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Am J Physiol Lung Cell Mol Physiol 293: L1437-L1443, 2007. First published September 28, 2007; doi:10.1152/ajplung.00239.2007
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Surfactant protein A regulates IgG-mediated phagocytosis in inflammatory neutrophils

Jessica A. Wofford and Jo Rae Wright

Department of Cell Biology, Duke University Medical Center, Durham, North Carolina

Submitted 20 June 2007 ; accepted in final form 25 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Surfactant proteins (SP)-A and SP-D have been shown to affect the functions of a variety of innate immune cells and to interact with various immune proteins such as complement and immunoglobulins. The goal of the current study is to test the hypothesis that SP-A regulates IgG-mediated phagocytosis by neutrophils, which are major effector cells of the innate immune response that remove invading pathogens by phagocytosis and by extracellular killing mediated by reactive oxygen and nitrogen. We have previously shown that SP-A stimulates chemotaxis by inflammatory, but not peripheral, neutrophils. To evaluate the ability of SP-A to modulate IgG-mediated phagocytosis, polystyrene beads were coated with BSA and treated with anti-BSA IgG. SP-A significantly and specifically enhanced IgG-mediated phagocytosis by inflammatory neutrophils, but it had no effect on beads not treated with IgG. SP-A bound to IgG-coated beads and enhanced their uptake via direct interactions with the beads as well as direct interactions with the neutrophils. SP-A did not affect reactive oxygen production or binding of IgG to neutrophils and had modest effects on polymerization of actin. These data suggest that SP-A plays an important role in mediating the phagocytic response of neutrophils to IgG-opsonized particles.

innate immunity; lung; collectin; host defense

NEUTROPHILS COMPRISE A MAJOR component of the innate immune response. During acute inflammation, neutrophils are recruited from the circulation into the tissue, where they act as one of the first lines of defense against foreign invaders (4, 26). One of their primary defense mechanisms is the removal of pathogens by phagocytosis. They also neutralize pathogens by producing nitrogen and oxygen radicals via the respiratory burst and by releasing potent antimicrobial compounds upon degranulation. Although neutrophils are powerful effector cells that are crucial in the host defense against infection, they also have the potential to damage host tissues since the molecules released to thwart invaders cannot differentiate host tissue from invading pathogens. Resolution of inflammation requires that neutrophils undergo apoptosis once they have fulfilled their roles. Neutrophil dysregulation has been implicated in a variety of pathological states in the lung, including asthma, adult respiratory distress syndrome, cystic fibrosis, and emphysema (26).

Surfactant protein (SP)-A is a member of the collectin family, a group of proteins defined by their NH2-terminal collagen-like regions and COOH-terminal lectin domains. SP-A is found in the surfactant layer at the alveolar air-tissue interface in the alveoli of the lungs and has been demonstrated to regulate immune cell functions (31). SP-A has been shown to regulate multiple functions of alveolar macrophages, the resident phagocytes in the normal, noninflamed lung. For example, SP-A independently induces alveolar macrophage cytokine release (11), chemotaxis (32), phagocytosis (28), and clearance of apoptotic neutrophils (24). SP-A has been shown to enhance macrophage phagocytosis of bacteria via Fc{gamma}R by acting synergistically with IgG (22) and via scavenger receptor-A (SR-A) by increasing cell surface expression of SR-A (15). Observations that SP-A can induce F-actin polymerization (29), alter the phosphotyrosine profile in macrophages (22), and stimulate casein kinase-2 activity (15) indicate that SP-A can directly influence signaling pathways in alveolar macrophages.

Only a few studies have investigated interactions between neutrophils and SP-A. For example, Schagat et al. (23) compared the effects of SP-A on functions of peripheral, unactivated neutrophils from the blood and on inflammatory neutrophils lavaged from inflamed rat lungs. In contrast to its effects on macrophages, SP-A alone was unable to elicit chemotaxis in either peripheral or inflammatory neutrophils. However, SP-A significantly, but differentially, altered the chemotactic responses of both peripheral and inflammatory neutrophils to known chemoattractants. Specifically, SP-A enhanced agonist-induced chemotaxis in inflammatory neutrophils but inhibited it in peripheral neutrophils. Kramer and colleagues (14) reported that SP-A stimulates neutrophil recruitment in the lungs of preterm lambs. In addition, Hartshorn et al. (12) showed that SP-A stimulates phagocytosis of certain bacteria by peripheral neutrophils.

Phagocytosis is one of the most important functions of neutrophils, and previous studies have shown that many molecules, particularly IgG, function as opsonins to enhance phagocytosis by neutrophils (4). Since we had previously demonstrated that SP-A synergistically enhanced IgG-mediated phagocytosis in macrophages (16, 22), we hypothesized that SP-A may have similar synergistic effects on IgG-mediated phagocytosis in inflammatory neutrophils. To address this question, polystyrene beads were covalently coated with BSA and treated with anti-BSA IgG, thereby presenting a target similar to an IgG-opsonized pathogen. We found that SP-A significantly and specifically enhanced IgG-mediated phagocytosis by inflammatory neutrophils but had no effect on uptake of beads not coated with IgG.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Dulbecco's PBS and HBSS were from GIBCO (Rockville, MD). Alexa Fluor 647-phalloidin, dihydrorhodamine (DHR) 123, and FITC were purchased from Molecular Probes (Eugene, OR). BSA and gelatin from porcine skin were purchased from Sigma (St. Louis, MO) as were all other reagents unless otherwise noted.

