Collectin Surfactant Protein D Binds Antibodies and Interlinks Innate and Adaptive Immune Systems

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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 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) contains 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 interact 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 understood. Collectins and antibodies are two major antigen-recognition 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 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 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 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 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 includ- ing 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 block- ing protein. These results indicate that SP-D specifically interacts 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). (C) ELISA results showing the dependent manner [8,14,15]. Therefore, to determine whether interaction between SP-D (1 lg/ml) and IgG (2 lg) in the presence of calcium ions are necessary for SP-D immunoglobulin interac- indicated concentrations of monosaccharides. ¤, maltose; h,mannose; m, DD-fucose. (D) Diagrammatic representation of proposed SP-D-IgG tions, we immobilized a fixed amount of immunoglobulins, interactions. Dodecameric SP-D (100 nm) and IgG molecules and incubated with SP-D in the presence of 5 mM CaCl2 or (10 nm) are drawn proportionately to show the relative dimensions EDTA. The ELISA results showed that SP-D bound to of these two molecules. J. Nadesalingam et al. / FEBS Letters 579 (2005) 4449–4453 4451 immunoglobulins only in the presence of calcium ions, but not and glucose, but not DD-fucose, in a calcium-dependent manner in EDTA (Fig. 1A), and neither magnesium nor manganese [6,11,14,16]. Immunoglobulin molecules are glycoproteins, and ions could substitute for calcium ions (Fig. 1B). Therefore, contain both N- and O-linked carbohydrate moieties [17].To SP-D requires calcium ions for binding to immunoglobulins. determine whether SP-D binds IgG via CRD-carbohydrate interactions, we conducted competition experiments. 3.3. SP-D binds to Fab and Fc regions SP-D bound to IgG in the presence of maltose, glucose or DD- To determine the domains of immunoglobulins that interact fucose (Fig. 2C). The same monosaccharides inhibited the with SP-D, we immobilized IgG, and Fc and Fab fragments of interactions between SP-D and sIgA, or IgM, to different de- IgG, and incubated them with 1 lg/ml of SP-D. ELISA results grees (data not shown). We also used surface plasmon reso- showed that SP-D recognized both Fc and Fab fragments in a nance analysis to determine the ability of the SP-D to bind calcium-dependent manner (Fig. 2A). These results indicate to carbohydrate ligands in the presence of increasing concen- that SP-D recognizes both Fc and Fab domains of IgG. trations of IgG. Up to 2 mg/ml of IgG did not inhibit the interaction between SP-D (1 lg/ml) and immobilized mannan (data 3.4. Globular domain of SP-D binds IgG not shown). Therefore, we conclude that SP-D binds IgG via To determine the domain of SP-D interacting with antibod- protein–protein interactions (Fig. 2D). ies, we incubated 60-kDa trimeric recombinant fragments of SP-D that contain the ‘‘neck region’’ and the CRDs (SP- 3.6. SP-D enhances the phagocytosis of IgG-coated beads D(n/CRD)). This recombinant protein also detected IgG, To determine the biological relevance of SP-D-antibody and Fc and Fab fragments of IgG. Interactions between SP- interactions, we coated streptavidin containing-fluorescent D(n/CRD) and IgG fragments were also dependent on calcium beads with biotinylated IgG, and determined their phagocyto- ions (Fig. 2B). These results showed that the antibody-binding sis by a murine macrophage cell line RAW 264.7 