human AM
ingestion of apoptotic human PMNs (Fig. 2A) and human Jurkat T
lymphocytes (Fig. 2B). C1q also increased human AM ingestion
of apoptotic human PMNs and Jurkat T cells, but this increase did not
reach statistical significance. Similarly, human SP-A, SP-D, and C1q
increased murine AM uptake of apoptotic human Jurkat T lymphocytes
(Fig. 2C). In contrast, neither SP-A, SP-D, nor C1q
augmented uptake of viable human PMNs by AMs (data not shown),
suggesting that the effect of collectins on phagocytosis is specific to
apoptotic cells. Therefore, SP-A, SP-D, and C1q enhanced AM ingestion
of apoptotic cells independent of the apoptotic cell type.
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Several collectin receptors have been identified that are
associated with phagocytosis of microorganisms (36, 37),
but recent evidence suggests that C1q and MBL drive ingestion of
apoptotic cells through interaction with a macrophage surface complex
composed of calreticulin and CD91 (5). Because SP-A and
SP-D are known to bind calreticulin, experiments were performed to test
whether SP-A and SP-D enhanced apoptotic cell uptake by AMs through
interaction with the calreticulin/CD91 complex. In these experiments,
Abs against calreticulin or CD91 inhibited SP-A- and SP-D-amplified
phagocytosis of apoptotic cells (Fig. 3).
The presence of calreticulin and CD91 on the surface of murine and
human AMs was confirmed by flow cytometry (data not shown).
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Phagocytosis of apoptotic cells occurs through a complex set of
interactions between multiple ligands and receptors, making it
difficult to test the involvement of a single ligand-receptor pair.
Recently, our laboratory has developed an assay system that allows
evaluation of particle (erythrocyte) uptake through specific
ligand-receptor interactions (35). Biotinylated
erythrocytes are coated with biotinylated ligands through an avidin
sandwich (Ebab-ligand). Erythrocytes coated in
this manner bind to, and are taken up by, HMDMs depending on the
ligand-receptor specificity. Because externally bound, but uningested,
erythrocytes can be removed with hypotonic lysis, both binding and
ingestion can be quantitated. The Ebab ingestion
assay was used to confirm the ability of SP-A and SP-D to drive
ingestion through interaction with the calreticulin/CD91 complex on
HMDMs. In these experiments, Ebab-SP-A (Fig. 4A) and
Ebab-SP-D (Fig. 4B) bound to, and were
ingested by, HMDMs, and Abs with activity against human calreticulin
and CD91 blocked these effects. In both experiments, erythrocytes
coated with BSA (Ebab-BSA) were neither bound,
nor ingested, by HMDMs (data not shown).
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Experiments were conducted to determine whether competitive
modulation of calreticulin or CD91 with known ligands would interfere
with SP-A- or SP-D-driven ingestion of erythrocytes by HMDMs. To test
this, HMDMs were adhered to wells that had been precoated with human
serum albumin (HSA; negative control), or to known ligands of
calreticulin (C1q collagenous tails) or CD91 (calreticulin and
2 macroglobulin). Importantly, plating HMDMs
on surfaces coated with ligands for calreticulin or CD91 has been shown
to remove these proteins from the upper surface of the macrophage,
where the interaction occurs between the phagocyte and apoptotic cell
(5). Modulation of calreticulin with human C1q tails
decreased binding and ingestion of Ebab-SP-A
(Fig. 5A) and
Ebab-SP-D (Fig. 5B) compared with HSA
controls. Similarly, competitive modulation of CD91 with either bovine
calreticulin or human 2 macroglobulin also
decreased binding and ingestion of Ebab-SP-A
(Fig. 5A) and Ebab-SP-D (Fig. 5B) compared with HSA controls. In contrast, binding and
ingestion of anti-CD32-coated erythrocytes were not impaired by any
of the ligands used to precoat wells (data not shown). Therefore, these
data suggest that calreticulin and CD91 are involved in phagocytosis
mediated by SP-A or SP-D.
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Experiments were performed to further explore the interaction
among the pulmonary collectins (SP-A and SP-D), calreticulin, and CD91.
