Surfactant protein (SP)-A and SP-D, immunoglobulins, and complement all modulate inflammation within the lung by regulating pathogen clearance. For example, SP-A binds to and opsonizes a variety of bacteria and viruses, thereby enhancing their phagocytosis by innate immune cells such as alveolar macrophages. Immunoglobulins, which bind to antigen and facilitate Fc receptor-mediated phagocytosis, can also activate complement, a family of soluble proteins with multiple host defense functions. Previous studies showed that SP-A and complement protein C1q interact. Since complement protein C1q binds to IgG and IgM immune complexes, the hypothesis tested in this study was that SP-A, which is structurally homologous to C1q, also binds to IgG and affects its functions. SP-A binds to the Fc, rather than the Fab, region of IgG. Binding is calcium dependent but not inhibited by saccharides known to bind to SP-A's carbohydrate recognition domain. The binding of SP-A does not inhibit the formation of immune complexes or the binding of IgG to C1q. In contrast, SP-A enhances the uptake of IgG-coated erythrocytes, suggesting that SP-A might be influencing Fc receptor-mediated uptake. In summary, this study shows a novel interaction between SP-A and IgG and a functional consequence of the binding.
- immune complex
- innate immunity
among the major organs of the human body, the lung is one of the most vulnerable to infection. With each inhalation, the lung is exposed to pathogens, allergens, and pollutants. These foreign particles are inhaled and may become deposited in the alveolar epithelium lining of the lung, leading to infection and inflammation. To protect the lung from damage due to inflammation, pathogen clearance by immune cells is facilitated by a variety of innate immune defense mechanisms, including the surfactant protein (SP)-A and SP-D, immunoglobulins, and complement.
SP-A and SP-D, which contain both a collagen-like region and a carbohydrate recognition domain (CRD), are members of the collectin family of proteins. Collectins are known for their role in innate immunity. For example, SP-A, synthesized by type II alveolar epithelial cells (20) and by the Clara cell of the upper airway (1), binds to a variety of gram-positive and gram-negative bacteria, fungi, yeast, and viruses. This binding typically results in opsonization and enhanced clearance of the bound pathogens by macrophages or neutrophils. The binding of SP-A to viruses can also result in viral neutralization (2). The crucial role of SP-A in host defense has been confirmed in animal models. Compared with wild-type controls, SP-A-null mice have delayed microbial clearance of group B Streptococcus (8), Haemophilus influenzae (11), respiratory syncytial virus (9), and Pseudomonas aeruginosa (10) from the lungs. Therefore, SP-A plays a key role in the defense against infection within the lung.
Immunoglobulins, or antibodies, interact specifically, and with high avidity, with specific antigen. IgG binds to specific antigen and forms an immune complex (IC). Formation of IC can result in a clustering, or opsonization, of antigen by IgG, which in turn facilitates the uptake of the bound antigen by phagocytic cells. Under normal conditions, macrophages rapidly clear the IC. However, under certain conditions, IC may continue to circulate and eventually become trapped in tissue. Deposition of IC can cause pneumonitis and pulmonary lesions in the alveoli (3). Within the lung, and throughout the entire body, the most prominent immunoglobulin class is IgG. IgG is a major effector molecule that is secreted by specialized B cells, known as plasma cells, during an immune response. IgG is important for immune memory and the secondary antibody response; IgG IC can also activate complement.
The complement system is comprised of a group of serum proteins that in combination with antibody lead to antigen lysis and destruction. The IgG-dependent sequence of events leading to complement activation is called the classical pathway. The classical pathway is initiated when an IgG-bound antigen is recognized and bound by complement protein C1q. Complement proteins C1q, C3, and C4 were detected by Western blot in human bronchoalveolar lavage fluid (BALF) (18). Furthermore, in human BALF, there were detectable levels of classical pathway activity, albeit at reduced levels compared with serum (18).
Recent work by Watford et al. (19) showed that SP-A inhibits complement activation, suggesting one way in which these two innate immunity defense pathways cooperate within the same lung environment. A previous study by Hattori et al. (6) reported that IgG copurified with SP-A. Therefore, our goal was to investigate further the potential interactions between SP-A and IgG and to test the hypothesis that SP-A interacts with IgG and modulates its functions.