Isolation of inflammatory rat neutrophils. Inflammatory neutrophils were obtained as previously described (23). In brief, male Sprague-Dawley rats, 250–400 g, from Taconic Farms (Germantown, NY) were instilled intratracheally with O26:B6 LPS (100 µg/kg in 0.9% saline) to induce acute inflammation in the lungs. After 12–14 h, the lungs were excised and lavaged with PBS containing 0.2 mM EGTA (pH 7.2) to collect a total of 50 ml of lavage fluid per rat. Cells were pelleted by centrifugation at 250 g for 10 min and resuspended in 5 ml of PBS per rat; when multiple rats were used on one day, all cell pellets were pooled before being separated into new tubes. For every 2.5 ml of cells, a five-step Percoll gradient was underlayed below the cells and consisted of step densities of 1.081, 1.085, 1.089, 1.093, and 1.097 g/ml of Percoll in 0.9% saline. Gradients were centrifuged at 500 g for 30 min at room temperature. Cells were collected from bands at the interfaces of 1.085–1.089 and 1.089–1.093 and washed in PBS. Cells were counted and resuspended at 5 x 106 cells/ml in a standard assay buffer of HBSS with 0.1% (wt/vol) gelatin unless otherwise stated. Cell purity was ascertained to be no less than 97% neutrophils. Each experiment was performed at least three times with neutrophil preparations isolated on different days from bronchoalveolar lavage fluid pooled from one to three rats. All experiments were approved by the Duke University Institutional Animal Care and Use Committee.

Proteins. Purified C1q was purchased from Advanced Research Technologies (San Diego, CA). Recombinant rat SP-D was purified as previously described (7). SP-A was purified from bronchoalveolar lavage fluid from alveolar proteinosis patients and treated to remove LPS (17). Rat IgG (Sigma) and SP-A were labeled with FITC according to the manufacturer's instructions.

Preparation of BSA-coated and IgG-coated beads. Yellow-green and bright blue polystyrene carboxylate microspheres (1-µm diameter) were purchased from Polysciences (Warrington, PA) and covalently conjugated with BSA as per the manufacturer's instructions, with the minor modification that beads were stored in a storage buffer containing gelatin in place of BSA and lacking azide. BSA-coated beads were stored at 4°C and used within 3 wk of preparation.

To make IgG-coated beads, an aliquot of BSA-coated beads was washed twice in PBS, resuspended in PBS, vortexed liberally, and separated into two tubes previously coated with 1% gelatin at 4°C overnight. Anti-BSA-IgG was added (1:25) to one tube, and both were incubated, shaking, at 37°C for 30–60 min, followed by overnight incubation at 4°C on a rotator to ensure end-over-end rotation. This was crucial to guarantee homogeneous suspensions of beads and minimal aggregation. On the day of an experiment, and only shortly before use, beads were washed twice in PBS, resuspended in a minimal volume of HBSS, and transferred to new tubes. It was necessary to determine the concentration of each bead preparation just before use in an experiment. Bead concentration was determined by measuring the fluorescence of a serial dilution of beads of known concentration (untreated stock from the manufacturer) compared with dilutions of the BSA- and IgG-coated beads. Each bead preparation was then diluted into the standard assay buffer. Initial studies revealed that an appropriate neutrophil:bead ratio was 1:25, and this ratio was used in all experiments unless otherwise noted. Yellow-green beads were used in all assays except for those investigating binding of FITC-SP-A to the beads; bright blue beads were used in these experiments.

Bead phagocytosis assays. Neutrophil phagocytosis of beads was assayed in suspension in 0.25–0.5 ml using 2 x 106 cells/ml and 5 x 107 beads/ml in standard assay buffer. Samples were incubated at 37°C with gentle shaking for 1 h in the presence and absence of SP-A, SP-D, or C1q. The reaction was stopped by addition of 0.75 ml of ice-cold PBS. Samples were washed twice in cold PBS and resuspended and fixed in 1% formaldehyde in PBS. Flow cytometry was used to analyze the samples, and cells were identified based on forward and side scatter profiles.

Phagocytosis by adherent neutrophils was investigated using two-chamber slides (Nunc, Naperville, IL) coated overnight at 4°C with 25 µg/ml SP-A or poly-D-lysine in 0.1 M carbonate buffer, pH 9.6. Before the addition of cells, slides were warmed to 37°C and washed twice with PBS. Approximately 2–3 x 106 neutrophils in RPMI media were added to each chamber and incubated for 60–90 min at 37°C with 5% CO2. Nonadherent cells were removed by washing, and BSA-coated or IgG-coated beads were added at the same concentration as in the suspension assays, 5 x 107 beads/ml, with or without 25 µg/ml SP-A. Cells and beads were incubated for 60 min at 37°C, washed, and then fixed in 1% paraformaldehyde for 10 min at room temperature. The chambers and gaskets were removed, and cells were stained with 1% Evans Blue in PBS for 7 min at room temperature. Cells were mounted in anti-fading Gel/Mount (Biomeda, Foster City, CA) and stored at 4°C.

