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• W2017479299 abstract "The intricate system of serum complement proteins provides resistance to infection. A pivotal step in the complement pathway is the assembly of a C3 convertase, which digests the C3 complement component to form microbial binding C3 fragments recognized by leukocytes. The spleen and C3 provide resistance against blood-borne S. pneumoniae infection. To better understand the mechanisms involved, we studied SIGN-R1, a lectin that captures microbial polysaccharides in spleen. Surprisingly, conditional SIGN-R1 knockout mice developed deficits in C3 catabolism when given S. pneumoniae or its capsular polysaccharide intravenously. There were marked reductions in proteolysis of serum C3, deposition of C3 on organisms within SIGN-R1+ spleen macrophages, and formation of C3 ligands. We found that SIGN-R1 directly bound the complement C1 subcomponent, C1q, and assembled a C3 convertase, but without the traditional requirement for either antibody or factor B. The transmembrane lectin SIGN-R1 therefore contributes to innate resistance by an unusual C3 activation pathway. The intricate system of serum complement proteins provides resistance to infection. A pivotal step in the complement pathway is the assembly of a C3 convertase, which digests the C3 complement component to form microbial binding C3 fragments recognized by leukocytes. The spleen and C3 provide resistance against blood-borne S. pneumoniae infection. To better understand the mechanisms involved, we studied SIGN-R1, a lectin that captures microbial polysaccharides in spleen. Surprisingly, conditional SIGN-R1 knockout mice developed deficits in C3 catabolism when given S. pneumoniae or its capsular polysaccharide intravenously. There were marked reductions in proteolysis of serum C3, deposition of C3 on organisms within SIGN-R1+ spleen macrophages, and formation of C3 ligands. We found that SIGN-R1 directly bound the complement C1 subcomponent, C1q, and assembled a C3 convertase, but without the traditional requirement for either antibody or factor B. The transmembrane lectin SIGN-R1 therefore contributes to innate resistance by an unusual C3 activation pathway. The intricate but elegant system of complement proteins is responsible for several innate and adaptive resistance mechanisms. A pivotal step is the formation of a C3 convertase, which catalyzes the proteolysis of complement component, C3. The C3 convertase is formed by (1) a “classical” pathway initiated by the binding of C1q to immunoglobulin (Ig) in immune complexes, (2) an “alternative” pathway triggered directly by certain microbial cell walls and catalyzed by factor B, and (3) a soluble mannose binding lectin pathway with many homologies to the classical C1q based pathway (reviewed in Fearon and Wong, 1983Fearon D.T. Wong W.W. Complement ligand-receptor interactions that mediate biological responses.Annu. Rev. Immunol. 1983; 1: 243-271Crossref PubMed Scopus (190) Google Scholar, Walport, 2001aWalport M.J. Complement.N. Engl. J. Med. 2001; 344: 1140-1144Crossref PubMed Scopus (1170) Google Scholar, Walport, 2001bWalport M.J. Complement.N. Engl. J. Med. 2001; 344: 1058-1066Crossref PubMed Scopus (2216) Google Scholar, Fujita, 2002Fujita T. Evolution of the lectin-complement pathway and its role in innate immunity.Nat. Rev. Immunol. 2002; 2: 346-353Crossref PubMed Scopus (531) Google Scholar). The C3 convertase first generates C3b that is further processed to C3bi and C3d. C3b, C3bi, and C3d serve as ligands or “opsonins” for cellular receptors on B cells, phagocytes, and follicular dendritic cells (FDCs). Receptor-mediated binding of these C3 opsonins leads to microbial clearance by phagocytes (Fearon and Wong, 1983Fearon D.T. Wong W.W. Complement ligand-receptor interactions that mediate biological responses.Annu. Rev. Immunol. 