1Fraunhofer Institute of Toxicology and Experimental Medicine; 2Department of Respiratory Medicine, Hannover Medical School; 4Department of Anatomy, Hannover Medical School, Hannover; 5Altana Pharma AG, Konstanz, Germany; 3Department of Cell Biology, Duke University Medical Center, Durham; and 6Environmental Protection Agency, Chapel Hill, North Carolina
Submitted 27 September 2004 ; accepted in final form 4 December 2004
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ABSTRACT |
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innate immunity; antigen processing; allergy; lung
Alveolar macrophages are the predominant cells in the alveolus that serve as the first line of defense against inhaled material (7, 18). They clear inhaled material from the lung by phagocytosis, and they release microbicidal and immunomodulatory mediators. In a previous study, Currie and coworkers (5) have shown that alveolar macrophages bind and phagocytose pollen-derived allergen particles. Allergen uptake was C-type lectin receptor mediated and induced upregulation of inducible nitric oxide synthase mRNA and release of nitric oxide.
Surfactant protein A (SP-A) and surfactant protein D (SP-D) belong to the family of C-type lectins that use C-type lectin receptors on alveolar macrophages to bind specifically to the cell surface (15, 22, 25). SP-A and SP-D play an important role in the innate immune defense because they bind to pathogens and modulate their phagocytosis and clearance by alveolar macrophages. For example, SP-D binds to Pseudomonas aeruginosa and stimulates phagocytosis by rat alveolar macrophages (26), whereas SP-A enhances the phagocytosis of respiratory syncytial virus, Haemophilus influenzae, Streptococcus pneumoniae, and group A Streptococcus (2, 31). Previous work has demonstrated that SP-A and -D bind to allergens as well (21, 32). In addition, potent anti-inflammatory effects of SP-A and SP-D have been observed in asthma animal models (20, 29). We hypothesized that SP-A and -D play a role in allergen clearance by binding to allergen and enhancing the phagocytosis by alveolar macrophages.
To test this hypothesis, PSG from Dactylis glomerata or Phleum pratense were isolated and incubated with rat and human alveolar macrophages in the presence of various surfactant components. We investigated the effect of recombinant human SP-A (rhSP-A), SP-A obtained from bronchoalveolar lavage (BAL) of patients with alveolar proteinosis (AP-SP-A), a recombinant fragment of human SP-D (rfhSP-D), recombinant rat SP-D (rrSP-D), as well as the natural surfactant preparations Alveofact and Curosurf, on binding and uptake of PSG by rat and human alveolar macrophages. Furthermore, the binding of SP-A and SP-D to PSG and the aggregation of PSG in the presence of surfactant components were examined.
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MATERIALS AND METHODS |
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Grass pollen (D. glomerata) was obtained from Biopol Laboratory (Spokane, WA); P. pratense pollen was obtained from Allergon (Ängelholm, Sweden). All other reagents, unless otherwise specified, were purchased from Sigma Chemical (St. Louis, MO).
Cell preparation. Male Sprague-Dawley rats were obtained from Charles River (Raleigh, NC) or Taconic Farms (Germantown, NY). The rats weighed 301470 g. Alveolar macrophages were obtained by BAL. Rats were killed by pentobarbital sodium injection, and the lungs were removed and lavaged eight times with 10 ml of PBS. The resulting BAL was centrifuged at 250 g for 10 min, and the supernatant was carefully removed. After being resuspended in 2 ml of culture medium (RPMI 1640; Cambrex, Walkersville, MD) supplemented with 10% heat-inactivated FCS, 1% penicillin-streptomycin, and 1% L-glutamine (GIBCO, Carlsbad, CA), the cells were counted in a Neubauer hemocytometer with trypan blue. Some experiments were performed in serum-free medium with or without supplementation of CaCl2.
We received human BAL cells from five healthy nonsmoking, nonatopic volunteers by performing BAL during fiber-optic bronchoscopy at the Environmental Protection Agency. Patients gave their written informed consent, and the procedure was approved by the institutional review board.