Cells were analyzed by confocal microscopy using a Zeiss LSM410 laser-scanning microscope with a krypton/argon laser, dual color settings, and a x63 lens. Images were collected for each condition and analyzed for the presence of neutrophils and beads. At least 100 cells were counted for each condition.

Assay for production of reactive oxygen species. The presence of reactive oxygen species (ROS) was analyzed using DHR as an indicator for the presence of ROS, particularly H2O2. DHR is cell-permeable and is reduced by ROS to membrane-impermeable rhodamine 123, which can be detected based on its fluorescent properties. The intensity of rhodamine 123 emission is indicative of the amount of ROS produced during incubation. DHR was resuspended in DMSO and stored at –80°C under N2 gas in single-use aliquots. Neutrophils were incubated with 1 µM DHR for 10 min at 37°C and then added to tubes containing the stimulating compound(s). Cells were stimulated at 37°C for 15–60 min depending on the stimulus, washed, and fixed. Samples were analyzed by flow cytometry within 2 h of the completion of the assay to minimize background due to dye leakage.

Quantification of F-actin. Alexa Fluor 647-conjugated phalloidin was used to quantify the amount of F-actin in cells. Following stimulation, cells were immediately permeabilized for 5 min by diluting them 1:2 in 3.7% formaldehyde with 1 µg/ml lysophosphatidylcholine in PBS without calcium and shaking at 37°C for 10 min. Cells were washed twice with calcium-free PBS, resuspended in 0.1 ml with 2 units of Alexa Fluor 647-phalloidin in calcium-free PBS, and incubated for 20 min at room temperature in the dark. Cells were washed and fixed before being analyzed by flow cytometry.

Statistical analysis. Data are expressed as means ± SE based on at least three independent experiments. Comparisons between two groups were based on Student's t-test, and variance was analyzed by one-way ANOVA. Statistical significance was assessed based on a minimal confidence interval of 95%.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
SP-A stimulates phagocytosis of IgG-coated beads. BSA-coated and IgG-coated beads were each incubated with neutrophils in suspension for 1 h, and phagocytosis was assessed by FACS. IgG-coated beads were phagocytosed significantly more than BSA-coated beads (percent positive cells 23.6 ± 3.3 vs. 15.5 ± 2.6, respectively).

To determine the effect of SP-A on neutrophil phagocytosis, SP-A was added to neutrophils with IgG- or BSA-coated beads. SP-A had no effect on baseline uptake of BSA-coated beads by neutrophils (15.5 ± 2.6 percent positive cells at baseline vs. 14.1 ± 2.3 percent positive cells with 25 µg/ml SP-A; Fig. 1A). However, SP-A significantly enhanced uptake of IgG-coated beads in an SP-A dose-dependent manner (Fig. 1A). At concentrations of 5, 10, 25, and 50 µg/ml, SP-A significantly stimulated IgG-coated bead phagocytosis (P < 0.04 by t-test, P < 0.0001 by 1-way ANOVA). In the presence of SP-A, more neutrophils phagocytosed IgG-coated beads (increased percent positive cells), and each neutrophil phagocytosed more beads (increased relative mean fluorescence; Fig. 1B). Thus, despite having no effect on baseline uptake of BSA-coated beads, SP-A stimulated IgG-mediated phagocytosis by neutrophils.


Figure 1
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  		Fig. 1. Effect of surfactant protein (SP)-A on phagocytosis of BSA-coated and IgG-coated beads. Rat inflammatory neutrophils were incubated in suspension with beads coated with BSA or IgG (1:25 cell:bead ratio) with increasing concentrations of SP-A. Uptake of BSA-coated beads with no SP-A was considered to represent baseline activity, and other samples were expressed as a percentage of this sample in each experiment. SP-A treatment significantly stimulated more cells to phagocytose IgG-coated beads, as represented by increasing relative percent positive cells (A), and an increase in number of IgG-coated beads phagocytosed by each cell (B), seen as increasing fluorescence. Means ± SE, n = 5. *By t-test, P < 0.05 compared with IgG-coated beads with no SP-A. Analysis of variance among the samples with IgG-coated beads indicated a significant effect of SP-A with P < 0.002.

 
SP-A stimulation of phagocytosis of IgG-coated beads is specific. The ability of other structurally or functionally related proteins to affect uptake of IgG-coated beads was determined. SP-D is another collectin shown to have immunomodulatory functions (31). In spite of its ability to enhance uptake of some pathogens, SP-D had no effect on uptake of BSA-coated or IgG-coated beads (Fig. 2). Complement protein C1q is structurally similar to SP-A and contains a collagen-like domain, although it lacks a lectin domain (6). C1q had no effect on neutrophil uptake of BSA-coated beads but inhibited phagocytosis of IgG-coated beads by ~65% (Fig. 2). This was not entirely surprising because C1q has been shown to bind the Fc portion of IgG molecules and may block the Fc interaction with the Fc{gamma}R (30). The inability of similar proteins to stimulate bead uptake indicated a specific effect for SP-A in neutrophil phagocytosis mediated by IgG.


