1Division of Pulmonary and Critical Care Medicine and 2Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
Submitted 18 December 2002 ; accepted in final form 3 June 2003
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ABSTRACT |
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lipopolysaccharide; innate immunity; collectin; apoptosis; transforming growth factor-1
Changes on the surface membrane of cells undergoing apoptosis evoke rapid recognition and uptake by phagocytes. The loss of cell membrane asymmetry for phosphatidylserine (PS) with exposure of PS on the cell surface is one of the most consistent markers of apoptosis. A specific PS receptor has been identified, and its role in apoptotic cell phagocytosis has been confirmed (10). Multiple cell surface ligands and receptors have been found to participate in the binding and subsequent internalization of apoptotic PMNs by macrophages and are thought to confer flexibility and tissue specificity to the rapid recognition by phagocytic cells of cells undergoing apoptosis (12, 28). Our laboratory recently found that surfactant protein (SP) A opsonizes apoptotic PMNs and stimulates their phagocytosis by AMs isolated from normal rat lungs (30). More recently, Vandivier et al. (32) demonstrated that calreticulin is an important AM cell surface mediator of the phagocytosis of SP-A-opsonized apoptotic cells. However, the cell membrane cell ligand(s) responsible for SP-A binding to apoptotic cells is unknown.
SP-A has important roles in the homeostasis of the alveolar lining fluid layer and the innate immune host defense within the lung (for review see Refs. 5 and 34). SP-A is a member of a family of immune proteins known as collectins; other members of this family include SP-D and the acute-phase protein mannose-binding lectin. The collectin family name derives from the common structure of an NH2-terminal collagen-like region and a COOH-terminal lectin domain (4). SP-A has been found to opsonize microorganisms and stimulate their phagocytosis by AMs and PMNs, enhance immune cell chemotaxis, and regulate cytokine production by AMs. Others have shown that SP-A inhibits LPS- and Candida-stimulated release of proinflammatory cytokines (including tumor necrosis factor- and macrophage inflammatory protein-2) from AMs and PMNs in vitro and in vivo (1, 21, 29). Furthermore, susceptibility to inflammation and infection induced by bacteria and viruses is enhanced in SP-A-deficient mice (17, 18), demonstrating that SP-A modulates immune function in vivo.
The related collectin SP-D has also been shown to modulate apoptotic cell clearance. Vandivier and co-workers (32) demonstrated that SP-D enhances apoptotic cell uptake by human and murine AMs in vitro; using SP-D-null and SP-D-overexpressing mice, they also showed that that changes in SP-D expression altered apoptotic cell clearance. Furthermore, Clark et al. (2) found that the administration of a truncated form of recombinant human SP-D reduced the number of apoptotic AMs isolated from SP-D-deficient mice. Thus both of the pulmonary collectins contribute to the removal of apoptotic cells.
Previous studies have shown that the phagocytosis of apoptotic PMNs by macrophages is associated with anti-inflammatory cytokine release, in contrast to the release of proinflammatory cytokines, which occurs with macrophage phagocytosis of microorganisms (14). Macrophage uptake of apoptotic cells stimulates the release of anti-inflammatory mediators, including transforming growth factor 1 (TGF
-1), interleukin-10, prostaglandin E2, and platelet-activating factor, and decreases the production of the PMN chemoattractant cytokines tumor necrosis factor-
and macrophage inflammatory-1
(8, 9, 20). Phagocytosis of apoptotic cells, thereby, can downregulate the macrophage inflammatory response.
We proposed to investigate the role of SP-A in AM apoptotic PMN uptake and cytokine release during the resolution of acute inflammation within the lung. In the present study, we found that SP-A enhances the phagocytosis of apoptotic PMNs by AMs isolated from normal lungs, as we previously observed (30), and from LPS-injured lungs. We also determined that LPS exposure, SP-A, and apoptotic cells modulate TGF-1 release from AMs.
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MATERIALS AND METHODS |
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Animals. Pathogen-free male Sprague-Dawley rats (250-400 g) were obtained from Taconic Farms (Germantown, NY). Animals were housed in a barrier facility and fed standard chow and water ad libitum.