1983; 1: 243-271Crossref PubMed Scopus (190) Google Scholar), stimulation of B cells (Carroll, 1998Carroll M.C. The role of complement and complement receptors in induction and regulation of immunity.Annu. Rev. Immunol. 1998; 16: 545-568Crossref PubMed Scopus (475) Google Scholar), and retention of antigens on FDCs (Pozdnyakova et al., 2003Pozdnyakova O. Guttormsen H.K. Lalani F.N. Carroll M.C. Kasper D.L. Impaired antibody response to group B streptococcal type III capsular polysaccharide in C3- and complement receptor 2-deficient mice.J. Immunol. 2003; 170: 84-90PubMed Google Scholar). C3 processing also releases the C3a peptide, which is an anaphylatoxin that causes smooth muscle contraction, mast cell histamine release, and local inflammation. Additionally, a lytic membrane attack complex of other complement components C5b, 6, 7, 8 is assembled on cellular targets. Therefore the complement system is a major pathway for resistance and pathology, and the formation of a C3 convertase is pivotal. The spleen clears microorganisms from the blood stream, with Streptococcus pneumoniae representing a major example (Amlot and Hayes, 1985Amlot P.L. Hayes A.E. Impaired human antibody response to the thymus-independent antigen DNP-Ficoll, after splenectomy: implications for post-splenectomy infections.Lancet. 1985; 1: 1008-1011Abstract PubMed Scopus (89) Google Scholar, Zandvoort and Timens, 2002Zandvoort A. Timens W. The dual function of the splenic marginal zone: essential for initiation of anti-TI-2 responses but also vital in the general first-line defense against blood-borne antigens.Clin. Exp. Immunol. 2002; 130: 4-11Crossref PubMed Scopus (161) Google Scholar). Individuals lacking a spleen are more susceptible to infection with S. pneumoniae (Schutze et al., 2002Schutze G.E. Mason Jr., E.O. Barson W.J. Kim K.S. Wald E.R. Givner L.B. Tan T.Q. Bradley J.S. Yogev R. Kaplan S.L. Invasive pneumococcal infections in children with asplenia.Pediatr. Infect. Dis. J. 2002; 21: 278-282Crossref PubMed Scopus (75) Google Scholar). Despite the development of effective treatments, this gram-positive coccus has remained a significant cause of morbidity and mortality (Garau, 2002Garau J. Treatment of drug-resistant pneumococcal pneumonia.Lancet Infect. Dis. 2002; 2: 404-415Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), being one of the most common causes of bacterial pneumonia, otitis media, meningitis, and septicemia (Musher, 1992Musher D.M. Infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity, and treatment.Clin. Infect. Dis. 1992; 14: 801-807Crossref PubMed Scopus (444) Google Scholar). Opsonin-dependent phagocytosis via complement plays an important role in host defense against pneumococci (Brown et al., 1982Brown E.J. Hosea S.W. Hammer C.H. Burch C.G. Frank M.M. A quantitative analysis of the interactions of antipneumococcal antibody and complement in experimental pneumococcal bacteremia.J. Clin. Invest. 1982; 69: 85-98Crossref PubMed Scopus (82) Google Scholar, Brown et al., 1983Brown E.J. Hosea S.W. Frank M.M. The role of antibody and complement in the reticuloendothelial clearance of pneumococci from the bloodstream.Rev. Infect. Dis. 1983; 5: S797-S805Crossref PubMed Google Scholar). Patients with deficiencies of early components of the classical pathway of complement and C3 are at high risk for pneumococcal disease (Picard et al., 2003Picard C. Puel A. Bustamante J. Ku C.L. Casanova J.L. Primary immunodeficiencies associated with pneumococcal disease.Curr. Opin. Allergy Clin. Immunol. 