Figure 2
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  		Fig. 2. Effect of different proteins on IgG-coated bead phagocytosis. IgG-coated beads were incubated with neutrophils in suspension with nothing, 25 µg/ml SP-A, 1 µg/ml SP-D, or 25 µg/ml C1q. Samples were standardized to the bead-alone condition. SP-D had no effect, but C1q significantly inhibited phagocytosis. The ability to enhance IgG-mediated phagocytosis was particular to SP-A. Means ± SE, n = 7. *By t-test, P < 0.01 vs. beads alone.

 
SP-A specifically binds IgG-coated beads. In attempting to reveal how SP-A might stimulate phagocytosis, several possible mechanisms were investigated. Initially, we asked whether opsonization of IgG-coated beads by SP-A enhanced their uptake. BSA-coated and IgG-coated beads were incubated for 1 h on a shaker at 37°C with or without SP-A, placed on ice, and analyzed by flow cytometry. On a dot plot of forward vs. side scatter, as bead aggregates increase in size, the population shifts toward the upper right. IgG-coated beads were observed to be spontaneously aggregated more than BSA-coated beads, as evidenced by greater forward and side scatter (Fig. 3A vs. 3D). IgG-coated bead aggregates increased in size in the presence SP-A, but not with SP-D or C1q (Fig. 3, D and E, and data not shown), whereas aggregation of BSA-coated beads was unaffected by the presence of SP-A (Fig. 3, A and B). When beads were incubated with FITC-labeled SP-A, IgG beads were almost 100% bound by FITC-SP-A (Fig. 3F, solid region). BSA-coated beads displayed varying degrees of binding, with 50–80% of beads positive for FITC (Fig. 3C, solid region). The relative mean fluorescence of beads incubated with FITC-SP-A was 35.6 ± 1.3 for BSA-coated beads and 1,423.9 ± 359.4 for IgG-coated beads (P < 0.02), indicating that FITC-SP-A bound to IgG-coated beads at a significantly higher rate. Thus, SP-A binds to and aggregates IgG-coated beads to a greater extent than it binds to and aggregates BSA-coated beads.


Figure 3
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  		Fig. 3. Effect of incubating beads with SP-A. BSA-coated (A–C) and IgG-coated beads (D–F) were incubated with 25 µg/ml SP-A (B and E) or 25 µg/ml FITC-SP-A (C and F) for 1 h on a shaker at 37°C and analyzed by flow cytometry. SP-A increased IgG-coated bead aggregation but not BSA-coated bead aggregates. FITC-SP-A bound more to IgG-coated beads than to BSA-coated beads; the white region in C and F depicts beads alone, and the solid black region depicts beads + FITC-SP-A. Graphs are representative samples, n = 3. FSC, forward scatter; SSC, side scatter.

 
Preincubating beads with SP-A does not affect phagocytosis. Since SP-A was found to bind IgG-coated beads, we next determined whether preincubating the beads with SP-A could affect bead uptake. BSA-coated and IgG-coated beads were incubated for 1 h at 37°C on a rotator with different amounts of SP-A, washed once, and then added to neutrophils in the phagocytosis assay. SP-A pretreatment at concentrations up to 50 µg/ml had no effect on neutrophil phagocytosis for either bead type (data not shown).

SP-A has no effect on binding of soluble IgG to neutrophils. It is possible that SP-A regulates neutrophil phagocytosis by directly enhancing binding of IgG to Fc{gamma}R on the neutrophil surface. To test this possibility, neutrophils were incubated with soluble FITC-IgG and different amounts of SP-A for 1 h at 4°C and 37°C. SP-A had no effect on binding of soluble FITC-IgG in all conditions tested (data not shown).

Preincubating neutrophils with SP-A enhanced phagocytosis of IgG-coated beads. Next, the direct effects of SP-A on neutrophils were investigated. Neutrophils were incubated in media with or without SP-A for 3 h, lightly shaking at 37°C, washed, and then used in the bead uptake assay. Phagocytosis of IgG-coated beads increased significantly from 20.3 ± 1.2 to 27.9 ± 2.4 percent positive cells following preincubation with SP-A (P < 0.03), but BSA-coated bead uptake was unaffected (Fig. 4).


Figure 4
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  		Fig. 4. Effect of SP-A preincubation on bead uptake. Neutrophils were incubated at 4 x 106 cells/ml in RPMI media with or without 25 µg/ml SP-A at 37°C for 3 h. Cells were diluted 10:1 with PBS and washed twice in 50 ml PBS before being added to the bead uptake assay. SP-A preincubation significantly enhanced IgG-coated bead phagocytosis but had little effect on BSA-coated bead uptake. Means ± SE, n = 3. *By t-test, P < 0.03.