Protein purification. SP-A was purified from the lung lavage fluid of patients with alveolar proteinosis as previously described (22). Briefly, the lavage fluid was treated with butanol to extract the SP-A, and the resulting pellet was sequentially solubilized in the detergent octylglucoside and 5 mM Tris, pH 7.4. The SP-A was then passed over a polymyxin B-agarose column to reduce endotoxin contamination. SP-A preparations had final endotoxin concentrations of <0.1 pg/µg SP-A as determined by the Limulus amoebocyte lysate assay (QCL-1000, BioWhittaker, Walkersville, MD).
Recombinant rat SP-D was purified from the media supernatant of cultured Chinese hamster ovary cells expressing a full-length rat SP-D cDNA in the expression vector pEE14. Medium was dialyzed, and SP-D was purified by maltose affinity chromatography as previously described (6). SP-D preparations had final endotoxin concentrations of <5 pg/µg SP-D as determined by the Limulus amoebocyte lysis assay (QCL-1000, BioWhittaker).
LPS lung injury model. After the rat was anesthetized in a halothane bell jar, LPS (Escherichia coli serotype 026:B6, 100 µg/kg body wt in 250 µl of sterile PBS) was instilled into the trachea using direct laryngoscopic visualization and a blunt 16-gauge feeding needle. Control animals underwent sham instillation and were housed in cages separate from the LPS-exposed animals. AMs were isolated after LPS injury.
Cell isolation. AMs were obtained (after the rats were killed by exsanguination under pentobarbital sodium anesthesia) by whole lung lavage. Lungs were lavaged with PBS without Ca2+ or Mg2+ + 0.2 mM EGTA (100 ml total volume). Lavage cells were collected by centrifugation at 230 g. Cells were resuspended in 3 ml of PBS and underlayed with a three-layer discontinuous Percoll gradient (1.065, 1.070, and 1.080). The gradient was centrifuged at 330 g for 20 min. AMs were aspirated from the PBS-1.065 and 1.065-1.070 interfaces. The AMs were then washed in PBS and resuspended in the appropriate buffer. The AMs collected were routinely >95% pure and >95% viable by hematoxylin differential and trypan blue exclusion staining, respectively.
Human peripheral PMNs were isolated by density gradient centrifugation after red blood cell (RBC) depletion by hetastarch sedimentation. Heparinized peripheral blood was mixed 2:1 with heparinized 6% hetastarch (25 U heparin/ml) and allowed to settle for 30 min at room temperature. The RBC-depleted supernatant was centrifuged at 230 g for 10 min. The cells were washed in PBS and resuspended in 2 ml of PBS. The cells were then underlayed with a six-layer discontinuous Percoll gradient (1.075, 1.081, 1.085, 1.089, 1.093, and 1.097). PMNs were aspirated from the 1.085-1.089 and 1.089-1.093 interfaces. The PMNs were washed in PBS and resuspended in the appropriate buffer. The human PMNs collected were routinely >90% pure and >95% viable by hematoxylin differential and trypan blue exclusion staining, respectively.
Peripheral rat PMNs were isolated from heparinized whole blood collected under anesthesia as previously described (30). Briefly, the jugular vein was cannulated under ketamine-xylazine anesthesia, and small volume exchanges (4-5 ml) were made of whole blood and heparinized 6% hetastarch. This exchange collection was continued until the rat died under anesthesia (typically 40-60 ml of diluted blood per 350-g rat). The diluted blood was allowed to settle at room temperature for 30 min. The RBC-depleted supernatant was then collected, and the PMNs were isolated by Percoll density gradient centrifugation as described for human PMNs. Rat PMN preparations were typically >90% pure, with RBCs being the main contaminant. Viability was also routinely >90% by trypan blue exclusion.
Cell culture. Human Jurkat cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in continuous culture in RPMI 1640 with L-glutamine supplemented with 10% heat-inactivated FBS and penicillin-streptomycin (100 U/ml and 100 µg/ml, respectively) at 37°C in 5% CO2.
Induction of apoptosis. Human and rat PMNs were induced to undergo apoptosis by UV irradiation (454 nm) for 10 min and 3 h of culture in IMDM with 0.1% BSA (low endotoxin, fraction V; Sigma). UV-irradiated PMNs were routinely >60% apoptotic as determined by FITC-annexin binding and vital dye exclusion.