In these experiments, HMDMs were adhered to wells that had been
precoated with HSA (negative control), or human SP-A or SP-D (to
competitively modulate calreticulin and CD91), and were then cocultured
with Ebab conjugated with anti-human
calreticulin Ab (Ebab-anti-calreticulin), or
anti-human CD91 Ab (Ebab-anti-CD91). SP-A
and SP-D specifically decreased binding and ingestion of
Ebab-anti-calreticulin (Fig. 6A) and
Ebab-anti-CD91 (Fig. 6B) in
comparison with HSA control. In contrast, binding and ingestion of
erythrocytes coated with anti-CD32 were not affected by surfaces
precoated with HSA, SP-A, or SP-D (data not shown). Therefore, these
results support the hypothesis that SP-A and SP-D specifically interact
with the calreticulin/CD91 complex to initiate uptake of coated
erythrocytes and apoptotic cells.
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Enhanced uptake of apoptotic cells by SP-A and SP-D in vitro
suggests that these collectins act as intercellular bridges between the
phagocyte and apoptotic cell. The alternative possibility, that SP-A
and SP-D enhance phagocytosis through activation of the phagocyte alone
(activation-ligation mechanism), is contradicted by receptor modulation
experiments, in which SP-A and SP-D down-regulated phagocytosis of
coated erythrocytes and apoptotic cells (Fig. 6
). In addition,
experiments were conducted to test whether SP-A and SP-D specifically
bind to apoptotic cells. Binding of SP-A and SP-D to viable and
apoptotic PMNs was quantitated using flow cytometry (Fig. 7A). These experiments showed
that FITC-labeled SP-A and SP-D bound to viable PMNs, and that the
amount of binding was unaffected by apoptosis. However, fluorescence
microscopy demonstrated that the pattern by which SP-A and SP-D bound
to PMNs was significantly altered by apoptosis; SP-A and SP-D binding
to viable PMNs was diffuse, whereas binding to apoptotic PMNs was
highly localized to patches (Fig. 7, B–E). Interestingly,
this pattern of binding has been seen earlier with C1q
(5). These data suggest that collectin aggregation on the
surface of apoptotic cells may be required to facilitate ingestion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C1q and SP-A deficiency had no effect on apoptotic cell removal from the naive lung. Given the redundancy of clearance mechanisms, these results were not surprising; however, additional factors may contribute to this lack of an in vivo effect. C1q is mainly produced by cells of the monocyte/macrophage lineage (39), is present at high concentrations in the plasma, but due to lung compartmentalization it is only present at low concentrations in the normal lung (39). Consequently, C1q largely moves into the lung only during times of inflammation with associated plasma leak. Our findings and the requirement for impaired vascular permeability suggest that C1q does not play a major role in homeostatic removal of apoptotic cells from the lung, but may play a larger role in apoptotic cell removal during episodes of severe pulmonary inflammation. In contrast, SP-A is present at high concentrations in the lung (17). High levels of pulmonary SP-A, however, are highly bound to surfactant lipids (38), and the effect of this protein/lipid interaction on the ability of SP-A to direct apoptotic cell clearance is, as yet, unknown. SP-A-deficient mice do not have reciprocal increases in pulmonary SP-D (25) nor increased numbers of AMs, either of which could potentially augment apoptotic cell clearance and mask any effect from SP-A deficiency. Therefore, while C1q and SP-A appear to contribute to the machinery available to drive removal of apoptotic cells from the lung, the redundancy of clearance mechanisms, and perhaps functionally low levels, appear to be sufficient to prevent C1q or SP-A deficiency from interfering with the overall ability of the naive murine lung to remove an apoptotic cell challenge.
In contrast, SP-D deficiency impaired clearance, and SP-D
overexpression enhanced clearance of apoptotic cells from the naive
lung, implying that a hierarchy exists for collectin potency in vivo.