MATERIALS AND METHODS
Isolation of SP-A.
SP-A was purified from the BALF of alveolar proteinosis patients as previously described (13). Studies were approved by the Institutional Review Board of Duke University. Briefly, SP-A was extracted from BALF with butanol and solubilized in octylglucoside and Tris-buffered water (pH 7.4). SP-A was treated with polymyxin B agarose beads to reduce endotoxin contamination.
Biotinylation of SP-A and IgG.
SP-A and rabbit anti-BSA IgG (Sigma, St. Louis, MO) were dialyzed into 2 liters of HEPES buffer (pH 6.4). The dialysis buffer was changed twice over a period of 24 h. Sulfo-N-hydroxysuccinimide-biotin (Pierce, Rockford, IL) was added to the proteins at 20 times molar excess. The solution was incubated at room temperature for 30 min. Finally, SP-A was dialyzed against Tris-buffered water (pH 7.4), and anti-BSA IgG against Tris-buffered saline (TBS, pH 7.45), to remove unincorporated biotin.
SP-A binding to immunoglobulins.
IgA (human), IgG (human and rabbit), or IgM (human) (Sigma) was diluted to 1–5 μg/ml in 0.1 M sodium bicarbonate buffer (pH 9.7) and then coated onto wells of a 96-well plate overnight at 4°C. The following day, wells were washed with Tris buffer containing 150 mM NaCl, 2 mM CaCl2, and 0.1% Tween 20 (TTBS). Wells were blocked with 1% BSA (Sigma). After the plate was washed, biotinylated SP-A in concentrations ranging from 0 to 25 μg/ml was added to the wells for 1 h at 37°C. The levels of bound SP-A were measured by the following colorimetric assay. After extensive washing with TTBS, streptavidin-horseradish peroxidase (HRP) (Sigma) was added to the wells. The bound biotin-labeled SP-A was detected with Sigma Fast o-phenylenediamine dihydrochloride (OPD) tablets (Sigma). The colorimetric reaction was stopped with 4 N sulfuric acid, and the optical density (OD) of solution in the wells, corresponding to the level of bound protein, was read at 490 nm in a microtiter plate reader (Bio-Rad, Hercules, CA).
To determine whether binding of SP-A to IgG was calcium dependent, binding of biotinylated SP-A to IgG was evaluated in TTBS containing either 2 mM CaCl2 or 10 mM EDTA, a calcium chelator, before addition to wells coated with 10 μg/ml IgG or human serum albumin (HSA). Washes were done with TTBS. The binding was assessed with HRP-streptavidin and OPD tablets.
Studies were also undertaken to determine whether mannan, a sugar that binds the CRD of SP-A, inhibits SP-A binding to IgG. Wells were coated with 10 μg/ml human IgG or BSA. Biotinylated SP-A and unlabeled mannan (50 or 100 mM) were added to the wells. The amount of bound biotinylated protein was measured as described above.
IgG fragment binding studies.
The ImmunoPure Fab Preparation Kit (Pierce) was used to obtain the Fab and Fc fragments of IgG. In brief, 10 mg of lyophilized human IgG (Sigma) was incubated in a digestion buffer containing cysteine-HCl in a phosphate buffer. This mixture was then added to a slurry of immobilized papain (cross-linked 6% agarose beads) and placed in a 37°C shaker water bath incubator. After 5 h, the solubilized Fab and Fc fragments and undigested IgG were recovered with a separator, which was provided in the kit; the digest was applied to a protein A column. The Fab fragments, which do not bind protein A, were collected in the eluate; a low-pH glycine-HCl buffer (Pierce) was used to elute the bound Fc fragments (as well as undigested IgG). Finally, Fc fragments were separated from intact IgG with a Sepharose 6 column (GE Healthcare/Life Sciences, Piscataway, NJ).
Fab or Fc fragments were diluted in 0.1 M sodium bicarbonate buffer (pH 9.7) and coated overnight onto wells of a 96-well plate. Biotinylated SP-A was added to wells. Binding was assessed by colorimetric assay as described above.