by confocal domain of SP-D is present in the globular C-terminal domain microscopy. Images showed that SP-D neither aggregated of the protein. blank beads nor enhanced their uptake to any significant de- gree (data not shown). When IgG-coated beads were incubated 3.5. SP-D binds IgG via protein–protein interactions with SP-D, but not BSA, the model immune complexes formed The globular domain of SP-D contains the CRDs, and is larger aggregates (Figs. 3a and d). Notably, macrophages known to recognize carbohydrate ligands such as maltose 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. 4452 J. Nadesalingam et al. / FEBS Letters 579 (2005) 4449–4453

(Figs. 3d–f). Most of the IgG-coated beads were taken up by [29], oligomeric sIgA and IgA isolated from nephropathy pa- macrophages in a time-dependent manner (Fig. 3). Therefore, tients via lectin carbohydrate interactions [30]. We found that we infer that SP-D aggregates IgG-coated beads and enhances MBL can effectively recognize IgM isolated from healthy hu- the phagocytosis of these model immune complexes. man 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 4. Discussion and enhances the phagocytosis of IgG-coated molecules, we consider SP-D as an important innate immune molecule that We found that SP-D effectively interacts with several classes uses both innate and adaptive immune pathways to effectively of antibody molecules, and these interactions involve the enhance the clearance of pathogens. neck/CRD of SP-D, and Fc and Fab regions of IgG Interestingly, collectins are evolutionarily more ancient mol- (Fig. 2D). SP-D binds to IgG coated model immune com- ecules than antibodies [31]. Therefore, higher organisms have plexes, aggregates them and enhances their phagocytosis. the advantage that collectins recognize both carbohydrate ar- Therefore, our results uncovered that the innate immune col- rays on microbes and can also utilize antibody molecules, to lectin SP-D directly binds immunoglobulins and links innate effectively defend the host against microbial pathogens. In and adaptive immune pathways. summary, our findings establish a novel pathway in which SP-D is known to modulate multiple functions but the mech- the innate immune collectin SP-D directly binds to antibodies anisms by which it exerts these effects are unclear and interlinks innate and adaptive immune pathways. [2,8,15,18,19]. Binding of SP-D to various classes of antibodies may explain some of the previously unknown pathways medi- Acknowledgements: We thank Jackie Shaw for maintaining RAW ated by this protein (Fig. 1). Different classes of antibodies are 264.7 cell cultures. P.N is a recipient of Wellcome Trust, UK and CIHR postdoctoral fellowships. 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 im- References mune-related functions. For example, IgG is the major antibody molecule present in the blood and opsonizes specific [1] Reid, K.B., Colomb, M., Petry, F. and Loos, M. (2002) microbes and form immune complexes. Recent studies indicate Complement component C1 and the collectins – first-line defense that SP-D is present in the blood, and its concentration in the molecules in innate and acquired immunity. Trends Immunol. 23, 115–117. serum is highly heritable [20,21]. Therefore, SP-D-IgG interac- [2] Palaniyar, N., Nadesalingam, J. and Reid, K.B. (2002) tions and subsequent enhancement of immune complex aggre- Pulmonary innate immune proteins and receptors that interact gation and phagocytosis provide an additional layer of with gram-positive bacterial ligands. Immunobiology 205, 575– immune response to amplify IgG-mediated responses (Figs. 2 594. [3] Palaniyar, N., Zhang, L., Kuzmenko, A., Ikegami, M., Wan, S., and 3). The exact mechanism(s) and receptors that regulate Wu, H., Korfhagen, T.R., Whitsett, J.A. and McCormack, F.X. SP-D-mediated uptake of immune complexes