Unlike SP-A, the majority of pulmonary SP-D is not complexed with
lipids (38) and may be more available in the lung to drive
removal of apoptotic cells. It is important in this study to note that
SP-D-deficient mice have 25% less SP-A, and 3-fold more saturated
phosphatidylcholine in their lungs compared with WT controls
(23). Lower SP-A levels, and perhaps more lipid-bound
SP-A, could have contributed to impaired apoptotic cell removal and
confounded the interpretation of delayed clearance in SP-D-deficient
mice, were it not for data showing that SP-D overexpression enhanced
removal of apoptotic cells. Still, it remains possible that lower
levels of SP-A could enhance the defect in apoptotic cell clearance
observed in SP-D-deficient mice. SP-D-deficient mice contain increased
numbers of AMs, some of which are enlarged, filled with phospholipid,
and foamy in appearance (23); this suggests the
possibility that AM ingestion of apoptotic cells in SP-D-deficient mice
is impaired due to a more general defect in phagocytosis. However, this
seems not to be the case because these abnormal macrophages ingest
IgG-opsonized PMNs through the Fc receptor, a receptor not
associated with uptake of apoptotic cells. Together, these findings
strongly imply that SP-D is directly involved with apoptotic cell
removal from the naive murine lung, and that, when tested individually,
SP-D is more potent than SP-A or C1q in vivo. However, during active
pulmonary inflammation, associated with altered levels of surfactant
and movement of C1q into the pulmonary compartment, SP-A and C1q may
become more important effectors of apoptotic cell removal in the
lung.
Human SP-A, SP-D, and C1q enhanced apoptotic cell ingestion by both human and murine AMs, confirming initial observations made by Schagat et al. (13), showing that SP-A and SP-D accelerate uptake of apoptotic rat PMNs by rat AMs. In contrast to their findings, our data also suggest that C1q enhances AM ingestion of apoptotic cells, albeit to a lesser degree than SP-A or SP-D. Differences in these results may be due to the higher concentration of C1q (75 µg/ml) used in our experiments vs that used by Schagat et al. (25 µg/ml). Alternatively, human C1q may more effectively drive ingestion of apoptotic cells by AMs of human and murine rather than rat origin.
Both SP-A and SP-D enhanced Ebab ingestion by HMDMs, confirming that macrophages derived from blood monocytes contain the surface recognition components necessary to interact with collectins primarily relegated to the lungs. It would seem likely then that, during an inflammatory response, macrophages migrating into the lung from the vascular space might remove dying, apoptotic inflammatory cells by a mechanism that uses the lung collectins. In addition, SP-D may participate in removal of apoptotic cells in sites other than the lung, because SP-D is also present in a variety of tissues (40). Schagat et al. found that SP-A did not increase ingestion of apoptotic cells by thioglycolate-elicited peritoneal macrophages. The reasons for these observed differences in macrophage response to SP-A are not clear, but most likely relate to pre-exposure of the peritoneal macrophages to MBL or C1q present in the inflammatory exudate, or due to up-regulation of other more dominant ingestion mechanisms in the peritoneal macrophage, such as the phosphatidylserine receptor.
Apoptosis altered the binding pattern, but not the overall attachment, of SP-A and SP-D to apoptotic PMNs. Our data demonstrated that SP-A and SP-D bound to both viable and apoptotic PMNs. However, the pattern by which SP-A and SP-D bound to PMNs was markedly influenced by apoptosis; SP-A and SP-D binding to viable PMNs was distributed evenly, while binding to apoptotic PMNs was localized to discrete patches. This binding pattern (i.e., equal intensity with altered distribution) of SP-A and SP-D is similar to that previously observed for C1q (5), and may be important in apoptotic cell interaction with the phagocyte.
C1q and MBL drive apoptotic cell ingestion through interaction with the calreticulin/CD91 complex on the phagocyte surface (5), suggesting a potential mechanism for SP-A- and SP-D-mediated apoptotic cell uptake. Apoptotic cells that are opsonized with C1q or MBL bind to calreticulin on the phagocyte surface by their collagenous tails. Calreticulin, in turn, has been shown to bind to CD91, which may then transduce an intracellular signal to initiate phagocytosis. Interestingly, the cytoplasmic tail of CD91 contains two NPXY endocytosis signal sequences. This tail region of CD91 also shares sequence homology with other receptors used for endocytosis, including ced-1, a gene from Caenorhabiditis elegans known to play a role in the phagocytosis of apoptotic cells (41). SP-A and SP-D also bind to calreticulin, suggesting that the calreticulin/CD91 complex is a common receptor mechanism for collectin and C1q-initiated apoptotic cell ingestion.