To determine whether SP-A could competitively inhibit C1q from binding the Fc fragment, SP-A (0–20 μg/ml) was preincubated in Fc-coated wells for 1 h at 37°C to allow for binding. After the preincubation, wells were washed with TTBS to remove the unbound SP-A, and then 10 μg/ml purified human C1q (Complement Technology, Tyler, TX) was added to the wells. Alternatively, SP-A was incubated with C1q for 30 min at room temperature before addition of the mixture to wells. SP-A and C1q were detected with a mouse monoclonal SP-A antibody (Chemicon, Temecula, CA) and a goat polyclonal C1q antibody (Calbiochem San Diego, CA), respectively, followed by a goat anti-mouse HRP-conjugated secondary antibody (Bio-Rad) for SP-A or a rabbit anti-goat HRP-conjugated secondary antibody (Calbiochem) for C1q detection. The levels of bound protein were measured as described above.
IC binding studies.
To test whether SP-A could competitively inhibit antigen from binding to its specific antibody, a BSA and rabbit anti-BSA IgG IC was used. BSA (10 μg/ml) or 1% gelatin (control) was coated onto wells of a 96-well plate overnight at 4°C. The wells were washed extensively with TTBS and then blocked with 1% gelatin for 1 h at 37°C. Biotinylated anti-BSA IgG and SP-A (5 μg/ml) were incubated together in TTBS for 1 h at room temperature before addition to the wells. After 1 h, the levels of bound biotinylated IgG were determined as described above.
Since SP-A was able to bind to both anti-BSA IgG and anti-HSA IgG, HSA-anti-HSA IgG IC were also used in experiments. IC were coated onto wells as previously described (19) with the following changes. HSA was dissolved in PBS (GIBCO, Gaithersburg, MD) at 10 mg/ml and heat aggregated at 56°C for 30 min. The heat-aggregated HSA (HA-HSA) was diluted 200-fold in 0.1 M sodium bicarbonate buffer (pH 9.7) and then added to wells. After extensive washing with TTBS, rabbit anti-HSA IgG (Sigma) was added to wells at 20 μg/ml for 1 h at 37°C. Wells were blocked with 3% dry milk-TTBS for 1 h at 37°C and then washed three times with TTBS. Next, wells were incubated with SP-A for 1 h at 37°C, after which wells were washed. SP-A was detected with a monoclonal anti-SP-A antibody followed by secondary HRP-conjugated antibody, as described above.
Phagocytosis of IC.
IC were prepared with rabbit anti-BSA IgG that had been labeled with Alexa Fluor 488 fluorescent dye (Pierce). Precipitating BSA-anti-BSA IgG IC were prepared at threefold antigen excess. Antigen and antibodies were incubated for 1 h at 37°C and 1 h at 4°C. After this, IC were centrifuged at 10,000 g for 5 min, and the pellet was resuspended to a final concentration of 1 mg antibody/ml. Fifty microliters of IC was preincubated with 0, 5, 10, or 25 μg/ml SP-A for 30 min at room temperature with agitation, washed twice to remove unbound SP-A, and then resuspended in PBS containing 1 mM CaCl2.
Freshly isolated alveolar macrophages were obtained from pathogen-free male rats (Taconic Farms, Germantown, NY). Rats were anesthetized with an overdose of pentobarbital sodium. The chest cavity was opened, and the trachea was cannulated. The lungs were removed and lavaged repeatedly with a total of ∼100 ml of warmed lavage buffer (PBS containing 0.2 mM EGTA). The BALF was centrifuged at 228 g for 10 min to obtain alveolar macrophages. Macrophages were resuspended at 1 × 106 cells/ml in phenol-free minimum essential medium containing l-glutamine (GIBCO). Two hundred microliters of cells were added to the wells of 96-well clear-bottom black polystyrene plates (Corning Costar, Cambridge, MA) and allowed to adhere for 2 h at 37°C and 5% CO2.
Before the phagocytosis assay, cells were washed and then incubated with the IC preopsonized with SP-A for 1 h at 37°C. Afterwards, cells were washed three times to remove uningested IC. Ingestion of IC was measured by reading plates in a Fluoro-count fluorescent plate reader (Packard Instrument, Meriden, CT).
Standardization of sheep erythrocytes.