Jurkat cells were induced to undergo apoptosis by 15 h of culture with anti-CD95 (Fas) monoclonal antibody (250 ng/ml at 1 x 106 Jurkat cells/ml). Jurkat cells were routinely >80% apoptotic as determined by FITC-annexin binding and vital dye exclusion.
Immunofluorescent cell labeling. Jurkat cells or rat PMNs were labeled with CTB by addition of 15 µM CTB to cultures of 1 x 106 cells/ml in RPMI 1640 with heat-inactivated FBS and antibiotics for 45 min at 37°C in 5% CO2. The cells were cultured for 30 min in fresh medium without CTB. Labeled cells were washed in warm CO2-equilibrated IMDM + 0.1% BSA and resuspended in IMDM + 0.1% BSA.
AMs were labeled with PKH2 according to the manufacturer's guidelines. Briefly, AMs were suspended at 2 x 107 cells/ml of diluent A and mixed with an equal volume of 4 µM PKH2 dye at room temperature for 5 min. Labeling was stopped with an equal volume of 1% BSA in PBS. Cells were then washed sequentially with 1% BSA in PBS and IMDM + 0.1% BSA. AMs were resuspended in IMDM + 0.1% BSA at the appropriate concentration.
FITC-labeled group B Streptococci (GBS) were prepared as previously described (19). Briefly, GBS were grown on nutrient agar and harvested, and the concentration was determined. Bacteria were killed by incubation at 95°C for 10 min, suspended in 0.1 M sodium carbonate, pH 9.0, and incubated with FITC (0.01 mg/ml) at room temperature for 1 h in the dark. Labeled GBS were washed with PBS four times to remove unbound FITC. Aliquots of labeled GBS were stored at -80°C until use.
Apoptotic cell phagocytosis assay. PKH2 green-labeled AMs were incubated in a ratio of 1:3 with CTB-labeled apoptotic cells (Jurkat cells or PMNs) in IMDM + 0.1% BSA (or HBSS + 0.1% BSA where indicated) at 37°C with gentle agitation for 1 h in the absence and presence of the study proteins. Labeled viable Jurkat cells were used instead of apoptotic cells where indicated. The cells were washed with iced PBS + 2 mM EDTA to remove unbound apoptotic cells and fixed with 1% formaldehyde + 0.1% BSA in PBS before flow cytometry. Cells were analyzed for both labels, and AMs that were PKH2 and CTB positive had phagocytosed apoptotic cells. Confocal microscopy has validated this approach (30). Inasmuch as the detected level of phagocytosis can vary between experimental preparations, positive and negative controls were repeated with each flow cytometric assay.
GBS phagocytosis assay. Unlabeled AMs were incubated with FITC-labeled bacteria at a ratio of 1:10 in IMDM + 0.1% BSA at 37°C with gentle agitation for 1 h. The cells were washed with iced PBS + 2 mM EDTA to remove unbound GBS and then fixed with 1% formaldehyde + 0.1% BSA in PBS. The AMs were analyzed by flow cytometry for incorporation of the FITC label.
AM release of TGF-1 assay. Freshly isolated AMs were adhered to 96-well culture plates (2.5 x 105 cells/well) for 2 h at 37°C in IMDM + 0.1% BSA. The wells were washed with PBS to remove nonadherent cells, and the medium was replaced. Apoptotic human PMNs (1.5 x 106 cells/well) were then coincubated with the plated AMs for 18 h in the presence and absence of SP-A. Appropriate controls were run in parallel with each assay, including wells containing only apoptotic PMNs (with and without SP-A) to assess the possible contribution of the apoptotic PMNs to the measured TGF-
1 release. The culture media from replicate wells were pooled and centrifuged at 140 g at 4°C for 10 min. The cell-free supernatants were aliquoted and frozen at -80°C. TGF-
1 levels were subsequently analyzed by ELISA (range 15.6-500 pg/ml) following the manufacturer's instructions. Briefly, the TGF-
1 ELISA is a sandwich assay using adhered human TGF-
1 receptor (sTGF-
RII) to capture active TGF-
1 from the samples followed by conjugated anti-human TGF-
1 antibody. Inasmuch as the sequence homology between rat and human TGF-
is >99%, the anti-human TGF-
1 antibody cross-reacts with rat TGF-
1. All culture media supernatant samples were acidified to activate any latent TGF-
1 before the samples were assayed. Thus our assay measured only total TGF-
1 released and did not distinguish latent from active forms of TGF-
1 in the culture media.