are unknown, (2002) The role of pulmonary collectin N-terminal domains in and are likely to be influenced by the phagocytes, and local surfactant structure, function, and homeostasis in vivo. J. Biol. concentrations of the collectin and size of the immune com- Chem. 277, 26971–26979. plexes (Nadesalingam et al., unpublished data). [4] Palaniyar, N., Ikegami, M., Korfhagen, T., Whitsett, J. and McCormack, F.X. (2001) Domains of surfactant protein A that For effective interlinking of innate and adaptive immune sys- affect protein oligomerization, lipid structure and surface tension. tems, SP-D should recognize the antibody molecules at the Fc Comp. Biochem. Physiol. A Mol. Integr. Physiol. 129, 109– regions, and perhaps parts of the Fab regions. This type of bind- 127. ing will allow Fab region of the IgG to recognize the antigens [5] Lu, J., Wiedemann, H., Timpl, R. and Reid, K.B. (1993) Similarity in structure between C1q and the collectins as judged while clustering the Fc regions as arrays in a given orientation. by electron microscopy. Behring Inst. Mitt., 6–16. This is the basis for the C1q binding to the immobilized IgGs [6] Crouch, E. and Wright, J.R. (2001) Surfactant proteins A and D and IgMs, and that globular regions of C1q make contact with and pulmonary host defense. Annu. Rev. Physiol. 63, 521– parts of Fc and Fab regions [22]. Our results are consistent with 554. similar modes of SP-D-IgG interactions (Fig. 2D). Notably, SP- [7] Palaniyar, N., Ridsdale, R.A., Holterman, C.E., Inchley, K., Possmayer, F. and Harauz, G. (1998) Structural changes of D-IgG interactions involve protein–protein interactions, and surfactant protein A induced by cations reorient the protein on not protein–carbohydrate interactions. Therefore, this interac- lipid bilayers. J. Struct. Biol. 122, 297–310. tion is unique and parallels the C1q–IgG interactions. [8] Wright, J.R. (2005) Immunoregulatory functions of surfactant It is well known that collectins such as SP-A, SP-D and proteins. Nat. Rev. Immunol. 5, 58–68. [9] Carroll, M.C. (2004) The complement system in regulation of MBL directly bind microbial surface carbohydrate structures adaptive immunity. Nat. Immunol. 5, 981–986. via their CRDs and enhance their phagocytosis [15,23]. Struc- [10] Nadesalingam, J., Bernal, A.L., Dodds, A.W., Willis, A.C., tural studies of C-terminal globular domains of collectins Mahoney, D.J., Day, A.J., Reid, K.B. and Palaniyar, N. (2003) [14,24–26] show that CRDs of each trimers are spaced at about Identification and characterization of a novel interaction between 50 A˚ , and this spatial organization fits well with terminal car- pulmonary surfactant protein D and decorin. J. Biol. Chem. 278, 25678–25687. bohydrate structures present in many microbial surfaces [27]. [11] Palaniyar, N., Nadesalingam, J., Clark, H., Shih, M.J., Dodds, Therefore, collectins can bind to the microbial surface carbo- A.W. and Reid, K.B. (2004) Nucleic acid is a novel ligand for hydrate arrays with high avidity. However, whether collectins innate, immune pattern recognition collectins surfactant proteins can bind to the native forms of carbohydrate moieties present A and D and mannose-binding lectin. J. Biol. Chem. 279, 32728– 32736. on the antibody molecules is not clearly understood. Although [12] Nadesalingam, J., Dodds, A.W., Reid, K.B. and Palaniyar, N. native octadecameric SP-A does not interact with IgG [28], (2005) Mannose-binding lectin recognizes peptidoglycan via the MBL can recognize aberrantly glycosylated forms of IgGs N-acetyl glucosamine moiety, and inhibits its proinflammatory J. Nadesalingam et al. / FEBS Letters 579 (2005) 4449–4453 4453