Sheep erythrocytes were prepared as follows. BSA was added to 1-wk-old sheep blood (Lampire Biological Laboratories, Pipersville, PA), to a final concentration of 0.5%, and then the blood was stored overnight at 4°C. The following day, sheep erythrocytes were isolated from the blood by spinning 10 ml of blood at 3,000 rpm for 5 min at 4°C and then aspirating off the supernatant and buffy coat layer. Sheep erythrocytes were resuspended in 20 ml of normal-ionic-strength (145 mM NaCl) veronal-buffered saline containing 1 mM MgCl2, 0.15 mM CaCl2, and 10 mM EDTA with gelatin (EDTA-GVBS++), collected by centrifugation, and washed once in EDTA-GVBS++. The suspension (in EDTA-GVBS++) was incubated in a 37°C water bath for 20 min with mixing. After being washed twice with 20 ml of GVBS++ (without EDTA) cells were standardized to 1 × 109 cells/ml by measuring the OD reading of a 1:50 dilution of cells at 541 nm and then adjusting the volume to obtain the desired OD reading of 0.210 (corresponding to 1 × 109 cells/ml) with the following equation: where E represents erythrocytes.
Sensitization of sheep erythrocytes with IgG.
IgG antibody-coated sheep erythrocytes (EA) were prepared by warming 10 ml of standardized erythrocytes to 30°C for 10 min. Separately, rabbit anti-sheep erythrocyte rabbit hemolysin was diluted 1:100 in 10 ml of GVBS++ and also warmed to 30°C for 10 min. The entire volume of antibody solution was added dropwise to the sheep erythrocytes with constant stirring. The cells were then incubated at 30°C for 15 min, with frequent mixing, to allow for sensitization with the antibody. After incubation, the sensitized sheep erythrocytes were spun at 3,000 rpm for 5 min at 4°C and washed once with GVBS++. Finally, cells were standardized to 5 × 108 cells/ml (an OD reading of 0.210 at 541 nm) by taking a 1:25 dilution and using the formula above. Antibody-sensitized sheep erythrocytes were stored in 60% low-ionic-strength (65 mM NaCl) veronal-buffered saline with dextrose and either 1 mM MgCl2 or 0.15 mM CaCl2 containing 5 mM sodium azide, at 4°C for up to two wk.
Phagocytosis of sensitized sheep erythrocytes.
Freshly isolated alveolar macrophages were obtained as described above. Macrophages were washed, collected by centrifugation. and resuspended at 1 × 106 cells/ml in RPMI 1640 (GIBCO) containing 0.1% BSA and 1 mM CaCl2 (phagocytosis buffer). One milliliter of the macrophage suspension was added to each chamber of a two-well chamber Permanox slide (Lab-Tek/Nunc, Rochester, NY). Cells were gently centrifuged onto slides for 3 min at the lowest setting (no brake), and then slides were incubated at 37°C with 5% CO2 for 2 h. Before the assay, cells were washed three times with buffer.
Erythrocytes and EA were washed and then diluted to a total of 1.6 × 108 cells in phagocytosis buffer and preincubated with 0, 5, or 25 μg/ml SP-A at room temperature for 30 min with agitation. Erythrocytes and EA were washed twice to remove unbound SP-A and then gently spun down onto adhered cells. Slides were incubated at 37°C with 5% CO2 for 2 h to allow for uptake to occur. After incubation, cells adhered to slides were washed twice with PBS and then stained with hematoxylin-eosin (EM Science, Gibbstown, NJ). Phagocytosis was assessed by light microscopy.
SP-A binds to IgG in a calcium-dependent manner.
The interaction between SP-A and IgG was investigated in vitro with a microtiter plate binding assay. SP-A bound to IgG-coated wells in a dose-dependent manner (Fig. 1). No further increase in binding was seen at higher concentrations of SP-A (15–25 μg/ml; results not shown). SP-A did not bind control wells (HSA) to a significant extent, nor did SP-A bind to wells containing buffer alone (results not shown). With SP-A concentrations similar to those used for the IgG binding experiments, we tested for, but did not observe, significant binding of SP-A to IgM or IgA (results not shown).
