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Collectin surfactant protein D binds antibodies and interlinks innate and adaptive immune systems
FEBS 29800
FEBS Letters 579 (2005) 4449–4453
Collectin surfactant protein D binds antibodies and interlinks innate and adaptive immune systems Jeya Nadesalingama, Kenneth B.M. Reida, Nades Palaniyara,b,c,* b
a MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom Lung Biology Research Program, Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8 c Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Canada
Received 5 May 2005; revised 28 June 2005; accepted 11 July 2005 Available online 22 July 2005 Edited by Barry Halliwell
Abstract Innate immune collectins, such as surfactant protein D (SP-D), contain fibrillar collagen-like regions and globular carbohydrate-recognition domains (CRDs). SP-D recognizes carbohydrate arrays present on microbial surfaces via its CRDs, agglutinates microbes and enhances their phagocytosis. In contrast, adaptive immune proteins such as immunoglobulins (Igs) recognize pathogens via binding to specific antigens. Here we show that: SP-D binds various classes of immunoglobins, including IgG, IgM, IgE and secretory IgA, but not serum IgA; the globular domains of SP-D bind both the Fab and Fc domains of IgG; SP-D recognizes IgG via calcium-dependent protein– protein interactions, aggregates IgG-coated beads and enhances their phagocytosis by murine macrophage RAW 264.7 cells. Therefore, we propose that SP-D effectively interlinks innate and adaptive immune systems. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Collectins; Surfactant protein D; Innate immunity; Adaptive immunity; Antibody recognition; Immune system evolution
1. Introduction Although the adaptive and innate immune systems have been intensely studied for the past several decades, the proteins that interlink these two immune systems are not clearly understood. Collectins and antibodies are two major antigen-recognition soluble proteins of the innate and adaptive immune systems, respectively [1,2]. Innate immune collectins such as surfactant proteins (SPs-) A and D, and mannose-binding lectin (MBL) contain globular C-type lectin domains and collagen-like regions, and bind arrays of carbohydrate moieties present on the surface of several microbes [2]. Three carbohydrate recognition domains (CRDs) of collectins are held together by amphipathic a-helical coiled-coil domains, and also by triple helical collagen-like regions. These trimeric subunits further assemble into higher order macromolecules, and intertrimeric disulfide bonds at N-terminal segments stabilize the
* Corresponding author. Fax: +1 416 813 5002. E-mail address: [email protected] (N. Palaniyar).
Abbreviations: CRD, carbohydrate-recognition domain; ELISA, enzyme-linked immunosorbant assay; MBL, mannose-binding lectin; SP-, surfactant protein
overall oligomeric structure of these collectins [2–4]. Oligomeric structures of these proteins provides a high avidity interaction between the collectins and the microbial surfaces by making multiple low affinity interactions between individual CRDs of the protein and the carbohydrate moieties [2,4]. Since SP-D assembles as an X- or ‘‘fuzzy ball’’-like structure and orients its CRDs in opposite, or multiple, directions, this protein effectively agglutinates various microbes [5,6]. However, SP-A, MBL and a complement protein C1q (a non-collectin) each assemble as a ‘‘bouquet of flowers’’, and do not aggregate bacteria, as effectively as SP-D [2,4,7], but they all act as opsonins and enhance the clearance of various microorganisms by leukocytes [8]. The adaptive immune system generates several classes of antibodies such as IgG, IgM, IgA and IgE against foreign antigens, or pathogens [1]. The secreted form of IgA (sIgA) contains an additional highly glycosylated non-immunoglobulin protein called secretory component. The major classes of antibodies differ in their structure, tissue distribution and effector functions. However, all the antibodies recognize their targets via their Fab domains while allowing their Fc regions to interact with signalling molecules such as C1q or Fc receptors [9]. We hypothesized that innate immune collectin SP-D binds antibodies and interlinks innate and adaptive immune systems. Here, we describe a pathway where SP-D directly recognizes various types of antibodies, aggregates IgG coated beads and enhances effective phagocytosis. 