Statistical analysis. Values are means ± SE. Statistical analysis was performed using two-tailed Student's t-test of unpaired samples.
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RESULTS |
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Schagat et al. (30) demonstrated that SP-A stimulates nearly threefold the in vitro phagocytosis of apoptotic PMNs by AMs isolated from normal lungs. Using AMs isolated from unexposed animals or from animals at various times after LPS instillation and a flow cytometric assay of phagocytosis (30), we examined the effect of LPS-induced inflammation on SP-A's enhancement of AM phagocytosis of apoptotic cells. We hypothesized that apoptotic cell phagocytosis would be maximal during the later phase of our LPS injury model (18-48 h after LPS injury) when large numbers of PMNs are undergoing apoptosis and lavage PMN cell counts are falling (3). As shown in Fig. 1, SP-A stimulates apoptotic cell phagocytosis by AMs isolated from normal and inflamed lungs by approximately threefold above the level of phagocytosis in the absence of SP-A. This effect of SP-A on AM phagocytosis is independent of the time after LPS instillation at which the AMs are collected. LPS injury itself (in the absence of SP-A) increased AM phagocytosis of apoptotic cells compared with normal AMs during the earliest phases of inflammation: 145 ± 15% at 4 h and 125 ± 21% at 12 h after LPS injury. However, by 18 h and as long as 96 h after LPS injury, the isolated AMs phagocytose apoptotic cells at the same level as unexposed AMs. Contrary to our hypothesis, apoptotic cell phagocytosis was not maximal during the later phase of inflammation. The highest level of apoptotic cell phagocytosis was found in the SP-A-treated AMs isolated at 4 and 12 h after LPS (424 and 383% of control at 50 µg/ml). The enhancing effects of SP-A are dose dependent over the range 0-50 µg/ml at all injury time points tested (Fig. 2; other data not shown). Thus SP-A enhances phagocytosis by normal and LPS-injured AMs threefold above the level in the absence of SP-A, regardless of their baseline capacity for apoptotic cell phagocytosis.
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SP-A augments the uptake of apoptotic PMNs and apoptotic Jurkat cells. We examined the specificity of SP-A's effect on AM apoptotic cell uptake by comparing different apoptotic cell types. SP-A's augmentation of apoptotic cell phagocytosis is notably consistent whether freshly isolated rat PMNs or cultured Jurkat cells were used as the apoptotic target (Fig. 3). Given the equivalence of the apoptotic targets, apoptotic Jurkat cells were used for the bulk of the studies to reduce the number of animals needed.
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To determine whether the SP-A-enhancing effect was specific for apoptotic cells, we compared the uptake of viable and apoptotic cells. Data in Fig. 3 demonstrate that neither SP-A nor LPS exposure stimulated the phagocytosis of viable Jurkat cells. In our phagocytosis assay, there is some association of viable cells with AMs. However, the association of viable cells is just above the sensitivity limits of the assay and at much lower levels than the baseline uptake of apoptotic Jurkat cells.
Other collectins or opsonic proteins have no effect on AM phagocytosis of apoptotic cells. We examined the specificity of SP-A's enhancement of apoptotic cell phagocytosis by comparing the effect of SP-A with that of several proteins found in the alveolar space under normal or inflammatory conditions. Despite structural and functional homology to SP-A, SP-D did not significantly enhance apoptotic cell phagocytosis by AMs under any condition tested (Table 1). SP-D at 0.5-5.0 µg/ml had no effect, whereas SP-A at 5-100 µg/ml enhanced apoptotic cell uptake. These different concentrations of SP-A and SP-D were selected on the basis of previously published reports (21, 30, 33), which demonstrate that the two proteins affect a variety of cell functions at these different concentrations. It was not possible to test higher concentrations of SP-D, because our purification procedure from Chinese hamster ovary cells yields relatively dilute protein, and concentrators that we have tested have very low recoveries.