effect and promotes chemokine production by macrophages. J. [22] Gaboriaud, C., Thielens, N.M., Gregory, L.A., Rossi, V., Immunol. 175, 1785–1794. Fontecilla-Camps, J.C. and Arlaud, G.J. (2004) Structure and [13] Palaniyar, N., Clark, H., Nadesalingam, J., Shih, M.J., Hawgood, activation of the C1 complex of complement: unravelling the S. and Reid, K.B. (2005) Innate immune collectin surfactant puzzle. Trends Immunol. 25, 368–373. protein D enhances the clearance of DNA by macrophages and [23] Gardai, S.J., Xiao, Y.Q., Dickinson, M., Nick, J.A., Voelker, minimizes anti-DNA antibody generation. J. Immunol. 174, D.R., Greene, K.E. and Henson, P.M. (2003) By binding 7352–7358. SIRPalpha or calreticulin/CD91, lung collectins act as dual [14] Shrive, A.K., Tharia, H.A., Strong, P., Kishore, U., Burns, I., function surveillance molecules to suppress or enhance inflam- Rizkallah, P.J., Reid, K.B. and Greenhough, T.J. (2003) High- mation. Cell 115, 13–23. resolution structural insights into ligand binding and immune cell [24] Hakansson, K., Lim, N.K., Hoppe, H.J. and Reid, K.B. (1999) recognition by human lung surfactant protein D. J. Mol. Biol. Crystal structure of the trimeric alpha-helical coiled-coil and the 331, 509–523. three lectin domains of human lung surfactant protein D. Struct. [15] Holmskov, U., Thiel, S. and Jensenius, J.C. (2003) Collections Fold Des. 7, 255–264. and ficolins: humoral lectins of the innate immune defense. Annu. [25] Head, J.F., Mealy, T.R., McCormack, F.X. and Seaton, B.A. Rev. Immunol. 21, 547–578. (2003) Crystal structure of trimeric carbohydrate recognition and [16] Lu, J., Teh, C., Kishore, U. and Reid, K.B. (2002) Collectins and neck domains of surfactant protein A. J. Biol. Chem. 278, 43254– ficolins: sugar pattern recognition molecules of the mammalian 43260. innate immune system. Biochim. Biophys. Acta 1572, 387–400. [26] Palaniyar, N., McCormack, F.X., Possmayer, F. and Harauz, G. [17] Rudd, P.M., Elliott, T., Cresswell, P., Wilson, I.A. and Dwek, (2000) Threedimensional structure of rat surfactant protein A R.A. (2001) Glycosylation and the immune system. Science 291, trimers in association with phospholipid monolayers. Biochemis- 2370–2376. try 39, 6310–6316. [18] Hickling, T.P., Clark, H., Malhotra, R. and Sim, R.B. (2004) [27] Drickamer, K. (1999) C-type lectin-like domains. Curr. Opin. Collectins and their role in lung immunity. J. Leukoc. Biol. 75, Struct. Biol. 9, 585–590. 27–33. [28] Hattori, A., Kuroki, Y., Takahashi, H., Sohma, H. and Akino, T. [19] McCormack, F.X. and Whitsett, J.A. (2002) The pulmonary (1997) Immunoglobulin G is associated with surfactant protein A collectins, SP-A and SP-D, orchestrate innate immunity in the aggregates isolated from patients with pulmonary alveolar pro- lung. J. Clin. Invest. 109, 707–712. teinosis. Am. J. Respir. Crit. Care Med. 155, 1785–1788. [20] Husby, S., Herskind, A.M., Jensenius, J.C. and Holmskov, U. [29] Malhotra, R., Wormald, M.R., Rudd, P.M., Fischer, P.B., Dwek, (2002) Heritability estimates for the constitutional levels of the R.A. and Sim, R.B. (1995) Glycosylation changes of IgG collectins mannan-binding lectin and lung surfactant protein D. A associated with rheumatoid arthritis can activate complement study of unselected like-sexed mono- and dizygotic twins at the via the mannose-binding protein. Nat. Med. 1, 237–243. age of 6–9 years. Immunology 106, 389–394. [30] Roos, A., Bouwman, L.H., van Gijlswijk-Janssen, D.J., Faber- [21] Leth-Larsen, R., Garred, P., Jensenius, H., Meschi, J., Hartshorn, Krol, M.C., Stahl, G.L. and Daha, M.R. (2001) Human IgA K., Madsen, J., Tornoe, I., Madsen, H.O., Sorensen, G., Crouch, activates the complement system via the mannan-binding lectin E. and Holmskov, U. (2005) A common polymorphism in the pathway. J. Immunol. 167, 2861–2868. SFTPD gene influences assembly, function, and concentration of [31] Fujita, T. (2002) Evolution of the lectin-complement pathway and surfactant protein D. J. Immunol. 174, 1532–1538. its role in innate immunity. Nat. Rev. Immunol. 2, 346–353.