 
SP-A has no effect on ROS production. The production of ROS in a respiratory burst is a major function of neutrophils during inflammation and is a downstream response to activation of Fc{gamma}R (19). To determine if SP-A might activate or enhance ROS production, production of H2O2 was monitored based on the reduction of DHR 123 to rhodamine 123. PMA and the bacterial peptide fMLP activate the respiratory burst by inducing formation of the NADPH oxidase, the complex responsible for production of ROS (1). Rhodamine 123 fluorescence increased with increasing doses of PMA (0.01–1 µg/ml) or fMLP (1 µM). SP-A had no effect on rhodamine 123 fluorescence, either alone or with PMA or fMLP (data not shown).

SP-A diminishes actin polymerization induced by fMLP. Major cytoskeletal rearrangements are required for many neutrophil functions, including phagocytosis, respiratory burst, and chemotaxis. Altering actin polymerization was one possible means by which SP-A might affect neutrophils. SP-A alone had no effect on F-actin content in neutrophils. Actin polymerization induced by fMLP (10 nM) was diminished by higher doses of SP-A (Fig. 5). This effect was significant (P < 0.00002 by ANOVA), although the maximal dose of SP-A, 100 µg/ml, was only able to inhibit fMLP-induced F-actin polymerization by roughly 10%. Phagocytosis requires rapid and extreme reorganization of the cytoskeleton (5); it is possible that a small decrease in actin polymerization may facilitate these rearrangements.


Figure 5
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  		Fig. 5. Effect of SP-A on actin polymerization. Neutrophils were incubated with 10 nM fMLP and several concentrations of SP-A for 30 min. F-actin was quantitated using fluorescence emission of Alexa 647-phalloidin and standardized to the fMLP-alone condition. SP-A diminished fMLP-stimulated actin polymerization. Although it was not a large effect, it was dose dependent and was significant by 1-way ANOVA (P < 0.0002). Means ± SE, n = 4. *By t-test, P < 0.05.

 
Adherence to SP-A-coated surface inhibits phagocytosis; inhibition is reversed by soluble SP-A. Adherence to a surface coated with a protein that binds to Fc{gamma}R, such as an antibody to the receptor, has been shown to inhibit neutrophil IgG-mediated phagocytosis and respiratory burst due to Fc{gamma}R activation, purportedly by sequestering receptors on the basal side of the cell (33). On the other hand, adherence to a surface coated with a protein that activates neutrophils without binding active receptors, such as various extracellular matrix proteins, has been shown to stimulate phagocytosis (3). To clarify if SP-A might alter receptor localization and/or neutrophil activation, neutrophils were allowed to adhere to chamber slides coated with lysine or SP-A. Adherent cells were then presented with BSA-coated or IgG-coated beads with or without soluble SP-A. Neutrophils adhered to lysine recapitulated the results from neutrophils in suspension: SP-A had no effect on BSA-coated bead uptake (16.0 ± 5.5 vs. 15.2 ± 4.1 percent positive cells, in the absence and presence of SP-A) and stimulated IgG-coated bead phagocytosis (32.0 ± 8.9 vs. 55.6 ± 4.0 percent positive cells; Fig. 6).


Figure 6
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  		Fig. 6. Effect of SP-A as an adherence substrate during neutrophil phagocytosis of beads. Neutrophils were allowed to adhere to lysine- or SP-A-coated chamber slides for 60–90 min. BSA-coated or IgG-coated beads were then added, with and without 25 µg/ml SP-A. Cells and beads were incubated together for 1 h. Cells were fixed and stained with Evans Blue before being analyzed by confocal microscopy. Using a x63 oil-immersion lens, cells were analyzed for bead internalization. A bead was considered to be inside of a cell when it was fully surrounded by the cell on all sides. Images were viewed with a minimum of 100 cells, and total bead and cell numbers were recorded. Neutrophils adherent to SP-A-coated slides phagocytosed significantly fewer BSA-coated and IgG-coated beads. Addition of soluble SP-A reversed this inhibition and restored phagocytosis to levels comparable to phagocytosis by cells adherent to lysine and treated with soluble SP-A. Percent positive cells refers to the number of cells having internalized at least 1 bead as a percentage of the total number of cells counted. Means ± SE, n = 3–5. *P < 0.06 compared with same condition lysine coat. #P < 0.008 compared with same condition (same slide coat, same bead type) without soluble SP-A.

 
Neutrophils adhered to SP-A phagocytosed significantly fewer beads compared with those adhered to lysine. This effect was observed for both BSA-coated and IgG-coated beads. Adherence of neutrophils to SP-A inhibited uptake of BSA-coated beads by 62.4 ± 8.5% and of IgG-coated beads by 37.9 ± 8.9%. When soluble SP-A was present, the inhibitory effect of adherence to SP-A was reversed, resulting in similar levels of phagocytosis by cells exposed to soluble SP-A and adherent to lysine or to SP-A.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Although SP-A is able to regulate multiple functions of alveolar macrophages and other immune cells, its ability to directly modulate neutrophil functions appears to be more limited. For example, SP-A can independently stimulate alveolar macrophage chemotaxis and uptake of multiple pathogens but is unable to stimulate the uptake of targets (polystyrene beads) in our experimental system. In contrast, we observed that SP-A does enhance the uptake of beads coated with IgG. These findings were interesting in the context of our previous studies showing that although SP-A did not directly stimulate neutrophil chemotaxis, it did alter responses to other chemoattractants such as fMLP and IL-8 (23). Thus, SP-A does not directly regulate neutrophil chemotaxis and phagocytosis but does augment effects elicited by other mediators.