2. Materials and methods 2.1. Biochemicals and buffers Unless otherwise stated all the reagents were purchased from Sigma– Aldrich. Human IgE, IgG, Fc and Fab were obtained from Biodesign. 2.2. Proteins Human SP-D was purified from the therapeutic lung washings from alveolar proteinosis patients as described previously [10,11]. Briefly, clarified bronchoalveolar lavage supernatant was calcified and mixed with maltose–agarose matrix, washed with 1 M NaCl in the binding buffer. SP-D was selectively eluted with 50 mM MnCl2, concentrated and further purified by Superose 6 column chromatography in an AKTA purifier (Amersham). SP-D(n/CRD) was expressed in Pechia pastoris and purified by SP-Shepherose, Maltose–agarose and Superose 6 column chromatographies as described previously [12]. 2.3. Enzyme-linked immunosorbant assay (ELISA) Different classes of immunoglobulins (1–5 lg/well) were immobilized on Maxisorp polystyrine ELISA plates (Nunc) in 0.1 M sodium bicarbonate buffer (pH 9.6) at 4 C for 18 h. The wells were washed three
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.07.012
4450 times with TSCT buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.05% (v/v) Tween 20) between each of the following steps, and all of the incubations were carried out at 37 C in TSCT. Free sites in the ELISA plates were blocked with 5% (w/v) BSA for 1 h, and incubated with SP-D (1 lg/ml) or SP-D(n/CRD) (50 lg/ml) for 2 h. In the experiments that analyzed the role of divalent cations, 5 mM CaCl2 was replaced with the same concentrations of MgCl2, MnCl2 or EDTA. In the experiments involving carbohydrate inhibition, indicated concentrations of the monosaccharides were present during SP-D (1 lg/ml) incubation step. We raised polyclonal antibody against recombinant SP-D(n/CRD) fragments in rabbits by a standard immunization protocol (Eurogentec,UK). The IgG fraction of the antiserum was purified using a Protein G column (Amersham), biotinylated (EZLink Sulfo-NHS-S-S-biotin, i.e., sulfosuccinimidyl 2-(biotinamido)ethyl-1, 3-dithiopropionate, Pierce) and used in the subsequent steps. Biotinylated rabbit-anti human SP-D (1 lg/ml) was incubated for 1– 2 h, followed by streptavidin-conjugted HRP (1:10,000 dilution; Sigma) for 20 min. A colour product was developed with TMB substrates as directed by the manufacturer (BioRad). The intensity of the colour was determined by a spectrophotometer at 450 nm. 2.4. Phagocytosis assay Streptavidin-containing Polystyrene Florescent beads (1 lm diameter; Molecular Probes) were incubated with biotinylated human IgG for 18 h at 23 C, free sites were blocked with 10 mM biotin for 2 h at 23 C, washed and resuspended in HANK balanced salt solution (ICN biochemicals). Blank beads or IgG-coated beads were mixed with 5 lg/ml BSA or SP-D for 10–15 min at 23 C and incubated with RAW 264.7 cells, at cell to beads ratio of 1:50 for 0, 15, 30 and 60 min at 37 C in 1.5 ml Eppendorf tubes as described previously [13]. The cells were washed with 1 ml PBS and fixed with 0.5% (v/v) formaldehyde, resuspended in PBS and deposited on slides by centrifugation at 500 · g for 1 min (Cytospin). Pre-fixation processing time of the procedure was about 5 min. The label distribution was analysed by confocal microscopy (Zeiss).
J. Nadesalingam et al. / FEBS Letters 579 (2005) 4449–4453
Fig. 1. SP-D binds to IgG, IgM, sIgA, IgE, but not IgA or BSA, in a calcium-dependent manner. (A) ELISA results showing the binding between SP-D (1 lg/ml) and different classes of Igs (5 lg) in the presence of CaCl2 (solid bars) or EDTA (open bars). (B) ELISA results showing the binding between SP-D (1 lg/ml) and IgG (5 lg) in the presence of 5 mM concentrations of CaCl2 (solid bars), MgCl2 (striped bars) MnCl2 (cross hatched bars) or EDTA (open bar).
2.5. Statistical analyses Mean and standard errors were calculated by Excel software. Analysis of variance (ANOVA), and DunnetÕs multiple mean comparisons (P < 0.05), was conducted using JMP Statistical Discovery Software (www.jmpdiscovery.com).