2003; 3: 451-459Crossref PubMed Scopus (129) Google Scholar). In contrast, the soluble mannose binding lectin binds poorly to S. pneumoniae (Neth et al., 2000Neth O. Jack D.L. Dodds A.W. Holzel H. Klein N.J. Turner M.W. Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition.Infect. Immun. 2000; 68: 688-693Crossref PubMed Scopus (449) Google Scholar, Krarup et al., 2005Krarup A. Sorensen U.B. Matsushita M. Jensenius J.C. Thiel S. Effect of capsulation of opportunistic pathogenic bacteria on binding of the pattern recognition molecules mannan-binding lectin, L-ficolin, and H-ficolin.Infect. Immun. 2005; 73: 1052-1060Crossref PubMed Scopus (155) Google Scholar), and lower lectin levels are only weakly associated with S. pneumoniae infections (Kronborg et al., 2002Kronborg G. Weis N. Madsen H.O. Pedersen S.S. Wejse C. Nielsen H. Skinhoj P. Garred P. Variant mannose-binding lectin alleles are not associated with susceptibility to or outcome of invasive pneumococcal infection in randomly included patients.J. Infect. Dis. 2002; 185: 1517-1520Crossref PubMed Scopus (97) Google Scholar, Atkinson et al., 2004Atkinson A.P. Cedzynski M. Szemraj J. St Swierzko A. Bak-Romaniszyn L. Banasik M. Zeman K. Matsushita M. Turner M.L. Kilpatrick D.C. L-ficolin in children with recurrent respiratory infections.Clin. Exp. Immunol. 2004; 138: 517-520Crossref PubMed Scopus (59) Google Scholar). Knockout mice have revealed that C3 and C4 are dominantly required for innate resistance to S. pneumoniae, but surprisingly, there is only a small or partial need for the traditional mediators of C3 fixation like factor B, mannose binding lectin, and immunoglobulin (Ig) (Brown et al., 2002Brown J.S. Hussell T. Gilliland S.M. Holden D.W. Paton J.C. Ehrenstein M.R. Walport M.J. Botto M. The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice.Proc. Natl. Acad. Sci. USA. 2002; 99: 16969-16974Crossref PubMed Scopus (276) Google Scholar, Kars et al., 2005Kars M. van Dijk H. Salimans M.M. Bartelink A.K. van de Wiel A. Association of furunculosis and familial deficiency of mannose-binding lectin.Eur. J. Clin. Invest. 2005; 35: 531-534Crossref PubMed Scopus (15) Google Scholar). Together, these findings indicate that there is likely to be an unusual complement fixation pathway that is independent of factor B, Ig, and mannose binding lectin but that nevertheless forms C3 opsonins for resistance to pneumococci by phagocytes and B cells. The spleen contains specialized regions called marginal zones (MZ), which lie at the junction of each white pulp nodule with the red pulp (Mebius and Kraal, 2005Mebius R.E. Kraal G. Structure and function of the spleen.Nat. Rev. Immunol. 2005; 5: 606-616Crossref PubMed Scopus (1267) Google Scholar). The MZ contains unique MZ B cells (Martin and Kearney, 2002Martin F. Kearney J.F. Marginal-zone B cells.Nat. Rev. Immunol. 2002; 2: 323-335Crossref PubMed Scopus (651) Google Scholar) enmeshed with MZ macrophages in a reticular cell network (Kraal and Janse, 1986Kraal G. Janse M. Marginal metallophilic cells of the mouse spleen identified by a monoclonal antibody.Immunology. 1986; 58: 665-669PubMed Google Scholar). In the MZ, there is a strongly reduced blood flow allowing intimate contact between antigens and effector cells (Guinamard et al., 2000Guinamard R. Okigaki M. Schlessinger J. Ravetch J.V. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response.Nat. Immunol. 