There was a reduction in the levels of SP-A bound to IgG-coated wells when binding studies were repeated in the presence of 10 mM EDTA, a calcium chelator, indicating that binding is at least partially calcium dependent (Fig. 2A). To determine whether the binding of SP-A to IgG involves the CRD of SP-A, we tested for the ability of mannan, which binds to the CRD, to inhibit the interaction of SP-A with IgG. As a control, it was shown that biotinylated SP-A bound to mannan-coated wells and that excess mannan in solution competed with immobilized mannan in binding SP-A. However, the binding of SP-A to IgG was not inhibited by the presence of either 50 or 100 mM mannan (Fig. 2B).
SP-A binds to the Fc fragment of IgG.
To determine whether SP-A binds specifically to either the Fc or Fab fragment of IgG, IgG was digested with papain to obtain the separate fragments. The amount of SP-A bound to wells coated with intact IgG and the Fc fragment was nearly two to three times the amount of SP-A bound to control (HSA) wells (Fig. 3). However, the binding of SP-A to Fab fragment-coated wells was not significant compared with control. Overall, the observed binding of SP-A to intact IgG was greater than the binding of SP-A to the Fc fragment.
SP-A does not inhibit binding of C1q to the Fc fragment of IgG.
In the microtiter plate assay utilized for these studies, maximal binding of C1q to Fc fragment-coated wells was seen at a 3-to-1 molar ratio of Fc fragment to C1q (results not shown). This ratio was used in the following experiments. Preincubation of SP-A in the Fc fragment-coated wells before addition of C1q did not affect binding of C1q to the Fc fragments (Fig. 4). In addition, preincubation of SP-A with C1q before the addition of both to the Fc-coated wells did not affect C1q binding to the Fc fragments. Likewise, SP-A did not inhibit C1q from binding to IC-coated wells (results not shown).
SP-A binds to IC.
To determine whether SP-A affects IC formation, BSA and anti-BSA IgG were used to generate IC in vitro. Preincubation of SP-A with labeled anti-BSA IgG did not inhibit or affect the ability of the antibody to bind to BSA-coated wells (Fig. 5A). SP-A alone did not bind to BSA-coated wells, nor did it bind to control wells coated with 1% gelatin. However, SP-A did bind to the preformed IC (HSA and anti-HSA IgG) themselves (Fig. 5B). SP-A did not bind to wells coated with HA-HSA alone or to control wells containing buffer alone (results not shown). These studies show that SP-A binds to preformed IC and does not prevent their formation.
SP-A does not affect the uptake of IgG IC by alveolar macrophages.
Despite having the ability to bind to BSA-anti-BSA IgG IC, SP-A, at concentrations of 5–25 μg/ml, did not have any affect on phagocytosis of IC by adherent macrophages (Fig. 6). As controls, adhered macrophages were shown to ingest an antibody-coated fluorescently labeled pathogen, Cryptococcus neoformans, which is a target known to be readily taken up by macrophages (unpublished results from our lab; for negative control, uncoated C. neoformans was used).
SP-A enhances the uptake of IgG-coated sheep red blood cells by alveolar macrophages.
Under normal conditions, alveolar macrophages do not ingest sheep erythrocytes. In contrast, sheep erythrocytes coated with an anti-sheep IgG (EA) are phagocytosed by macrophages. SP-A, at a concentration of 5 μg/ml, significantly enhanced the number of macrophages ingesting EA targets by 100% (Fig. 7, left). A higher dose of SP-A (25 μg/ml) resulted in a 250% increase in the uptake of EA. Similarly, at 5 and 25 μg/ml SP-A, the phagocytic index, defined by the number of EA ingested/cell, increased 55.7% and 76.3%, respectively (Fig. 7, right). The addition of SP-A to both erythrocytes and EA caused aggregates to form (results not shown). However, SP-A enhanced the uptake of only the EA, suggesting that SP-A induced aggregation alone is not sufficient to enhance uptake.
SP-A binds to the Fc region of IgG in a calcium-dependent manner that is not inhibited by mannan.
Before this study, Kuroki et al. (7) observed that IgG copurified with SP-A isolated from patients with pulmonary alveolar proteinosis or interstitial pulmonary fibrosis. Hattori et al. (6) later detected trace amounts of IgG associated with an abnormal multimerized form of SP-A isolated from alveolar proteinosis patients. Both studies suggested that IgG may associate with SP-A during a diseased state, but the studies did not determine whether SP-A copurified with IgG or bound to IgG. In addition, it was not clear whether this association was only observed with SP-A isolated from diseased lungs. Previous studies by Schagat et al. (16) showed that SP-A isolated from the BALF of alveolar proteinosis patients contained a very small amount (<1% by weight) of IgG.