3. Results 3.1. SP-D binds to IgG, IgM, sIgA and IgE, but not to serum IgA and BSA To determine the interaction between collectins and immunoglobulins we used ELISA. Different classes of antibodies were immobilized on ELISA plates, and incubated with 1 lg/ ml of SP-D. SP-D that interacted with these immunoglobulins was detected by biotinylated SP-D-specific antibodies, streptavidin-conjugated HRP and chromogenic substrates. We found that SP-D bound well to different classes of antibodies including IgG, IgM, sIgA and IgE, compared with control wells containing BSA (P < 0.05; Fig. 1A). SP-D, however, did not bind to serum IgA or to BSA that was used as a non-specific blocking protein. These results indicate that SP-D specifically interacts with several, but not all, classes of antibodies. 3.2. SP-D binds immunoglobulins in a calcium ion-dependent manner SP-D is known to bind to many of its ligands in a calciumdependent manner [8,14,15]. Therefore, to determine whether calcium ions are necessary for SP-D immunoglobulin interactions, we immobilized a fixed amount of immunoglobulins, and incubated with SP-D in the presence of 5 mM CaCl2 or EDTA. The ELISA results showed that SP-D bound to
Fig. 2. Globular C-terminal domains of SP-D bind IgG via both Fc and Fab regions by protein–protein interactions. ELISA results showing the binding of (A) SP-D (1 lg/ml), or (B) SP-D(n/CRD) (50 lg/ml), with 2 lg of IgG, Fc and Fab, in the presence of CaCl2 (solid bars) or EDTA (open bars). (C) ELISA results showing the interaction between SP-D (1 lg/ml) and IgG (2 lg) in the presence of indicated concentrations of monosaccharides. ¤, maltose; h, mannose; m, D -fucose. (D) Diagrammatic representation of proposed SP-D-IgG interactions. Dodecameric SP-D (100 nm) and IgG molecules (10 nm) are drawn proportionately to show the relative dimensions of these two molecules.
J. Nadesalingam et al. / FEBS Letters 579 (2005) 4449–4453
immunoglobulins only in the presence of calcium ions, but not in EDTA (Fig. 1A), and neither magnesium nor manganese ions could substitute for calcium ions (Fig. 1B). Therefore, SP-D requires calcium ions for binding to immunoglobulins. 3.3. SP-D binds to Fab and Fc regions To determine the domains of immunoglobulins that interact with SP-D, we immobilized IgG, and Fc and Fab fragments of IgG, and incubated them with 1 lg/ml of SP-D. ELISA results showed that SP-D recognized both Fc and Fab fragments in a calcium-dependent manner (Fig. 2A). These results indicate that SP-D recognizes both Fc and Fab domains of IgG. 3.4. Globular domain of SP-D binds IgG To determine the domain of SP-D interacting with antibodies, we incubated 60-kDa trimeric recombinant fragments of SP-D that contain the ‘‘neck region’’ and the CRDs (SPD(n/CRD)). This recombinant protein also detected IgG, and Fc and Fab fragments of IgG. Interactions between SPD(n/CRD) and IgG fragments were also dependent on calcium ions (Fig. 2B). These results showed that the antibody-binding domain of SP-D is present in the globular C-terminal domain of the protein. 3.5. SP-D binds IgG via protein–protein interactions The globular domain of SP-D contains the CRDs, and is known to recognize carbohydrate ligands such as maltose
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and glucose, but not D -fucose, in a calcium-dependent manner [6,11,14,16]. Immunoglobulin molecules are glycoproteins, and contain both N- and O-linked carbohydrate moieties [17]. To determine whether SP-D binds IgG via CRD-carbohydrate interactions, we conducted competition experiments. SP-D bound to IgG in the presence of maltose, glucose or D fucose (Fig. 2C). The same monosaccharides inhibited the interactions between SP-D and sIgA, or IgM, to different degrees (data not shown). We also used surface plasmon resonance analysis to determine the ability of the SP-D to bind to carbohydrate ligands in the presence of increasing concentrations of IgG. Up to 2 mg/ml of IgG did not inhibit the interaction between SP-D (1 lg/ml) and immobilized mannan (data not shown). Therefore, we conclude that SP-D binds IgG via protein–protein interactions (Fig. 2D). 3.6. SP-D enhances the phagocytosis of IgG-coated beads To determine the biological relevance of SP-D-antibody interactions, we coated streptavidin containing-fluorescent beads with biotinylated IgG, and determined their phagocytosis by a murine macrophage cell line RAW 264.7 by confocal microscopy. Images showed that SP-D neither aggregated blank beads nor enhanced their uptake to any significant degree (data not shown). When IgG-coated beads were incubated with SP-D, but not BSA, the model immune complexes formed larger aggregates (Figs. 3a and d). Notably, macrophages avidly recognize these SP-D-induced IgG bead aggregates
Fig. 3. SP-D aggregates IgG-coated beads and enhances phagocytosis. Confocal microscopy images showing the effect of 5 lg/ml of BSA (a–c) or SP-D (d–f) for 0 (A,D), 15 (b,e) and 30 (c,f) min on the uptake of IgG-coated fluorescent beads (green) by RAW 264.7 cells. About 5 min pre-fixation processing time should be added to each of these time points.
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(Figs. 3d–f). Most of the IgG-coated beads were taken up by macrophages in a time-dependent manner (Fig. 3). Therefore, we infer that SP-D aggregates IgG-coated beads and enhances the phagocytosis of these model immune complexes.
4. Discussion We found that SP-D effectively interacts with several classes of antibody molecules, and these interactions involve the neck/CRD of SP-D, and Fc and Fab regions of IgG (Fig. 2D). SP-D binds to IgG coated model immune complexes, aggregates them and enhances their phagocytosis. Therefore, our results uncovered that the innate immune collectin SP-D directly binds immunoglobulins and links innate and adaptive immune pathways. SP-D is known to modulate multiple functions but the mechanisms by which it exerts these effects are unclear [2,8,15,18,19]. Binding of SP-D to various classes of antibodies may explain some of the previously unknown pathways mediated by this protein (Fig. 1). Different classes of antibodies are selectively localized in different tissues, and modulate different types of immune responses. Since SP-D interacts with various classes of antibodies, this collectin may exert a variety of immune-related functions. For example, IgG is the major antibody molecule present in the blood and opsonizes specific microbes and form immune complexes. Recent studies indicate that SP-D is present in the blood, and its concentration in the serum is highly heritable [20,21]. Therefore, SP-D-IgG interactions and subsequent enhancement of immune complex aggregation and phagocytosis provide an additional layer of immune response to amplify IgG-mediated responses (Figs. 2 and 3). The exact mechanism(s) and receptors that regulate SP-D-mediated uptake of immune complexes are unknown, and are likely to be influenced by the phagocytes, and local concentrations of the collectin and size of the immune complexes (Nadesalingam et al., unpublished data). For effective interlinking of innate and adaptive immune systems, SP-D should recognize the antibody molecules at the Fc regions, and perhaps parts of the Fab regions. This type of binding will allow Fab region of the IgG to recognize the antigens while clustering the Fc regions as arrays in a given orientation. This is the basis for the C1q binding to the immobilized IgGs and IgMs, and that globular regions of C1q make contact with parts of Fc and Fab regions [22]. Our results are consistent with similar modes of SP-D-IgG interactions (Fig. 2D). Notably, SPD-IgG interactions involve protein–protein interactions, and not protein–carbohydrate interactions. Therefore, this interaction is unique and parallels the C1q–IgG interactions. It is well known that collectins such as SP-A, SP-D and MBL directly bind microbial surface carbohydrate structures via their CRDs and enhance their phagocytosis [15,23]. Structural studies of C-terminal globular domains of collectins [14,24–26] show that CRDs of each trimers are spaced at about ˚ , and this spatial organization fits well with terminal car50 A bohydrate structures present in many microbial surfaces [27]. Therefore, collectins can bind to the microbial surface carbohydrate arrays with high avidity. However, whether collectins can bind to the native forms of carbohydrate moieties present on the antibody molecules is not clearly understood. Although native octadecameric SP-A does not interact with IgG [28], MBL can recognize aberrantly glycosylated forms of IgGs
J. Nadesalingam et al. / FEBS Letters 579 (2005) 4449–4453
[29], oligomeric sIgA and IgA isolated from nephropathy patients via lectin carbohydrate interactions [30]. We found that MBL can effectively recognize IgM isolated from healthy human sera (data not shown). Therefore, some of the other collectins may also interact with certain classes of antibodies. Since SP-D binds to several classes of antibody molecules and enhances the phagocytosis of IgG-coated molecules, we consider SP-D as an important innate immune molecule that uses both innate and adaptive immune pathways to effectively enhance the clearance of pathogens. Interestingly, collectins are evolutionarily more ancient molecules than antibodies [31]. Therefore, higher organisms have the advantage that collectins recognize both carbohydrate arrays on microbes and can also utilize antibody molecules, to effectively defend the host against microbial pathogens. In summary, our findings establish a novel pathway in which the innate immune collectin SP-D directly binds to antibodies and interlinks innate and adaptive immune pathways. Acknowledgements: We thank Jackie Shaw for maintaining RAW 264.7 cell cultures. P.N is a recipient of Wellcome Trust, UK and CIHR postdoctoral fellowships.