Isolation and fluorescence labeling of PSG. PSG were isolated from whole grass pollen (D. glomerata or P. pratense) based on a protocol that was previously described by Currie et al. (5). In brief, 200 mg of pollen were added to 20 ml of deionized, autoclaved water containing 0.05% (vol/vol) Tween 20 in a 50-ml tube. The suspension was vortexed for 3 min and rotated for 2 h at 4°C. Whole pollen and pollen fragments were removed by centrifugation at 50 g for 4 min, and the supernatant was passed through a 20-µm nylon mesh (VWR International, Hannover, Germany). The filtrate was centrifuged at 2,500 g for 10 min, and the pellet was resuspended in 20 ml of sterile deionized water. This volume was passed through a polycarbonate membrane with a pore size of 3 µm (Osmonics, Minnetonka, MN) using a 20-ml syringe and a filter holder. The filtrate was centrifuged as before, and the resulting pellet was resuspended in 1 ml of sterile PBS and stored at 4°C. To determine the number of PSG, an aliquot was diluted in PBS (1:100) and counted in an improved Neubauer chamber. The isolation procedure yielded 12.0 x 10819.2 x 108 PSG. Analysis of PSG by scanning electron microscopy revealed a monodisperse particle distribution with a maximum diameter of 1.34 ± 0.03 µm (D. glomerata) and 1.50 ± 0.04 µm (P. pratense).
PSG were fluorescently labeled with Alexa Fluor 488 or Alexa Fluor 546 fluorescent dyes (Molecular Probes, Eugene, OR). For the staining procedure 4 x 108 PSG were resuspended in 500 µl of PBS containing 0.1 M sodium bicarbonate. The suspension was transferred into the vial of reactive dye, the stir bar was removed, and the vial was rotated for 1 h at room temperature in the dark. After the staining procedure the volume was transferred into a 15-ml tube, diluted to a final volume of 15 ml, and centrifuged at 2,500 g for 12 min. The supernatant was removed, and the pellet was resuspended in 15 ml of PBS and centrifuged as before. After removing the supernatant, we resuspended the pellet in 1 ml of PBS, counted the PSG again, and added the appropriate amount to the assays.
Phagocytosis assay. Alveolar macrophages (2 x 105) in 1 ml of culture medium were placed into polypropylene tubes (Becton Dickinson, Franklin Lakes, NY). The surfactant preparations (Curosurf, Alveofact, rhSP-A, AP-SP-A, rfhSP-D, rrSP-D) were added immediately, and the cells were incubated for 40 min at 37°C in 5% CO2. One sample without surfactant was placed at 4°C as a negative control. After 40 min, Alexa Fluor 488-labeled PSG were added to the cells at ratios of 10:1 or 20:1. Rat BAL cells were then incubated for 8 h at 37°C (4°C for the negative control). Due to differences in basal phagocytic activity compared with rat cells, human cells were incubated only for 4 h. After the incubation, the cells were centrifuged at 250 g for 5 min, and the supernatant was removed. The cells were fixed with 2 ml of a fixative solution containing 2% formaldehyde and 12 mM NaN3 for 20 min at room temperature. The fixed cells were centrifuged again as before and resuspended in 350 µl of PBS for analysis by flow cytometry or confocal microscopy. A nuclear stain was performed with 7-amino-actinomycin D (Beckmann-Coulter, Krefeld, Germany).
Determination of intracellular fate of phagocytosed PSG. To examine the intracellular fate of phagocytosed PSG, we preincubated 2 x 105 rat alveolar macrophages with Alexa Fluor 488-labeled PSG in culture medium at a ratio of 1:20 for 8 h in the presence or absence of rrSP-D (1 µg/ml). After this preincubation period, cells were spun through a 5%/68% Percoll step gradient to remove free PSG, thereby preventing further phagocytosis of PSG. The Percoll separation reduced the ratio of cells to extracellular PSG from 1:20 to 1:0.5 ± 0.2. After two washes with PBS, cells were resuspended in 0.5 ml of culture medium and incubated for another 0 (immediately fixed), 24, 48, or 96 h. After each incubation, vitality of cells was determined by trypan blue staining, and cells were immediately fixed with 2 ml of fixative solution, stained with TO-PRO3 (Molecular Probes), and measured by flow cytometry. Cytospin preparations were used for evaluation with the confocal laser scanning microscope.