2000; 1: 31-36Crossref PubMed Scopus (426) Google Scholar). The MZ thus has several functions, including the generation of antigen-specific B cell responses to T cell-independent antigens (Humphrey, 1985Humphrey J.H. Splenic macrophages: antigen presenting cells for T1–2 antigens.Immunol. Lett. 1985; 11: 149-152Crossref PubMed Scopus (36) Google Scholar) and the capture of blood-borne pathogens (Humphrey and Grennan, 1981Humphrey J.H. Grennan D. Different macrophage populations distinguished by means of fluorescent polysaccharides. Recognition and properties of marginal-zone macrophages.Eur. J. Immunol. 1981; 11: 221-228Crossref PubMed Scopus (165) Google Scholar, Kang et al., 2004Kang Y.S. Kim J.Y. Bruening S.A. Pack M. Charalambous A. Pritsker A. Moran T.M. Loeffler J.M. Steinman R.M. Park C.G. The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen.Proc. Natl. Acad. Sci. USA. 2004; 101: 215-220Crossref PubMed Scopus (148) Google Scholar) such as S. pneumoniae (Lanoue et al., 2004Lanoue A. Clatworthy M.R. Smith P. Green S. Townsend M.J. Jolin H.E. Smith K.G. Fallon P.G. McKenzie A.N. SIGN-R1 contributes to protection against lethal pneumococcal infection in mice.J. Exp. Med. 2004; 200: 1383-1393Crossref PubMed Scopus (118) Google Scholar). SIGN-R1, a C-type lectin related to DC-SIGN (Park et al., 2001Park C.G. Takahara K. Umemoto E. Yashima Y. Matsubara K. Matsuda Y. Clausen B.E. Inaba K. Steinman R.M. Five mouse homologues of the human dendritic cell C-type lectin, DC-SIGN.Int. Immunol. 2001; 13: 1283-1290Crossref PubMed Scopus (162) Google Scholar), is an uptake receptor expressed at high levels by spleen MZ and lymph node macrophages (Geijtenbeek et al., 2002Geijtenbeek T.B.H. Groot P.C. Nolte M.A. van Vliet S.J. Gangaram-Panday S.T. van Duijnhoven G.C.F. Kraal G. van Oosterhout A.J.M. van Kooyk Y. Marginal zone macrophages express a murine homologue of DC-SIGN that captures blood-born antigens in vivo.Blood. 2002; 100: 2908-2916Crossref PubMed Scopus (148) Google Scholar, Kang et al., 2003Kang Y.-S. Yamazaki S. Iyoda T. Pack M. Bruening S. Kim J.Y. Takahara K. Inaba K. Steinman R.M. Park C.G. SIGN-R1, a novel C-type lectin expressed by marginal zone macrophages in spleen, mediates uptake of the polysaccharide dextran.Int. Immunol. 2003; 15: 177-186Crossref PubMed Scopus (135) Google Scholar). SIGN-R1 is the principal receptor for bacterial dextrans as well as the capsular pneumococcal polysaccharide (CPS) of S. pneumoniae, so that deletion of SIGN-R1 ablates clearance of these polysaccharides by MZ macrophages (Geijtenbeek et al., 2002Geijtenbeek T.B.H. Groot P.C. Nolte M.A. van Vliet S.J. Gangaram-Panday S.T. van Duijnhoven G.C.F. Kraal G. van Oosterhout A.J.M. van Kooyk Y. Marginal zone macrophages express a murine homologue of DC-SIGN that captures blood-born antigens in vivo.Blood. 2002; 100: 2908-2916Crossref PubMed Scopus (148) Google Scholar, Kang et al., 2003Kang Y.-S. Yamazaki S. Iyoda T. Pack M. Bruening S. Kim J.Y. Takahara K. Inaba K. Steinman R.M. Park C.G. SIGN-R1, a novel C-type lectin expressed by marginal zone macrophages in spleen, mediates uptake of the polysaccharide dextran.Int. Immunol. 2003; 15: 177-186Crossref PubMed Scopus (135) Google Scholar, Kang et al., 2004Kang Y.S. Kim J.Y. Bruening S.A. Pack M. Charalambous A. Pritsker A. Moran T.M. Loeffler J.M. Steinman R.M. Park C.G. The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen.Proc. Natl. Acad. Sci. USA. 2004; 101: 215-220Crossref PubMed Scopus (148) Google Scholar, Lanoue et al., 2004Lanoue A. Clatworthy M.R. Smith P. Green S. Townsend M.J. Jolin H.E. Smith K.G. Fallon P.G. McKenzie A.N. SIGN-R1 contributes to protection against lethal pneumococcal infection in mice.J. Exp. Med. 2004; 200: 1383-1393Crossref PubMed Scopus (118) Google Scholar). Our approach to conditional deletion of SIGN-R1 involves administration of the monoclonal antibody 22D1. Treatment with this mAb selectively ablates SIGN-R1 expression, but not MZ macrophages, as monitored with a combination of antibodies to a distinct epitope to SIGN-R1 and other MZ receptors (Kang et al., 2004Kang Y.S. Kim J.Y. Bruening S.A. Pack M. Charalambous A. Pritsker A. Moran T.M. Loeffler J.M. Steinman R.M. Park C.G. The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen.Proc. Natl. Acad. Sci. USA. 2004; 101: 215-220Crossref PubMed Scopus (148) Google Scholar). Furthermore, Lanoue et al. used genetic deletion of SIGN-R1 to demonstrate the contribution of this lectin to protection of mice against pneumococcal septicemia (Lanoue et al., 2004Lanoue A. Clatworthy M.R. Smith P. Green S. Townsend M.J. Jolin H.E. Smith K.G. Fallon P.G. McKenzie A.N. SIGN-R1 contributes to protection against lethal pneumococcal infection in mice.J. Exp. Med. 2004; 200: 1383-1393Crossref PubMed Scopus (118) Google Scholar). In our continuing studies of 22D1 anti-SIGN-R1-treated mice, we found surprisingly that, in addition to a lack of CPS and dextran uptake in SIGN-R1+ MZ macrophages, the polysaccharides were no longer deposited on SIGN-R1− spleen FDCs. This suggested a link between SIGN-R1 and the formation of C3 opsonins for FDCs. We will show that this membrane bound lectin initiates a classical but Ig-independent pathway for C3 fixation by binding C1q and depositing C3 on bound polysaccharides and S. pneumoniae. We suggest that this new pathway for C3 fixation by SIGN-R1 bound polysaccharides contributes to the role of the spleen in resistance to certain encapsulated organisms. To study the previously defined interaction of capsular pneumococcal polysaccharide (CPS) with the SIGN-R1 lectin on marginal zone (MZ) macrophages of spleen (Kang et al., 2004Kang Y.S. Kim J.Y. Bruening S.A. Pack M. Charalambous A. Pritsker A. Moran T.M. Loeffler J.M. Steinman R.M. Park C.G. The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen.Proc. Natl. Acad. Sci. USA. 2004; 101: 215-220Crossref PubMed Scopus (148) Google Scholar), we injected mice with 100 μg of CPS14 i.v. and localized it with anti-CPS14 antibody in sections of spleen 10 min to 1 hr later. Within minutes, the CPS localized to SIGN-R1+ MZ macrophages, which were identified with anti-SIGN-R1 antibodies (Kang et al., 2003Kang Y.-S. Yamazaki S. Iyoda T. Pack M. Bruening S. Kim J.Y. Takahara K. Inaba K. Steinman R.M. Park C.G. SIGN-R1, a novel C-type lectin expressed by marginal zone macrophages in spleen, mediates uptake of the polysaccharide dextran.Int. Immunol. 2003; 15: 177-186Crossref PubMed Scopus (135) Google Scholar, Kang et al., 2004Kang Y.S. Kim J.Y. Bruening S.A. Pack M. Charalambous A. Pritsker A. Moran T.M. Loeffler J.M. Steinman R.M. Park C.G. The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen.Proc. Natl. Acad. Sci. USA. 2004; 101: 215-220Crossref PubMed Scopus (148) Google Scholar). However, by 1 hr the CPS was also found in the B cell areas or follicles of the white pulp (Figure 1A, white arrows). The follicle-associated CPS colocalized to follicular dendritic cells (FDCs), which are known to express CD21/CD35 complement receptors but not SIGN-R1 (Figure 1B, yellow staining). In SIGN-R1-transient knockout mice generated by mAb 22D1 treatment (Kang et al., 2004Kang Y.S. Kim J.Y. Bruening S.A. Pack M. Charalambous A. Pritsker A. Moran T.M. Loeffler J.M. Steinman R.M. Park C.G. The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen.Proc. Natl. Acad. Sci. USA. 2004; 101: 215-220Crossref PubMed Scopus (148) Google Scholar), CPS14 was not taken up by MZ macrophages as expected, but in addition the CPS did not localize to follicles (Figure 1A) or FDCs (Figure 1B). This surprising finding suggested that SIGN-R1 on MZ macrophages is needed for the deposition of CPS14 on SIGN-R1− FDCs. Since prior results (Harms et al., 1996Harms G. Hardonk M.J. Timens W. In vitro complement-dependent binding and in vivo kinetics of pneumococcal polysaccharide TI-2 antigens in the rat spleen marginal zone and follicle.Infect. Immun. 