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FEBS Letters 579 (2005) 4449–4453
Collectin surfactant protein D binds antibodies and interlinks innate and adaptive immune systems Jeya Nadesalingama, Kenneth B.M. Reida, Nades Palaniyara,b,c,* b
a MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom Lung Biology Research Program, Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8 c Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Canada
Received 5 May 2005; revised 28 June 2005; accepted 11 July 2005 Available online 22 July 2005 Edited by Barry Halliwell
Abstract Innate immune collectins, such as surfactant protein D (SP-D), contain fibrillar collagen-like regions and globular carbohydrate-recognition domains (CRDs). SP-D recognizes carbohydrate arrays present on microbial surfaces via its CRDs, agglutinates microbes and enhances their phagocytosis. In contrast, adaptive immune proteins such as immunoglobulins (Igs) recognize pathogens via binding to specific antigens. Here we show that: SP-D binds various classes of immunoglobins, including IgG, IgM, IgE and secretory IgA, but not serum IgA; the globular domains of SP-D bind both the Fab and Fc domains of IgG; SP-D recognizes IgG via calcium-dependent protein– protein interactions, aggregates IgG-coated beads and enhances their phagocytosis by murine macrophage RAW 264.7 cells. Therefore, we propose that SP-D effectively interlinks innate and adaptive immune systems. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Collectins; Surfactant protein D; Innate immunity; Adaptive immunity; Antibody recognition; Immune system evolution
1. Introduction Although the adaptive and innate immune systems have been intensely studied for the past several decades, the proteins that interlink these two immune systems are not clearly understood. Collectins and antibodies are two major antigen-recognition soluble proteins of the innate and adaptive immune systems, respectively [1,2]. Innate immune collectins such as surfactant proteins (SPs-) A and D, and mannose-binding lectin (MBL) contain globular C-type lectin domains and collagen-like regions, and bind arrays of carbohydrate moieties present on the surface of several microbes [2]. Three carbohydrate recognition domains (CRDs) of collectins are held together by amphipathic a-helical coiled-coil domains, and also by triple helical collagen-like regions. These trimeric subunits further assemble into higher order macromolecules, and intertrimeric disulfide bonds at N-terminal segments stabilize the
* Corresponding author. Fax: +1 416 813 5002. E-mail address: [email protected] (N. Palaniyar).
Abbreviations: CRD, carbohydrate-recognition domain; ELISA, enzyme-linked immunosorbant assay; MBL, mannose-binding lectin; SP-, surfactant protein
overall oligomeric structure of these collectins [2–4]. Oligomeric structures of these proteins provides a high avidity interaction between the collectins and the microbial surfaces by making multiple low affinity interactions between individual CRDs of the protein and the carbohydrate moieties [2,4]. Since SP-D assembles as an X- or ‘‘fuzzy ball’’-like structure and orients its CRDs in opposite, or multiple, directions, this protein effectively agglutinates various microbes [5,6]. However, SP-A, MBL and a complement protein C1q (a non-collectin) each assemble as a ‘‘bouquet of flowers’’, and do not aggregate bacteria, as effectively as SP-D [2,4,7], but they all act as opsonins and enhance the clearance of various microorganisms by leukocytes [8]. The adaptive immune system generates several classes of antibodies such as IgG, IgM, IgA and IgE against foreign antigens, or pathogens [1]. The secreted form of IgA (sIgA) contains an additional highly glycosylated non-immunoglobulin protein called secretory component. The major classes of antibodies differ in their structure, tissue distribution and effector functions. However, all the antibodies recognize their targets via their Fab domains while allowing their Fc regions to interact with signalling molecules such as C1q or Fc receptors [9]. We hypothesized that innate immune collectin SP-D binds antibodies and interlinks innate and adaptive immune systems. Here, we describe a pathway where SP-D directly recognizes various types of antibodies, aggregates IgG coated beads and enhances effective phagocytosis. 