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We also examined the ability of the opsonins C1q and IgG, which are known to enhance the phagocytosis of microbes by AMs under inflammatory circumstances, to augment apoptotic cell phagocytosis by AMs. In contrast to SP-A, neither C1q nor IgG stimulated the uptake of apoptotic cells when tested with normal or LPS-exposed AM uptake (Table 1). Thus, under these experimental conditions, SP-A appears to have specific action mediating the enhancement of apoptotic cell phagocytosis by AMs.
LPS injury alters AM phagocytosis functions independently. To assess whether our time-course observations (Fig. 1) represented a generalized activation of AM function 4-12 h after LPS injury, we studied the ability of AMs isolated after LPS instillation to phagocytose GBS in the absence and presence of SP-A. Data in Fig. 4 show that, in the absence of SP-A, AMs isolated after LPS injury take up GBS in vitro significantly more than AMs from normal animals. Interestingly, AM phagocytosis of GBS increases twofold early after LPS injury (4 and 12 h) compared with normal AMs and increases further 18 and 48 h after LPS, in contrast to AM phagocytosis of apoptotic cells, which returned to control (e.g., normal AM) levels. SP-A enhances AM phagocytosis of GBS comparably in all AM populations studied (1.6-fold above the level in the absence of SP-A). SP-A augments GBS uptake by AMs in a dose-dependent manner (0-50 µg/ml), with saturation at 25-50 µg/ml (data not shown). These data support the concept that SP-A augments the phagocytic capacity of AMs even after the AMs have been activated by LPS injury. The data further demonstrate that the mechanisms for postinflammatory phagocytosis of apoptotic cells and bacteria are regulated independently.
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SP-A stimulates TGF-1 release by normal and LPS-exposed AMs. Given that SP-A enhances apoptotic cell phagocytosis by AMs and apoptotic cell uptake has been linked to anti-inflammatory cytokine release, we sought to determine the effect of SP-A on release of TGF-
1 by AMs alone and in the presence of apoptotic cells. We demonstrate that SP-A alone can enhance the release of TGF-
1 from cultured AMs isolated from normal and LPS-exposed animals. AMs isolated after LPS injury have a nearly twofold higher baseline level of TGF-
1 release than AMs from uninjured animals (Fig. 5). SP-A enhances TGF-
1 release from normal AMs in a dose-dependent manner (0-100 µg/ml, maximal release 269% of basal level). This SP-A effect is less clearly seen with LPS-exposed AMs.
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Similar to findings in other macrophage populations (11, 16), the presence of apoptotic PMNs enhances TGF-1 release from the normal cultured AMs (179% of non-LPS-injured AM control), although apoptotic PMNs do not significantly stimulate TGF-
1 release from LPS-exposed AMs (Fig. 6). Controls run in parallel with every experiment indicate that the apoptotic PMNs were not a significant source of TGF-
1 in the cell culture supernatants and that SP-A had no detectable effect on release of TGF-
1 by apoptotic PMNs. The addition of SP-A (50 µg/ml) and apoptotic PMNs to non-LPS-exposed AMs and LPS-injured AMs at 18 h yielded comparable levels of released TGF-
1 (330 and 309% of non-LPS-injured AM control, respectively). Thus basal and SP-A-induced TGF-
1 release was higher in AMs studied at 18 h after LPS injury than in normal AMs, but normal and LPS-exposed AMs respond equivalently to apoptotic PMNs. Furthermore, in the presence of apoptotic PMNs and SP-A, normal and LPS-exposed AMs release similar amounts of TGF-
1. There was no significant difference between the effects of SP-A or SP-A + apoptotic PMNs on TGF-
1 release by either AM population, despite the observation that SP-A stimulates a threefold increase in the phagocytosis of apoptotic cells. This surprising finding implies that TGF-
1 release in response to apoptotic cell phagocytosis is not directly proportional to the degree of apoptotic cell uptake. Furthermore, these findings indicate that LPS lung injury itself can induce sustained changes in AM anti-inflammatory cytokine release.