Aggregation and binding assay. For the aggregation assay, PSG (2 x 106) were incubated with the surfactant proteins (rhSP-A at 30 µg/ml, AP-SP-A at 10 µg/ml, rfhSP-D at 30 µg/ml, and rrSP-D at 1 µg/ml) in culture medium at 37°C (or 4°C) for 8 h. The PSG were then centrifuged and fixed as described for the cells and subsequently analyzed by flow cytometry and confocal microscopy. For the evaluation by flow cytometry, the size of the granule aggregates was measured in the forward scatter (FSC) channel. A similar method was previously described for cells (28) or bacteria (10). For the calculation, the intensity in the FSC of the PSG incubated at 37°C in the absence of surfactant protein was set to 100%.
For the binding assay, the surfactant proteins (AP-SP-A and rrSP-D) were fluorescently labeled with FITC (n = 2) or Alexa Fluor 488 (n = 1) as described previously (27). The FITC-labeled surfactant proteins (AP-SP-A at 10 µg/ml, rrSP-D at 1 µg/ml) were then added to 2 x 106 Alexa Fluor 546-stained PSG in 1 ml of culture medium, incubated for 8 h, and prepared as in the previous experiments.
Flow cytometry, confocal microscopy. Cells were analyzed by flow cytometry with a FACSCalibur (Becton Dickinson) equipped with a 488-nm argon-ion laser and a 635-nm red diode laser. A minimum of 5,000 cells (gated on positive nuclear stain) was counted from each tube. The percentage of cells that bound/phagocytosed PSG was calculated as the number of positive cells in relation to the total number of cells analyzed. For the analysis of aggregation of the granules, the mean FSC intensities of all counted granules of each sample were compared. The mean FSC of the control at 37°C was set to 100%, and the change of size/aggregation of the PSG was expressed as a percentage of this control.
Confocal microscopy was performed on a Zeiss LSM 510 META run by LSM 510 software. To determine whether the PSG were adherent to the surface or located inside the cells, three-dimensional images were taken from fixed cells in solution (PBS) in a chamber slide after staining with Evans blue. A minimum of 100 cells was counted from representative samples.
Statistical analysis. The unpaired, two-tailed t-test was used for statistical analysis of the data. A Bonferroni correction was used throughout. P values <0.05 were considered to be significant. All values are given as means ± SE.
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RESULTS |
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The uptake/binding of PSG by macrophages was enhanced dose dependently by rrSP-D. At concentrations of 0.25, 0.5, 1, 2.5, and 5 µg/ml, the percentage of positive cells was increased to 25.9 ± 4.3, 33.7 ± 4.5, 40.7 ± 6.3, 54.7 ± 8.9, and 65.3 ± 10.9%, respectively. Increasing concentrations of AP-SP-A from 1 to 50 µg/ml showed no effect (data not shown).
After incubation with PSG from P. pratense pollen, 25.9 ± 6.2% of the cells were positive after incubation at 37°C, and rrSP-D increased the percentage of positive cells up to 50.3 ± 6.6%.
To determine whether rrSP-D increased only binding of PSG to the surface of macrophages or only uptake of PSG by macrophages or both binding and uptake, we performed additional experiments at 4°C, a temperature that disables macrophages to phagocytose PSG. At 4°C rrSP-D (1 µg/ml) enhanced binding of PSG to the cells from 7.1 ± 1.1% to 14.4 ± 3.3% (P < 0.01, Fig. 2), but the percentage of PSG-positive cells was much more increased at 37°C (40.7 ± 6.3%, P < 0.01), indicating that rrSP-D enhances binding as well as uptake at this temperature. These observations were confirmed by evaluation of 100 cells in three-dimensional images obtained by confocal microscopy from one representative experiment. After incubation at 37°C without rrSP-D, PSG were found on the surface of 11 ± 1.0% of the cells and inside of 13 ± 1.9% of the cells (cells that showed PSG inside and on the surface were only counted as inside). The rrSP-D increased the percentage of cells with PSG on the surface to 14 ± 1.7% and cells with ingested PSG to 28 ± 2.2% (Fig. 3). These data suggest that rrSP-D enhanced both binding and phagocytosis of PSG at 37°C.