Using SP-A isolated from patients with alveolar proteinosis, we consistently observed that SP-A binds to IgG in a microtiter plate assay. The binding was significantly higher than the levels of SP-A bound to control wells (HSA or BSA) or to wells containing buffer alone. The binding was dose dependent at concentrations ranging from 0.5 to 10 μg/ml and reached saturation at ∼10 μg/ml; significant binding was observed at 2 μg/ml SP-A. By our calculations, this suggests that maximal binding occurs at approximately a 1-to-4 molar ratio of SP-A (650 kDa) to IgG (150 kDa).
SP-A bound to IgG in a calcium-dependent manner, suggesting that binding may be mediated by the CRD of SP-A. However, binding was not inhibited by mannan, a sugar that also binds the CRD. This suggests that the residues involved in binding to mannan are not involved in the interactions between SP-A and IgG.
Nadesalingam et al. (14) recently reported that SP-D binds to IgG via protein-protein interactions. SP-D also bound to IgE, secretory IgA, and IgM, unlike SP-A, which bound only to IgG. Thus both SP-A and SP-D bind to IgG via a mechanism that is not blocked by carbohydrates and therefore most likely does not involve the CRD; instead, the binding appears to involve protein-protein interactions. Malhotra et al. (12) noted mannan-binding protein bound to an unusual glycosylated form of IgG, but not to the native form, which is consistent with the possibility that collectin interactions with aberrant forms of IgG may occur via the collectin CRD.
Binding of SP-A to IgG does not impede, and appears to be independent of, IgG interactions with C1q.
Since C1q binds to the CH2 domain within the Fc fragment of IgG (4, 5), experiments were conducted to determine whether SP-A inhibits C1q binding to IgG. In our studies, SP-A did not decrease C1q binding to wells coated with the Fc fragment or IC. In fact, both SP-A and C1q bound to IC-coated wells at the same time. There are several possible explanations for this observation: 1) SP-A and C1q are bound concurrently to different regions of IgG; 2) C1q is bound to SP-A, which, in turn, is bound to IgG; or 3) as seen previously by Watford et al. (19), SP-A can bind C1q and C1q binds to IgG.
We originally proposed that the interactions between SP-A and IgG would prevent C1q from binding to IC; this, in turn, could have an effect on the classical, or antibody-dependent pathway, leading to complement activation. Previous studies (12) confirmed that binding of a collectin, mannose-binding protein, to an aberrant glycosylated form of IgG can lead to complement activation via C1q-independent pathways. Although SP-A was previously shown to inhibit the classical pathway leading to complement activity (19), our results thus far suggest this inhibition is not due to the interaction between SP-A and IgG because, even in the presence of SP-A, C1q was still able to bind to IC-coated wells. Although SP-A bound to IC in a dose-dependent manner, the levels of C1q bound to the IC did not show a corresponding change.
Binding of SP-A to IgG does not inhibit IC formation.
SP-A binding to IgG does not inhibit the binding of IgG to antigen. As we observed, SP-A does not bind to the antigen-binding (Fab) region but instead binds to the Fc region of IgG. However, the level of binding of SP-A to the Fc fragment was less than the level of binding to intact IgG. This observation is consistent with the possibility that some property of intact IgG is required for optimal SP-A binding. IgG consists of two heavy and two light chains held together by disulfide bonds and noncovalent interactions. Papain digestion produces two identical halves of the Fab portion and the Fc portion, consisting of the constant region of two heavy chains plus the hinge region that separates the Fab and Fc portions. Papain digest might have disrupted interactions between the Fab and Fc regions that are needed to stabilize the binding to SP-A. However, the Fab fragment was unable to bind to SP-A, suggesting that the majority of binding interactions occur in the hinge, CH2, or CH3 regions of IgG.
SP-A enhances the uptake of antibody-sensitized sheep erythrocytes.