Three independent criteria were used to test whether a common receptor complex composed of calreticulin and CD91 was responsible for SP-A- and SP-D-mediated phagocytosis; these included: 1) Ab inhibition of calreticulin or CD91; 2) competitive modulation of macrophage calreticulin or CD91 using known ligands; and 3) competitive modulation of macrophage calreticulin or CD91 using SP-A or SP-D. Together, these experiments demonstrated that SP-A and SP-D enhanced phagocytosis of coated Ebab and apoptotic cells through a mechanism dependent on macrophage surface calreticulin and CD91. Because a similar relationship has been described for C1q and MBL (5), these data also support the concept that calreticulin and CD91 compose a common receptor complex for apoptotic cell ingestion by all collectins and C1q. However, because blockade of calreticulin or CD91 did not completely inhibit Ebab ingestion mediated through SP-A or SP-D, other receptors such as gp340 or C1qRp may still contribute to this process (36).
The specific form of phagocytosis induced by SP-A or SP-D was not investigated. However, recent evidence suggests that a form of stimulated phagocytosis, macropinocytosis, is the mode of uptake associated with ligation of calreticulin or CD91 (5), stimulation of the phosphatidylserine receptor (35), and more broadly the ingestion of apoptotic cells (5, 35). Therefore, ingestion via macropinocytosis would be the anticipated consequence of SP-A- or SP-D-driven phagocytosis through the calreticulin/CD91 complex.
Is there a relationship between deficiency of surfactant proteins, impaired clearance of apoptotic cells, and the development of disease in the lung? The phenotype of SP-D-deficient mice (i.e., spontaneous emphysema) suggests that a link may indeed exist, especially in view of the finding that apoptotic cells are increased in the alveolar septa of patients with emphysema (42). Chronic airway inflammatory diseases, such as cystic fibrosis, have also been associated with decreased SP-A and SP-D (43), and have been found to have high numbers of airway apoptotic cells (26). Protease/antiprotease imbalance in these diseases may also contribute to impaired collectin-mediated removal of apoptotic cells, either through direct degradation of collectins (44), or through degradation of their receptor. In this vein, we have observed that elastase cleaves calreticulin and CD91 (our unpublished observation), as well as the phosphatidylserine receptor (26). Therefore, impaired clearance of apoptotic cells, either through actual or functional collectin deficiency, may contribute to the development of emphysema or airway inflammation in cystic fibrosis.
The collectins and C1q are proteins of the innate immune system with suggested roles in the clearance of microorganisms and modulation of the inflammatory response. Data reported in this work support the concept that the extended collectin family is also broadly involved in another process for control of inflammation, namely the removal of cells dying by apoptosis. In this context, it would appear that SP-D, and perhaps SP-A, is integral to the maintenance of homeostasis in the naive lung, through the orderly removal of dying cells before they have the opportunity to release internal contents and initiate an inflammatory response. These results may have particularly important implications for human diseases, such as cystic fibrosis, in which clearance of apoptotic cells is defective, control of inflammation is dysregulated, and levels of SP-A and SP-D are decreased.
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Acknowledgments |
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2 R.W.V. and C.A.O. contributed equally to the preparation of this manuscript.
3 Address correspondence and reprint requests to Dr. R. William Vandivier, National Jewish Medical and Research Center, 1400 Jackson Street, Room D505, Denver, CO 80262. E-mail address: vandivierb{at}njc.org
4 Abbreviations used in this paper: SP-A, surfactant protein A; SP-D, surfactant protein D; AM, alveolar macrophage; Ebab, single-ligand particle; HMDM, human monocyte-derived macrophage; HSA, human serum albumin; KO, knockout; MBL, mannose-binding lectin; SP-D OE, SP-D-overexpressing; PMN, neutrophil; WT, wild type.
Received for publication April 3, 2002. Accepted for publication July 25, 2002.
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N. Kaminski, J. A. Belperio, P. B. Bitterman, L. Chen, S. W. Chensue, A. M.K. Choi, S. Dacic, J. H. Dauber, R. M. du Bois, J. J. Enghild, et al. Idiopathic Pulmonary Fibrosis Am. J. Respir. Cell Mol. Biol., September 1, 2003; 29(3): S1 - 105. [Full Text] [PDF]
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I. Ghiran, L. B. Klickstein, and A. Nicholson-Weller Calreticulin Is at the Surface of Circulating Neutrophils and Uses CD59 as an Adaptor Molecule J. Biol. Chem., May 30, 2003; 278(23): 21024 - 21031. [Abstract] [Full Text] [PDF]
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