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DISCUSSION |
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AMs have been shown to be less efficient at phagocytosing apoptotic cells than blood monocytes or bone marrow-derived macrophages. However, these studies have tested the AMs in the absence of SP-A (15, 26). We have found that SP-A enhances phagocytosis by normal and LPS-injured AMs threefold above baseline (i.e., no SP-A), regardless of their baseline capacity for apoptotic cell phagocytosis. In the presence of SP-A, the differences in phagocytosis between AM and other macrophage populations would be presumed to be diminished, although we have not specifically tested this presumption in the present study. This view is supported by the finding that SP-A-stimulated normal AM phagocytosis of apoptotic PMNs was similar to that of thioglycolate-elicited peritoneal macrophages (30).
We originally hypothesized that the AMs isolated during the resolution phase of LPS-induced lung injury would have higher capacity to take up apoptotic cells, but we did not find this to be the case. However, we found in our system the same baseline and SP-A-enhanced capacity to phagocytose apoptotic cells in AMs isolated from normal animals and from animals in the resolution phase of LPS lung injury (18-72 h after LPS). This may be an actual reflection of the AM behavior during acute inflammation; i.e., AM capacity to take up apoptotic cells is unaffected by the inflammatory state. However, our data at 4 and 12 h suggest that inflammation can alter the baseline capacity of the AMs to phagocytose apoptotic cells.
This finding raises the question whether our ex vivo assay fully assesses the functional changes in AMs that occur within the lung. One possibility is that different levels of SP-A are associated with the AMs isolated from normal or LPS-injured rats. These different amounts of SP-A in the AM preparations might influence the functional behavior of the AMs in vitro. Thus we performed Western blot analysis for SP-A in AM preparations isolated and purified according to the usual experimental protocol. Comparable levels of SP-A were detected in the control and LPS-exposed macrophages (data not shown). Furthermore, the AMs isolated at different times after LPS treatment and then studied in our phagocytosis assay have been exposed previously to apoptotic cells in vivo. Prior exposure to apoptotic cells may alter subsequent AM apoptotic cell uptake; Erwig et al. (7) reported decreased apoptotic cell phagocytosis in vitro in bone marrow-derived macrophages subjected to successive exposures to apoptotic cells.
Our finding that the capacity of AMs to phagocytose apoptotic cells varies after LPS injury could also be explained by differences in apoptotic cell phagocytic capacity between resident AMs and AMs newly recruited into the alveoli by the LPS inflammation. Early during inflammation (4 and 12 h after LPS), the resident AMs predominate in our isolated AM fractions. At later times after LPS injury, the newly recruited AMs predominate in our isolated AM population. Thus we may be studying the behavior of heterogeneous cell populations. Taylor et al. (31), studying apoptotic cell uptake in a murine model of sterile peritonitis, found an increased percentage of phagocytosed apoptotic cells in resident peritoneal macrophages compared with macrophages isolated 96 h after thioglycollate-induced peritonitis (31). However, extrapolation of functional properties of one macrophage population to other types of macrophages has proven to be problematic. Differentiation of the capacities of resident vs. newly recruited AMs for apoptotic cell phagocytosis from post-LPS lung lavage is beyond the scope of the present study. Nonetheless, the AM populations studied in our ex vivo system are the lavageable AMs within the lung at various times after lung injury and, thus, reflect the capacity to phagocytose apoptotic cells at that time.
In testing the specificity of SP-A on AM phagocytosis of apoptotic cells, we found that AMs phagocytose different apoptotic cells similarly but do not phagocytose viable cells. The equivalence in the uptake of the two different apoptotic cell types induced into apoptosis via different methods (UV irradiation vs. anti-CD95 antibody) indicates that AMs specifically recognize apoptotic cells, rather than any changes induced only by the experimental treatment. SP-A enhances AM apoptotic cell uptake specifically, in that neither SP-A nor LPS exposure stimulated the phagocytosis of viable Jurkat cells. The low level of viable cell uptake likely reflects the normal amount of injured or dying cells in the Jurkat population (viability consistently 95% by trypan blue exclusion).
SP-A also appears to have specific action mediating the enhancement of apoptotic cell phagocytosis by AMs. In our rat AM system, neither C1q, IgG, nor SP-D enhanced apoptotic cell phagocytosis.