There are at least two basic means by which SP-A may affect neutrophil phagocytosis mediated by IgG. The first is by affecting the bead targets and how they are presented to the cells. This may occur via opsonization of the beads and/or aggregation of beads. SP-A is known to aggregate various bacteria (12, 28) and has been reported to bind to immunoglobulins (13, 16). We found that SP-A increased aggregation of IgG-coated but not BSA-coated beads and that SP-A bound to both kinds of beads, but much more to IgG-coated beads. These findings are consistent with the recently published studies of Lin and Wright (16) who reported that SP-A binds to the Fc region of IgG and enhances uptake of IgG-coated red blood cells, although the mechanism of enhanced uptake did not involve aggregation of the IgG-coated red blood cells. Nadesalingam and coworkers (18) recently demonstrated that SP-D binds to IgG, aggregates IgG-coated beads, and enhances their uptake by murine macrophage RAW 264.7 cells, although SP-D did not affect neutrophil phagocytosis in our assays. Preincubating the beads with SP-A did not have any effect in these experiments, but this does not rule out an important bead-SP-A interaction, since the necessary washing steps may have washed off or reduced any bead-associated SP-A. It is possible that the affinity of SP-A for the beads is rather low and/or that SP-A binding to the beads is not important for bead uptake. Kuronuma et al. (15) found that the ability of SP-A to stimulate uptake of Streptococcus pneumoniae by alveolar macrophages was independent of its binding to the bacteria, although SP-A did bind to these bacteria. SP-A's inability to alter binding of soluble IgG molecules to neutrophils does not rule out an effect of SP-A in binding of IgG-coated beads, since the lower affinity Fc{gamma}R (Fc{gamma}RII and Fc{gamma}RIII) require IgG complexes for binding. These data do indicate that SP-A does not affect IgG binding to the high-affinity receptor Fc{gamma}RI, known to be expressed upon activation of neutrophils from mice and humans (8). Based on these observations, SP-A's ability to alter how the beads are presented to the neutrophils may or may not be significant in stimulating IgG-mediated phagocytosis.

The second way SP-A may affect phagocytosis is by altering the state of activation of the neutrophils. The lack of effect of SP-A in fMLP- or PMA-induced production of ROS and the small effect on fMLP-induced F-actin polymerization indicate that SP-A is not having a generalized activation effect that merely enhances all inflammatory functions measured. We have shown that SP-A enhances fMLP- and macrophage inflammatory protein-2-induced chemotaxis in inflammatory neutrophils (23). The finding that SP-A enhanced IgG-mediated phagocytosis when preincubated with neutrophils suggests that SP-A may have a direct effect on neutrophil phagocytosis of particles opsonized by IgG. Hartshorn and colleagues (12) demonstrated that SP-A preincubation also stimulated neutrophil uptake of various kinds of bacteria, yet washing SP-A-treated bacteria diminished the effect of SP-A to stimulate bacteria phagocytosis. As we found that SP-A had no effect on uptake of BSA-coated beads, the ability of SP-A to stimulate bacterial phagocytosis by neutrophils indicates that SP-A enhances phagocytosis when the target presents either endogenous ligands (such as on the surface of a bacterium) or opsonin (such as IgG) to which the neutrophil can bind.

Our results showed that adherence of neutrophils to SP-A-coated surfaces inhibited phagocytosis of both BSA- and IgG-coated beads. These findings suggest there is a significant direct interaction between SP-A and neutrophils and that this effect is dependent on whether the cells are adherent to SP-A or exposed to SP-A in suspension, as in the preincubation experiments. Interestingly, this inhibitory effect is in contrast to the previously reported finding that uptake of IgG-coated targets by monocyte-derived macrophages is enhanced by adherence to SP-A- and C1q-coated surfaces (27). The generalized inhibitory effect we found was surprising since SP-A enhanced uptake of IgG-coated, but not BSA-coated, beads when SP-A was added to neutrophils in suspension or adhered to lysine-coated surfaces. In cells adherent to lysine control slides, soluble SP-A had the same effect as for cells in suspension: enhancing uptake of IgG-coated beads but not affecting uptake of BSA-coated beads. This indicates that adherence in and of itself does not alter the ability of neutrophils to phagocytose targets. Additionally, soluble SP-A was able to reverse the inhibitory effect of SP-A as an adherence substrate: phagocytosis of each bead type was returned to levels comparable to those observed in samples adherent to lysine and treated with soluble SP-A, returning BSA-coated bead uptake to baseline levels, and stimulating uptake of IgG-coated beads. These observations indicate that SP-A directly affects neutrophils, probably via more than one mechanism.