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Enhanced uptake/binding of PSG by human alveolar macrophages in the presence of rrSP-D but not rfhSP-D. Human BAL cells (n = 5) were used to test whether the effect of rrSP-D might enhance the uptake of PSG by human alveolar macrophages as well. Because of the increased capability of human macrophages to phagocytose/bind the PSG compared with rat macrophages, an incubation time of 4 h was used for all experiments with human macrophages. The incubation of human macrophages together with PSG at a ratio of 1:10 at 37°C showed that 38.0% (± 4.7%) of the cells bound/phagocytosed PSG after 4 h, which was significantly enhanced by rrSP-D (69.5 ± 9.7%, P < 0.05). In contrast, there was no significant difference in the percentage of cells that bound/phagocytosed PSG in the presence of 250 µg/ml Curosurf (37.8 ± 7.9%), 250 µg/ml Alveofact (39.1 ± 6.2%), 30 µg/ml rhSP-A (41.0 ± 10.5%), 10 µg/ml AP-SP-A (44.0 ± 7.1%), or 30 µg/ml rfhSP-D (43.1 ± 13.0%).
Further experiments with rrSP-D at concentrations of 0.25, 1, and 5 µg/ml showed a dose dependency of this effect (data not shown).
Phagocytosed PSG remain inside alveolar macrophages.
To investigate the fate of phagocytosed PSG, macrophages were separated from extracellular PSG by Percoll gradient centrifugation after 8 h of preincubation and taken back into culture for up to 96 h. Without rrSP-D, the percentage of alveolar macrophages that bound/phagocytosed PSG was stable at 10% for 96 h (Fig. 4). In the presence of rrSP-D, >60% of the cells were PSG positive. After an initial decrease of about 13% during the first 24 h, the percentage remained stable at 45% up to 96 h (Fig. 4). Similar results were obtained by evaluation of mean fluorescence values of PSG-positive cells, indicating that the majority of PSG remained inside the cells (data not shown).
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PSG are aggregating in the presence of rrSP-D and to a lesser extent in the presence of AP-SP-A. Confocal microscopy reveals that the PSG were aggregating in the presence of rrSP-D. We further investigated this phenomenon by incubating the PSG with the different surfactant preparations without macrophages.
The incubation of PSG with rrSP-D at a concentration of 1 µg/ml induced an aggregation of the PSG up to 2,500% compared with the control at 37°C (set to 100%), as determined by the mean FSC intensity (Fig. 5A). Detectable, but significantly less, aggregation was also found in the presence of AP-SP-A at a concentration of 10 µg/ml, whereas no aggregation was found in the presence of rhSP-A or rfhSP-D. Aggregation of the PSG in the presence of rrSP-D was completely blocked by addition of EDTA (10 mM). A significant reduction of PSG aggregation was achieved by adding maltose at a concentration of 10 mM, but the aggregation was not significantly reduced by 10 mM galactose (Fig. 5A). Addition of EDTA to the PSG in the presence of AP-SP-A reduced the aggregation, which did not achieve statistical significance (data not shown). Flow cytometric evaluation of PSG aggregation in binding/uptake experiments with rrSP-D reveals that there was no correlation between the degree of aggregation of the PSG and the percentage of PSG-positive cells (Spearman r = 0.4818, P = 0.13).
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DISCUSSION |
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SP-D, like SP-A, belongs to the group III C-type lectin family. SP-D is composed of 43-kDa monomers, each consisting of four major domains: a short cysteine-containing amino-terminal region, a triple helical collagenous domain, a trimeric coiled neck region, and a globular carboxy-terminal CRD (4). These monomers are assembled into trimers, and four of these trimeric subunits undergo disulfide cross-linking within their amino-terminal domains to form a cruciform dodecameric structure. The binding of allergen by the CRD on one side of the molecule and the binding to the cell by the CRD on the other side of the molecule might promote phagocytosis. This might be a possible explanation of our findings, since previous studies have shown that surfactant proteins bind allergen (21, 32) as well as alveolar macrophages in a Ca2+-dependent manner through their CRDs (15). The effect of SP-D appears to be species independent since rrSP-D enhanced phagocytosis by both rat and human alveolar macrophages.