Our finding that C1q does not enhance apoptotic Jurkat cell uptake by AMs does not conflict with the well-recognized role of C1q as an opsonin and enhancer of apoptotic cell phagocytosis by monocyte-derived macrophages (24, 25, 27, 31). Given the very low levels of C1q in the lung, even under inflammatory conditions (33), AMs are unlikely to use C1q opsonization of apoptotic cells as a mechanism to promote apoptotic cell clearance, in contrast to monocyte-derived or peritoneal macrophages, which are much more likely to be exposed to C1q (24). This finding is also consistent with prior work in our laboratory that found no C1q stimulation of apoptotic PMN uptake by AMs (30).
In our present study, we found no enhancing effect of SP-D on apoptotic Jurkat cell uptake by rat AMs, although, for technical reasons, SP-D was tested at a 25-fold lower concentration than SP-A. This differs from previously published effects of SP-D on apoptotic cell uptake. In their study of rat AMs and apoptotic rat PMNs, Schagat et al. (30) found a small but significant SP-D effect (125 ± 7% of no-protein control). Vandivier et al. (32) reported that SP-D enhanced the uptake of apoptotic Jurkat cells by murine and human AMs, although murine AMs were much more responsive to SP-D than were human AMs. Recently, using SP-D-deficient mice, Clark et al. (2) demonstrated that SP-D bound to apoptotic AMs in vitro and promoted the in vivo clearance of apoptotic AMs. We cannot provide an unequivocal explanation for our divergent results, but species specificity may contribute to the different findings. We used recombinant rat SP-D, whereas the other studies used recombinant human SP-D. Furthermore, different populations of AMs (mouse, rat, or human) as well as different apoptotic cell targets (human PMNs, rat PMNs, and Jurkat cells) were used in all these studies. Differences in SP-D binding to the various apoptotic cells or AMs may alter the findings. SP-D may also modulate the uptake of apoptotic cells to a greater or lesser extent on the basis of the species of AM. This possibility is supported by the fact that Vandivier et al. found that murine AMs were more responsive to recombinant human SP-D than human AMs.
We also examined the effect of SP-A-enhanced apoptotic cell phagocytosis on AM TGF-1 release and found 1) enhancement of TGF-
1 release from cultured normal AMs and LPS-injured AMs by SP-A alone and 2) higher levels of TGF-
1 secretion in AMs isolated after LPS lung injury than in AMs isolated from normal animals.
Our TGF-1 ELISA measured only total TGF-
1 levels, without distinguishing between latent and active forms of TGF-
1 released. Thus there may be differences in the activity of the different forms of TGF-
1 released by SP-A or apoptotic PMN uptake. Additionally, we have not discriminated between release of preformed and newly synthesized TGF-
1 in response to any stimuli.
The higher basal TGF-1 release from the LPS-injured AMs and the finding that the addition of SP-A or apoptotic PMNs does not significantly increase this level of release further imply that the LPS-exposed AMs are activated in vivo to release more TGF-
1 in culture. The normal AMs, however, have not been activated and retain the ability to respond to SP-A or apoptotic PMNs. We speculate that the prior in vivo exposure to and subsequent phagocytosis of apoptotic PMNs likely contribute to the higher levels of TGF-
1 release from the cultured LPS-exposed AMs. Apoptotic PMNs have been detected in lung lavage fluid as early as 6-12 h after LPS instillation (3, 23); thus AMs studied at 18 h after LPS injury were exposed to apoptotic PMNs before ex vivo culture.
This post-LPS sustained release of TGF-1 has implications for the resolution of inflammation. TGF-
1 has been shown to stimulate apoptotic cell clearance in bone marrow-derived macrophages in vitro and in vivo (11, 16). Phagocytosis of apoptotic cells enhances TGF-
1 release, which in turn stimulates further macrophage phagocytosis of apoptotic cells and, thereby, diminishes inflammation. SP-A-enhanced apoptotic PMN uptake (3-fold higher than without SP-A) significantly increases TGF-
1 release from the normal and postinflammatory AMs compared with basal release. Thus SP-A enhances both ends of this feedback loop. These findings provide evidence for SP-A's involvement with the resolution of inflammation in the alveolar space.
In summary, we have demonstrated that SP-A stimulates apoptotic cell uptake by normal as well as inflammatory AMs, and SP-A itself modulates TGF-1 release as well as the amount of TGF-
1 released when AMs phagocytose apoptotic cells. In this way, SP-A promotes the resolution of acute inflammation within the alveolar space.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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