The concentration of SP-A in the rat lung is estimated to be in the range of 0.3–1.8 mg/ml (31), with ~2% of this being lipid-free (2, 21), indicating that the 25 µg/ml SP-A used in this study is within the physiological range. Phospholipids not only bind to SP-A but have also been demonstrated to affect phagocytosis of alveolar macrophages by competing with bacteria for engulfment (25). While Golioto and Wright (10) reported that lipids inhibited alveolar macrophage association with bacteria, they found that 25 µg/ml SP-A was sufficient to stimulate phagocytosis above the control condition (no SP-A, no lipids) when lipids were present at concentrations up to 250 µg/ml. Thus, while phospholipids may diminish the potency of SP-A effects, by binding to SP-A, competing with targets for uptake, or some other mechanism, it is clear that physiologically relevant concentrations of SP-A are capable of stimulating phagocytosis by alveolar macrophages even in the presence of surfactant lipids. To the best of our knowledge, the effects of lipids directly on neutrophil responses and on SP-A regulation of neutrophil functions have not been investigated and are important topics of future studies.

In conclusion, inflammatory neutrophils respond to SP-A in a more specific and limited manner than alveolar macrophages. A molecular mechanism for the effects of SP-A in regulating macrophage immune functions remains unknown, although several receptors and intracellular mediators have been implicated; these include SIRP{alpha}, CD91 (9), Toll-like receptor 4 (11), Toll-like receptor 2 (20), and casein kinase II (15). In these studies, we have found that SP-A has different effects on inflammatory lung neutrophils than those observed with alveolar macrophages. On the basis of the different responses to SP-A, it appears that SP-A may have different or only partially overlapping mechanisms for affecting alveolar macrophages and inflammatory neutrophils, possibly by binding to different receptors and/or by affecting different intracellular signaling pathways. The differential effects of SP-A in regulating macrophages and neutrophils may provide a clue as to the means by which SP-A modulates host responses in the lung.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-30923 and HL-51134.


