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Department of Cell Biology, Duke University Medical Center, Durham, North Carolina
Submitted 31 May 2006 ; accepted in final form 21 June 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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collectin; immune complex; innate immunity; lung
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.
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MATERIALS AND METHODS |
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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 x 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 x 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 x 109 cells/ml) with the following equation:
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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 x 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 x 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 x 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.
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RESULTS |
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DISCUSSION |
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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.
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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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activity from LPS-stimulated macrophages. Am J Physiol Lung Cell Mol Physiol 271: L310–L319, 1996.This article has been cited by other articles:
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  P. Haberzettl, R. P. F. Schins, D. Hohr, V. Wilhelmi, P. J. A. Borm, and C. Albrecht Impact of the Fc{gamma}II-receptor on quartz uptake and inflammatory response by alveolar macrophages Am J Physiol Lung Cell Mol Physiol, June 1, 2008; 294(6): L1137 - L1148. [Abstract] [Full Text] [PDF]
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J. A. Wofford and J. R. Wright Surfactant protein A regulates IgG-mediated phagocytosis in inflammatory neutrophils Am J Physiol Lung Cell Mol Physiol, December 1, 2007; 293(6): L1437 - L1443. [Abstract] [Full Text] [PDF]
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) or human serum albumin (HSA, 
) was coated onto microtiter plate wells. Biotinylated SP-A was then added to the wells. After several washes, the levels of bound SP-A were determined with o-phenylenediamine dihydrochloride (OPD) tablets. The colorimetric assay was stopped with 4 N sulfuric acid, and the optical density of solution in the wells, corresponding to the amount of bound protein, was read in a microplate reader. The results are representative of 3 separate experiments. *P < 0.05 compared with HSA wells (Student's paired t-test).
of EDTA or HSA in the absence (
of EDTA was coated onto the wells of a microtiter plate. Biotinylated SP-A was incubated in the wells in the presence of calcium or EDTA, a calcium chelator. After several washes, the bound protein was measured as described in 
). Biotinylated SP-A and 100 mM mannan were also added to IgG-coated wells (
) or gelatin (
