Collectin Surfactant Protein D Binds Antibodies and Interlinks Innate and Adaptive Immune Systems
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CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector 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,* a MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom b 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 overall oligomeric structure of these collectins [2–4]. Oligo- Abstract Innate immune collectins, such as surfactant protein D (SP-D), contain fibrillar collagen-like regions and globular meric structures of these proteins provides a high avidity inter- carbohydrate-recognition domains (CRDs). SP-D recognizes action between the collectins and the microbial surfaces by carbohydrate arrays present on microbial surfaces via its CRDs, making multiple low affinity interactions between individual agglutinates microbes and enhances their phagocytosis. In con- CRDs of the protein and the carbohydrate moieties [2,4]. Since trast, adaptive immune proteins such as immunoglobulins (Igs) SP-D assembles as an X- or ‘‘fuzzy ball’’-like structure and ori- recognize pathogens via binding to specific antigens. Here we ents its CRDs in opposite, or multiple, directions, this protein show that: SP-D binds various classes of immunoglobins, includ- effectively agglutinates various microbes [5,6]. ing IgG, IgM, IgE and secretory IgA, but not serum IgA; the However, SP-A, MBL and a complement protein C1q (a globular domains of SP-D bind both the Fab and Fc domains non-collectin) each assemble as a ‘‘bouquet of flowers’’, and of IgG; SP-D recognizes IgG via calcium-dependent protein– do not aggregate bacteria, as effectively as SP-D [2,4,7], but protein interactions, aggregates IgG-coated beads and enhances their phagocytosis by murine macrophage RAW 264.7 cells. they all act as opsonins and enhance the clearance of various Therefore, we propose that SP-D effectively interlinks innate microorganisms by leukocytes [8]. and adaptive immune systems. The adaptive immune system generates several classes of Ó 2005 Federation of European Biochemical Societies. Published antibodies such as IgG, IgM, IgA and IgE against foreign anti- by Elsevier B.V. All rights reserved. gens, or pathogens [1]. The secreted form of IgA (sIgA) con- tains an additional highly glycosylated non-immunoglobulin Keywords: Collectins; Surfactant protein D; Innate immunity; protein called secretory component. The major classes of anti- Adaptive immunity; Antibody recognition; Immune system bodies differ in their structure, tissue distribution and effector evolution functions. However, all the antibodies recognize their targets via their Fab domains while allowing their Fc regions to inter- act with signalling molecules such as C1q or Fc receptors [9]. We hypothesized that innate immune collectin SP-D binds 1. Introduction antibodies and interlinks innate and adaptive immune systems. Here, we describe a pathway where SP-D directly recognizes Although the adaptive and innate immune systems have various types of antibodies, aggregates IgG coated beads and been intensely studied for the past several decades, the proteins enhances effective phagocytosis. that interlink these two immune systems are not clearly under- stood. Collectins and antibodies are two major antigen-recog- nition soluble proteins of the innate and adaptive immune 2. Materials and methods systems, respectively [1,2]. Innate immune collectins such as 2.1. Biochemicals and buffers surfactant proteins (SPs-) A and D, and mannose-binding lec- Unless otherwise stated all the reagents were purchased from Sigma– tin (MBL) contain globular C-type lectin domains and colla- Aldrich. Human IgE, IgG, Fc and Fab were obtained from Biodesign. gen-like regions, and bind arrays of carbohydrate moieties present on the surface of several microbes [2]. Three carbohy- 2.2. Proteins drate recognition domains (CRDs) of collectins are held to- Human SP-D was purified from the therapeutic lung washings from gether by amphipathic a-helical coiled-coil domains, and also alveolar proteinosis patients as described previously [10,11]. Briefly, by triple helical collagen-like regions. These trimeric subunits clarified bronchoalveolar lavage supernatant was calcified and mixed with maltose–agarose matrix, washed with 1 M NaCl in the binding further assemble into higher order macromolecules, and inter- buffer. SP-D was selectively eluted with 50 mM MnCl2, concentrated trimeric disulfide bonds at N-terminal segments stabilize the 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 Supe- rose 6 column chromatographies as described previously [12]. *Corresponding author. Fax: +1 416 813 5002. E-mail address: [email protected] (N. Palaniyar). 2.3. Enzyme-linked immunosorbant assay (ELISA) Abbreviations: CRD, carbohydrate-recognition domain; ELISA, Different classes of immunoglobulins (1–5 lg/well) were immobilized enzyme-linked immunosorbant assay; MBL, mannose-binding lectin; on Maxisorp polystyrine ELISA plates (Nunc) in 0.1 M sodium bicar- SP-, surfactant protein bonate buffer (pH 9.6) at 4 °C for 18 h. The wells were washed three 0014-5793/$30.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.07.012 4450 J. Nadesalingam et al. / FEBS Letters 579 (2005) 4449–4453 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 incu- bated 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, indi- cated 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 immu- nization protocol (Eurogentec,UK). The IgG fraction of the antiserum was purified using a Protein G column (Amersham), biotinylated (EZ- Link 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; Sig- ma) for 20 min. A colour product was developed with TMB substrates as directed by the manufacturer (BioRad). The intensity of the colour Fig. 1. SP-D binds to IgG, IgM, sIgA, IgE, but not IgA or BSA, in a was determined by a spectrophotometer at 450 nm. 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 2.4. Phagocytosis assay showing the binding between SP-D (1 lg/ml) and IgG (5 lg) in the Streptavidin-containing Polystyrene Florescent beads (1 lm diame- presence of 5 mM concentrations of CaCl2 (solid bars), MgCl2 (striped ter; Molecular Probes) were incubated with biotinylated human IgG bars) MnCl2 (cross hatched bars) or EDTA (open bar). 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 proce- dure was about 5 min. The label distribution was analysed by confocal microscopy (Zeiss). 2.5. Statistical analyses Mean and standard errors were calculated by Excel software. Anal- ysis 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 immu- noglobulins 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, strepta- vidin-conjugated HRP and chromogenic substrates. We found that SP-D bound well to different classes of antibodies includ- ing IgG, IgM, sIgA and IgE, compared with control wells con- taining 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 block- ing protein. These results indicate that SP-D specifically inter- acts with several, but not all, classes of antibodies. Fig. 2. Globular C-terminal domains of SP-D bind IgG via both Fc 3.2. SP-D binds immunoglobulins in a calcium ion-dependent 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) manner (50 lg/ml), with 2 lg of IgG, Fc and Fab, in the presence of CaCl2 SP-D is known to bind to many of its ligands in a calcium- (solid bars) or EDTA (open bars).