2. Materials and methods 2.1. Biochemicals and buffers Unless otherwise stated all the reagents were purchased from Sigma– Aldrich. Human IgE, IgG, Fc and Fab were obtained from Biodesign. 2.2. Proteins Human SP-D was purified from the therapeutic lung washings from alveolar proteinosis patients as described previously [10,11]. Briefly, clarified bronchoalveolar lavage supernatant was calcified and mixed with maltose–agarose matrix, washed with 1 M NaCl in the binding buffer. SP-D was selectively eluted with 50 mM MnCl2, concentrated and further purified by Superose 6 column chromatography in an AKTA purifier (Amersham). SP-D(n/CRD) was expressed in Pechia pastoris and purified by SP-Shepherose, Maltose–agarose and Superose 6 column chromatographies as described previously [12]. 2.3. Enzyme-linked immunosorbant assay (ELISA) Different classes of immunoglobulins (1–5 lg/well) were immobilized on Maxisorp polystyrine ELISA plates (Nunc) in 0.1 M sodium bicarbonate buffer (pH 9.6) at 4 C for 18 h. The wells were washed three
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.07.012
4450 times with TSCT buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.05% (v/v) Tween 20) between each of the following steps, and all of the incubations were carried out at 37 C in TSCT. Free sites in the ELISA plates were blocked with 5% (w/v) BSA for 1 h, and incubated with SP-D (1 lg/ml) or SP-D(n/CRD) (50 lg/ml) for 2 h. In the experiments that analyzed the role of divalent cations, 5 mM CaCl2 was replaced with the same concentrations of MgCl2, MnCl2 or EDTA. In the experiments involving carbohydrate inhibition, indicated concentrations of the monosaccharides were present during SP-D (1 lg/ml) incubation step. We raised polyclonal antibody against recombinant SP-D(n/CRD) fragments in rabbits by a standard immunization protocol (Eurogentec,UK). The IgG fraction of the antiserum was purified using a Protein G column (Amersham), biotinylated (EZLink Sulfo-NHS-S-S-biotin, i.e., sulfosuccinimidyl 2-(biotinamido)ethyl-1, 3-dithiopropionate, Pierce) and used in the subsequent steps. Biotinylated rabbit-anti human SP-D (1 lg/ml) was incubated for 1– 2 h, followed by streptavidin-conjugted HRP (1:10,000 dilution; Sigma) for 20 min. A colour product was developed with TMB substrates as directed by the manufacturer (BioRad). The intensity of the colour was determined by a spectrophotometer at 450 nm. 2.4. Phagocytosis assay Streptavidin-containing Polystyrene Florescent beads (1 lm diameter; Molecular Probes) were incubated with biotinylated human IgG for 18 h at 23 C, free sites were blocked with 10 mM biotin for 2 h at 23 C, washed and resuspended in HANK balanced salt solution (ICN biochemicals). Blank beads or IgG-coated beads were mixed with 5 lg/ml BSA or SP-D for 10–15 min at 23 C and incubated with RAW 264.7 cells, at cell to beads ratio of 1:50 for 0, 15, 30 and 60 min at 37 C in 1.5 ml Eppendorf tubes as described previously [13]. The cells were washed with 1 ml PBS and fixed with 0.5% (v/v) formaldehyde, resuspended in PBS and deposited on slides by centrifugation at 500 · g for 1 min (Cytospin). Pre-fixation processing time of the procedure was about 5 min. The label distribution was analysed by confocal microscopy (Zeiss).
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Fig. 1. SP-D binds to IgG, IgM, sIgA, IgE, but not IgA or BSA, in a calcium-dependent manner. (A) ELISA results showing the binding between SP-D (1 lg/ml) and different classes of Igs (5 lg) in the presence of CaCl2 (solid bars) or EDTA (open bars). (B) ELISA results showing the binding between SP-D (1 lg/ml) and IgG (5 lg) in the presence of 5 mM concentrations of CaCl2 (solid bars), MgCl2 (striped bars) MnCl2 (cross hatched bars) or EDTA (open bar).
2.5. Statistical analyses Mean and standard errors were calculated by Excel software. Analysis of variance (ANOVA), and DunnetÕs multiple mean comparisons (P < 0.05), was conducted using JMP Statistical Discovery Software (www.jmpdiscovery.com).