   ACKNOWLEDGMENTS
 
We thank Kathy Evans for preparation of SP-A and assistance with data collection.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. Wright, Box 3709, Dept. of Cell Biology, Duke Univ. Medical Center, Durham, NC 27710 (e-mail: j.wright{at}cellbio.duke.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Babior B. NADPH oxidase: an update. Blood 93: 1464–1476, 1999.[Free Full Text]
  2. Baritussio A, Alberti A, Quaglino D, Pettenazzo A, Dalzoppo D, Sartori L, Pasquali-Ronchetti I. SP-A, SP-B, and SP-C in surfactant subtypes around birth: reexamination of alveolar life cycle of surfactant. Am J Physiol Lung Cell Mol Physiol 266: L436–L447, 1994.[Abstract/Free Full Text]
  3. Borgquist J, Quinn M, Swain S. Adhesion to extracellular matrix proteins modulates bovine neutrophil responses to inflammatory mediators. J Leukoc Biol 71: 764–774, 2002.[Abstract/Free Full Text]
  4. Burg N, Pillinger M. The neutrophil: function and regulation in innate and humoral immunity. Clin Immunol 99: 7–17, 2001.[CrossRef][Web of Science][Medline]
  5. Castellano F, Chavrier P, Caron E. Actin dynamics during phagocytosis. Semin Immunol 13: 347–355, 2001.[CrossRef][Web of Science][Medline]
  6. Crouch E. Collectins and pulmonary host defense. Am J Respir Cell Mol Biol 19: 177–201, 1998.[Abstract/Free Full Text]
  7. Dong Q, Wright J. Degradation of surfactant protein D by alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 274: L97–L105, 1998.[Abstract/Free Full Text]
  8. Flesch B, Neppert J. Functions of the Fc receptors for immunoglobulin G. J Clin Lab Anal 14: 141–156, 2000.[CrossRef][Web of Science][Medline]
  9. Gardai S, Xiao Y, Dickinson M, Nick J, Voelker D, Greene K, Henson P. By binding SIRPalpha or calreticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation. Cell 115: 13–23, 2003.[CrossRef][Web of Science][Medline]
  10. Golioto A, Wright JR. Effects of surfactant lipids and surfactant protein A on host defense functions of rat alveolar macrophages. Pediatr Res 51: 220–227, 2002.[CrossRef][Web of Science][Medline]
  11. Guillot L, Balloy V, McCormack F, Golenbock D, Chignard M, Si-Tahar M. Cutting edge: the immunostimulatory activity of the lung surfactant protein A involves Toll-like receptor 4. J Immunol 168: 5989–5992, 2002.[Abstract/Free Full Text]
  12. Hartshorn K, Crouch E, White M, Colamussi M, Kakkanatt A, Tauber B, Shepherd V, Sastry K. Pulmonary surfactant proteins A and D enhance neutrophil uptake of bacteria. Am J Physiol Lung Cell Mol Physiol 274: L958–L969, 1998.[Abstract/Free Full Text]
  13. Hattori A, Kuroki Y, Takahashi H, Sohma H, Akino T. Immunoglobulin G is associated with surfactant protein A aggregate isolated from patients with pulmonary alveolar proteinosis. Am J Respir Crit Care Med 155: 1785–1788, 1997.[Abstract]
  14. Kramer BW, Jobe AH, Bachurski CJ, Ikegami M. Surfactant protein A recruits neutrophils into the lungs of ventilated preterm lambs. Am J Respir Crit Care Med 163: 158–165, 2001.[Abstract/Free Full Text]
  15. Kuronuma K, Sano H, Kato K, Kudo K, Hyakushima N, Yokota S, Takahashi H, Fujii N, Suzuki H, Kodama T, Abe S, Kuroki Y. Pulmonary surfactant protein A augments the phagocytosis of Streptococcus pneumoniae by alveolar macrophages through a casein kinase 2-dependent increase of cell surface localization of scavenger receptor A. J Biol Chem 279: 21421–21430, 2004.[Abstract/Free Full Text]
  16. Lin PM, Wright JR. Surfactant protein A binds to IgG and enhances phagocytosis of IgG-opsonized erythrocytes. Am J Physiol Lung Cell Mol Physiol 291: L1199–L1206, 2006.[Abstract/Free Full Text]
  17. McIntosh J, Mervin-Blake S, Conner E, Wright J. Surfactant protein A protects growing cells and reduces TNF-{alpha} activity from LPS-stimulated macrophages. Am J Physiol Lung Cell Mol Physiol 271: L310–L319, 1996.[Abstract/Free Full Text]
  18. Nadesalingam J, Reid KB, Palaniyar N. Collectin surfactant protein D binds antibodies and interlinks innate and adaptive immune systems. FEBS Lett 579: 4449–4453, 2005.[CrossRef][Web of Science][Medline]
  19. Pricop L, Gokhale J, Redecha P, Ng S, Salmon J. Reactive oxygen intermediates enhance Fc-gamma receptor signaling and amplify phagocytis capacity. J Immunol 162: 7041–7048, 1999.[Abstract/Free Full Text]
  20. Sato M, Sano H, Iwaki D, Kudo K, Konishi M, Takahashi H, Takahashi T, Imaizumi H, Asai Y, Kuroki Y. Direct binding of Toll-like receptor 2 to zymosan, and zymosan-induced NF-kappa B activation and TNF-alpha secretion are down-regulated by lung collectin surfactant protein A. J Immunol 171: 417–425, 2003.[Abstract/Free Full Text]
  21. Savov J, Wright JR, Young SL. Incorporation of biotinylated SP-A into rat lung surfactant layer, type II cells, and Clara cells. Am J Physiol Lung Cell Mol Physiol 279: L118–L126, 2000.[Abstract/Free Full Text]
  22. Schagat T, Tino M, Wright J. Regulation of protein phosphorylation and pathogen phagocytosis by surfactant protein A. Infect Immun 67: 4693–4699, 1999.[Abstract/Free Full Text]
  23. Schagat T, Wofford J, Greene K, Wright J. Surfactant protein A differentially regulates peripheral and inflammatory neutrophil chemotaxis. Am J Physiol Lung Cell Mol Physiol 284: L140–L147, 2003.[Abstract/Free Full Text]
  24. Schagat T, Wofford J, Wright J. Surfactant protein A enhances alveolar macrophage phacocytosis of apoptotic neutrophils. J Immunol 166: 2727–2733, 2001.[Abstract/Free Full Text]
  25. Sherman MP, D'Ambola JB, Aeberhard EE, Barrett CT. Surfactant therapy of newborn rabbits impairs lung macrophage bactericidal activity. J Appl Physiol 65: 137–145, 1988.[Abstract/Free Full Text]
  26. Sibille Y, Marchandise FX. Pulmonary immune cells in health and disease: polymorphonuclear neutrophils. Eur Respir J 6: 1529–1543, 1993.[Abstract]
  27. Tenner A, Robinson S, Borchelt J, Wright J. Human pulmonary surfactant protein A (SP-A), a protein structurally homologous to C1q, can enhance FcR- and CR1-mediated phagocytosis. J Biol Chem 264: 13923–13928, 1989.[Abstract/Free Full Text]
  28. Tino M, Wright J. Surfactant protein A stimulates phagocytosis of specific pulmonary pathogens by alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 270: L677–L688, 1996.[Abstract/Free Full Text]
  29. Tino M, Wright J. Surfactant proteins A and D specifically stimulate directed actin-based responses in alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 276: L164–L174, 1999.[Abstract/Free Full Text]
  30. Volanakis J, Frank M. Human Complement System in Health and Disease. Dekker, 1998, p. 656.
  31. Wright J. Immunomodulatory functions of surfactant. Physiol Rev 77: 931–962, 1997.[Abstract/Free Full Text]
  32. Wright J, Youmans D. Pulmonary surfactant protein A stimulates chemotaxis of alveolar macrophage. Am J Physiol Lung Cell Mol Physiol 264: L338–L344, 1993.[Abstract/Free Full Text]
  33. Zhou M, Lublin D, Link D, Brown E. Distinct tyrosine kinase activation and Triton X-100 insolubility upon Fc-gamma RII or Fc-gamma RIIIB ligation in human polymorphonuclear leukocytes. J Biol Chem 270: 13553–13560, 1995.[Abstract/Free Full Text]



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