In addition to interacting with macrophages by its CRD, SP-D binding has also been shown via its collagen-like region (9). Thus SP-D might bind to PSG via its CRD and to alveolar macrophages via its collagen-like region. This could also explain why the effect is exclusively seen in the presence of full-length dodecameric rrSP-D but not rfhSP-D, which lacks the collagen-like domain and cysteine-rich region. In addition, SP-A did not enhance phagocytosis, but it increased aggregation of PSG although to a lesser extent compared with SP-D, which might indicate that SP-D-specific binding to alveolar macrophages is causing the increase of phagocytosis.
A phenomenon that was observed during the incubation of PSG with rrSP-D was the aggregation of the PSG. Interestingly, there was no aggregation of the PSG in the presence of rfhSP-D, which suggests that the aggregation of the PSG by SP-D is also dependent on the quaternary structure of the molecule. Previous studies have shown that SP-D aggregates various pathogens (8, 11, 16, 34). These studies also demonstrate that only dodecameric SP-D but not trimeric variants leads to agglutination of the bacteria and that the NH2-terminal and collagen domains of SP-D seem to be necessary for bacterial aggregation. Our results are consistent with these findings, and we conclude that the aggregation of PSG and bacteria by full-length dodecameric SP-D occurs via similar mechanisms.
AP-SP-A but not rhSP-A was also able to aggregate the PSG, however, to a much lesser extent compared with rrSP-D. Although no binding of AP-SP-A was visible in our confocal images, our data suggest that AP-SP-A has a low but significant potency to aggregate the PSG. The observed differences between SP-A and SP-D in their ability to enhance uptake and aggregation might be due to different binding affinities to the carbohydrate structures on the surface of PSG. Previous studies have shown that binding of SP-A and SP-D to pathogens via the CRD is dependent on the type of carbohydrate ligand present on the surface of the pathogens (1, 19).
Inhibition experiments demonstrated that CRD-mediated functions of SP-A and SP-D can be completely blocked by EDTA and competitively inhibited by various sugars with varying potency. Maltose, for example, is one of the most potent inhibitors for CRD-mediated functions of SP-D, whereas galactose inhibits these functions to a much lesser degree (24). In our experiments, EDTA completely blocked the uptake and aggregation of the PSG, indicating a calcium ion dependency of the process. Moreover, rrSP-D enhanced binding and uptake of PSG in experiments with serum-free medium only after supplementation of calcium. In addition, maltose and, to a lesser extent, galactose competitively inhibited the enhanced uptake and aggregation of the PSG in the presence of rrSP-D. Together, these data suggest that the described processes are dependent on the affinity of the surfactant proteins for specific carbohydrate structures on the surface of the PSG.
Previous studies have shown that aggregation of pathogens does not necessarily lead to enhanced uptake by alveolar macrophages (1) or that aggregation is a phenomenon that can occur independently of phagocytosis (8). Moreover, for some pathogens, aggregation by surfactant proteins might even impair their phagocytosis (34). Although we observed large aggregates of PSG after incubation with rrSP-D, the vast majority of the granules that were found intracellularly were solitary. In addition, there was no correlation between the degree of PSG aggregation and the percentage of PSG-positive cells in experiments performed with rrSP-D. Therefore, we assume that SP-D mediates both aggregation and phagocytosis but that aggregation might not be necessary to enhance phagocytosis.
To determine the fate of phagocytosed PSG, alveolar macrophages were preincubated with PSG and followed up to 96 h after separation of extracellular PSG. The percentage of cells that had ingested PSG in the presence of rrSP-D initially decreased 24 h after Percoll separation but remained stable from 24 to 96 h. Therefore, we assume that the majority of PSG that are phagocytosed by alveolar macrophages remain inside the cells where they undergo either degradation (14) or macrophage-mediated clearance via the bronchotracheal route (17, 23).
In conclusion, our results demonstrate that dodecameric SP-D enhances phagocytosis and aggregation of pollen allergen, which might contribute to an increased allergen elimination from the lung. Thereby, SP-D might reduce allergen load and reduce allergic inflammation in patients with allergic asthma.
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GRANTS |
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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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