3. Results 3.1. SP-D binds to IgG, IgM, sIgA and IgE, but not to serum IgA and BSA To determine the interaction between collectins and immunoglobulins we used ELISA. Different classes of antibodies were immobilized on ELISA plates, and incubated with 1 lg/ ml of SP-D. SP-D that interacted with these immunoglobulins was detected by biotinylated SP-D-specific antibodies, streptavidin-conjugated HRP and chromogenic substrates. We found that SP-D bound well to different classes of antibodies including IgG, IgM, sIgA and IgE, compared with control wells containing BSA (P < 0.05; Fig. 1A). SP-D, however, did not bind to serum IgA or to BSA that was used as a non-specific blocking protein. These results indicate that SP-D specifically interacts with several, but not all, classes of antibodies. 3.2. SP-D binds immunoglobulins in a calcium ion-dependent manner SP-D is known to bind to many of its ligands in a calciumdependent manner [8,14,15]. Therefore, to determine whether calcium ions are necessary for SP-D immunoglobulin interactions, we immobilized a fixed amount of immunoglobulins, and incubated with SP-D in the presence of 5 mM CaCl2 or EDTA. The ELISA results showed that SP-D bound to
Fig. 2. Globular C-terminal domains of SP-D bind IgG via both Fc and Fab regions by protein–protein interactions. ELISA results showing the binding of (A) SP-D (1 lg/ml), or (B) SP-D(n/CRD) (50 lg/ml), with 2 lg of IgG, Fc and Fab, in the presence of CaCl2 (solid bars) or EDTA (open bars). (C) ELISA results showing the interaction between SP-D (1 lg/ml) and IgG (2 lg) in the presence of indicated concentrations of monosaccharides. ¤, maltose; h, mannose; m, D -fucose. (D) Diagrammatic representation of proposed SP-D-IgG interactions. Dodecameric SP-D (100 nm) and IgG molecules (10 nm) are drawn proportionately to show the relative dimensions of these two molecules.
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immunoglobulins only in the presence of calcium ions, but not in EDTA (Fig. 1A), and neither magnesium nor manganese ions could substitute for calcium ions (Fig. 1B). Therefore, SP-D requires calcium ions for binding to immunoglobulins. 3.3. SP-D binds to Fab and Fc regions To determine the domains of immunoglobulins that interact with SP-D, we immobilized IgG, and Fc and Fab fragments of IgG, and incubated them with 1 lg/ml of SP-D. ELISA results showed that SP-D recognized both Fc and Fab fragments in a calcium-dependent manner (Fig. 2A). These results indicate that SP-D recognizes both Fc and Fab domains of IgG. 3.4. Globular domain of SP-D binds IgG To determine the domain of SP-D interacting with antibodies, we incubated 60-kDa trimeric recombinant fragments of SP-D that contain the ‘‘neck region’’ and the CRDs (SPD(n/CRD)). This recombinant protein also detected IgG, and Fc and Fab fragments of IgG. Interactions between SPD(n/CRD) and IgG fragments were also dependent on calcium ions (Fig. 2B). These results showed that the antibody-binding domain of SP-D is present in the globular C-terminal domain of the protein. 3.5. SP-D binds IgG via protein–protein interactions The globular domain of SP-D contains the CRDs, and is known to recognize carbohydrate ligands such as maltose
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and glucose, but not D -fucose, in a calcium-dependent manner [6,11,14,16]. Immunoglobulin molecules are glycoproteins, and contain both N- and O-linked carbohydrate moieties [17]. To determine whether SP-D binds IgG via CRD-carbohydrate interactions, we conducted competition experiments. SP-D bound to IgG in the presence of maltose, glucose or D fucose (Fig. 2C). The same monosaccharides inhibited the interactions between SP-D and sIgA, or IgM, to different degrees (data not shown). We also used surface plasmon resonance analysis to determine the ability of the SP-D to bind to carbohydrate ligands in the presence of increasing concentrations of IgG. Up to 2 mg/ml of IgG did not inhibit the interaction between SP-D (1 lg/ml) and immobilized mannan (data not shown). Therefore, we conclude that SP-D binds IgG via protein–protein interactions (Fig. 2D). 3.6. SP-D enhances the phagocytosis of IgG-coated beads To determine the biological relevance of SP-D-antibody interactions, we coated streptavidin containing-fluorescent beads with biotinylated IgG, and determined their phagocytosis by a murine macrophage cell line RAW 264.7 by confocal microscopy. Images showed that SP-D neither aggregated blank beads nor enhanced their uptake to any significant degree (data not shown). When IgG-coated beads were incubated with SP-D, but not BSA, the model immune complexes formed larger aggregates (Figs. 3a and d). Notably, macrophages avidly recognize these SP-D-induced IgG bead aggregates
Fig. 3. SP-D aggregates IgG-coated beads and enhances phagocytosis. Confocal microscopy images showing the effect of 5 lg/ml of BSA (a–c) or SP-D (d–f) for 0 (A,D), 15 (b,e) and 30 (c,f) min on the uptake of IgG-coated fluorescent beads (green) by RAW 264.7 cells. About 5 min pre-fixation processing time should be added to each of these time points.