To test for a functional consequence of SP-A interactions with IgG, the effect of SP-A on phagocytosis of IgG-coated particles by alveolar macrophages was assessed. Two IgG-coated targets, BSA-IgG anti-BSA IC and IgG-coated sheep red blood cells, were tested. Whereas SP-A appeared to have no effect on the uptake of IC, SP-A did enhance the uptake of IgG-sensitized erythrocytes. The reasons explaining this discrepancy may be due to the inherent nature of each target. It is also possible that the detection methods were of varying degrees of sensitivity. Microscopy, rather than fluorescent signal intensity, may be a better way to measure changes in phagocytic activity of adherent macrophages.
Alveolar macrophages do not phagocytose sheep erythrocytes unless they are coated with IgG. Opsonization of the IgG-coated sheep erythrocytes with SP-A significantly enhanced both the number of macrophages internalizing the erythrocytes and the number of erythrocytes per cell, compared with the level of uptake in the absence of SP-A. The enhancement is most likely due to interactions between SP-A and IgG and not a consequence of aggregation caused by the addition of SP-A, since macrophages failed to phagocytose aggregates of non-antibody-opsonized erythrocytes. These findings are consistent with previous studies from our laboratory in which SP-A was shown to enhance the uptake of IgG-coated particles, albeit via completely different mechanisms. Schagat et al. (16) reported that IgG and SP-A added in solution had a synergistic effect in enhancing the uptake of S. pneumoniae. Under those conditions, uptake was dependent on tyrosine kinases, protein kinase C, and actin polymerization, but not microtubule activity. Recent studies showing that SP-D binds to various immunoglobulins, including IgG, aggregates IgG-coated beads, and enhances their phagocytosis (14) are also consistent with our findings.
In this study, a direct binding interaction between SP-A and IgG has been shown to correspond to an increase in phagocytosis of IgG-opsonized erythrocytes. However, the mechanism by which this enhancement occurs has yet to be fully elucidated. Tenner et al. (17) reported that macrophages and monocytes plated on SP-A- or C1q-coated surfaces exhibited an increase in uptake of IgG-coated erythrocytes, suggesting that SP-A acts as an activation ligand to enhance Fc receptor-mediated uptake. Together, these studies suggest that SP-A can enhance uptake of IgG-opsonized particles via a variety of mechanisms, in solution with IgG, as an activation ligand when coated on a surface, and as an opsonin as we have shown here.
The observed binding of SP-A to the Fc portion of IgG raises the interesting possibility of an interaction between SP-A and the Fc receptors on phagocytic cells. Although it would be ideal to identify the specific phagocytic receptors involved in SP-A binding, the differential expression of Fc receptors across species and the lack of available reagents for investigations of rat Fc receptors make these studies untenable at this time. Furthermore, studies with blocking antibodies could be confounded by the interaction of SP-A with the antibody itself.
In summary, SP-A binds to the Fc portion of IgG and enhances the uptake of IgG-bound sheep erythrocytes. However, SP-A did not impair the formation of IC or enhance the uptake of IC. Because SP-A binds to the Fc region of IgG, the possibility exists that SP-A could block interaction of IgG with the Fc receptor, or, alternatively, SP-A could enhance the uptake of IgG-coated particles, possibly via an SP-A receptor. Our results show that SP-A enhances the uptake of IgG-coated particles by alveolar macrophages.
The interactions between SP-A and IgG may play an important role in preventing inflammation within the lung. IgG-opsonized particles are taken up by macrophages via the Fc receptor under normal conditions. However, in adverse conditions, such as during an infection, macrophages may be overwhelmed by the high number of phagocytic targets; the constant signaling through Fc receptor-mediated pathways could instigate high levels of inflammation, which could damage the lung. By enhancing uptake of IgG-opsonized particles, SP-A helps speed the rate of clearance of these particles. In addition, SP-A could also physically prevent IgG-bound particle complexes from encountering the lung epithelium. Both pathways are worth exploring because they demonstrate new roles by which SP-A modulates and protects the lung from inflammation.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-51134.
The authors thank Kathy Evans for the preparation of surfactant protein A, Dr. Haixiang Jiang for help regarding the preparation of the antibody-sensitized erythrocytes, and Dr. Steven Giles for generous help with the preparation of this manuscript.
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.
- Copyright © 2006 the American Physiological Society