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(Figs. 3d–f). Most of the IgG-coated beads were taken up by macrophages in a time-dependent manner (Fig. 3). Therefore, we infer that SP-D aggregates IgG-coated beads and enhances the phagocytosis of these model immune complexes.
4. Discussion We found that SP-D effectively interacts with several classes of antibody molecules, and these interactions involve the neck/CRD of SP-D, and Fc and Fab regions of IgG (Fig. 2D). SP-D binds to IgG coated model immune complexes, aggregates them and enhances their phagocytosis. Therefore, our results uncovered that the innate immune collectin SP-D directly binds immunoglobulins and links innate and adaptive immune pathways. SP-D is known to modulate multiple functions but the mechanisms by which it exerts these effects are unclear [2,8,15,18,19]. Binding of SP-D to various classes of antibodies may explain some of the previously unknown pathways mediated by this protein (Fig. 1). Different classes of antibodies are selectively localized in different tissues, and modulate different types of immune responses. Since SP-D interacts with various classes of antibodies, this collectin may exert a variety of immune-related functions. For example, IgG is the major antibody molecule present in the blood and opsonizes specific microbes and form immune complexes. Recent studies indicate that SP-D is present in the blood, and its concentration in the serum is highly heritable [20,21]. Therefore, SP-D-IgG interactions and subsequent enhancement of immune complex aggregation and phagocytosis provide an additional layer of immune response to amplify IgG-mediated responses (Figs. 2 and 3). The exact mechanism(s) and receptors that regulate SP-D-mediated uptake of immune complexes are unknown, and are likely to be influenced by the phagocytes, and local concentrations of the collectin and size of the immune complexes (Nadesalingam et al., unpublished data). For effective interlinking of innate and adaptive immune systems, SP-D should recognize the antibody molecules at the Fc regions, and perhaps parts of the Fab regions. This type of binding will allow Fab region of the IgG to recognize the antigens while clustering the Fc regions as arrays in a given orientation. This is the basis for the C1q binding to the immobilized IgGs and IgMs, and that globular regions of C1q make contact with parts of Fc and Fab regions [22]. Our results are consistent with similar modes of SP-D-IgG interactions (Fig. 2D). Notably, SPD-IgG interactions involve protein–protein interactions, and not protein–carbohydrate interactions. Therefore, this interaction is unique and parallels the C1q–IgG interactions. It is well known that collectins such as SP-A, SP-D and MBL directly bind microbial surface carbohydrate structures via their CRDs and enhance their phagocytosis [15,23]. Structural studies of C-terminal globular domains of collectins [14,24–26] show that CRDs of each trimers are spaced at about ˚ , and this spatial organization fits well with terminal car50 A bohydrate structures present in many microbial surfaces [27]. Therefore, collectins can bind to the microbial surface carbohydrate arrays with high avidity. However, whether collectins can bind to the native forms of carbohydrate moieties present on the antibody molecules is not clearly understood. Although native octadecameric SP-A does not interact with IgG [28], MBL can recognize aberrantly glycosylated forms of IgGs
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[29], oligomeric sIgA and IgA isolated from nephropathy patients via lectin carbohydrate interactions [30]. We found that MBL can effectively recognize IgM isolated from healthy human sera (data not shown). Therefore, some of the other collectins may also interact with certain classes of antibodies. Since SP-D binds to several classes of antibody molecules and enhances the phagocytosis of IgG-coated molecules, we consider SP-D as an important innate immune molecule that uses both innate and adaptive immune pathways to effectively enhance the clearance of pathogens. Interestingly, collectins are evolutionarily more ancient molecules than antibodies [31]. Therefore, higher organisms have the advantage that collectins recognize both carbohydrate arrays on microbes and can also utilize antibody molecules, to effectively defend the host against microbial pathogens. In summary, our findings establish a novel pathway in which the innate immune collectin SP-D directly binds to antibodies and interlinks innate and adaptive immune pathways. Acknowledgements: We thank Jackie Shaw for maintaining RAW 264.7 cell cultures. P.N is a recipient of Wellcome Trust, UK and CIHR postdoctoral fellowships.
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