1996; 64: 4220-4225Crossref PubMed Google Scholar) showed that CPS binding to the MZ and FDCs was dependent on complement, probably a C3 fragment, we compared C3−/− and wild-type mice. We found that CPS14 was taken up by SIGN-R1+ MZ macrophages in wild-type and C3−/− mice (Figure 1C). However, CPS14 did not bind to FDCs in C3−/− mice (Figure 1C). Thus SIGN-R1 and C3 are somehow both needed for CPS binding to FDCs, suggesting that this lectin might contribute to C3 fixation. To pursue the suggestion that SIGN-R1 was required for the formation of C3 ligands, we injected CPS or mitomycin-C treated (growth inactivated) S. pneumoniae into control and 22D1-treated mice (controls included mice given hamster Ig, N418 anti-CD11c, and anti-Marco, a scavenger receptor on MZ macrophages). Over the ensuing hour, we monitored C3 processing in blood by immunoblotting serum samples with polyclonal anti-C3 antibody. This antibody reacts with the two components of native C3, αC3, and βC3 as well as the fragments of αC3 that are generated during C3 processing by C3 convertases (Figure 2A shows information for human C3) (Law and Dodds, 1997Law S.K. Dodds A.W. The internal thioester and the covalent binding properties of the complement proteins C3 and C4.Protein Sci. 1997; 6: 263-274Crossref PubMed Scopus (275) Google Scholar). In the steady state, C3 convertase is active so that two C3 fragments (iC3b; 70 kDa and 43 kDa) are always evident in serum. However, when pneumococci or CPS were given i.v., there was rapid processing of αC3 (but not βC3, which serves as a loading control for the immunoblotting), such that the serum sample lost most of the detectable αC3 as well as the 70 kDa iC3b but accumulated the 43 kDa iC3b (see the two left lanes of Figures 2B and 2C). These data in vivo are consistent with previous results in vitro (Campbell et al., 1991Campbell J.R. Baker C.J. Edwards M.S. Deposition and degradation of C3 on type III group B streptococci.Infect. Immun. 1991; 59: 1978-1983Crossref PubMed Google Scholar). Pneumococci, but not several other bacteria that we tested, rapidly activated C3 in vivo (Figure S1). The processing of C3 was more active with CPS from type 14 S. pneumoniae than CPS23 and CPS26 (data not shown), and it is known that CPS14 binds with higher affinity to SIGN-R1 relative to CPS23 and CPS26 (Kang et al., 2004Kang Y.S. Kim J.Y. Bruening S.A. Pack M. Charalambous A. Pritsker A. Moran T.M. Loeffler J.M. Steinman R.M. Park C.G. The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen.Proc. Natl. Acad. Sci. USA. 2004; 101: 215-220Crossref PubMed Scopus (148) Google Scholar). Importantly, the activation of C3 in response to bacteria (Figure 2B) or CPS (Figure 2C) was blocked in 22D1-treated mice, indicating that SIGN-R1 is in large part responsible for rapid C3 convertase formation induced by S. pneumoniae in mice. These results were unexpected because in vitro, C3 can be activated via the alternative pathway when S. pneumoniae (but not CPS) is added to serum (Winkelstein and Tomasz, 1977Winkelstein J.A. Tomasz A. Activation of the alternative pathway by pneumococcal cell walls.J. Immunol. 1977; 118: 451-454PubMed Google Scholar), as we confirmed (Figure 2D, left; data for CPS not shown). We also verified that C3 processing in vitro in response to bacteria occurred similarly when we compared serum from wild-type mice or from mice treated with hamster Ig or 22D1 anti-SIGN-R1 mAb 24 hr earlier (Figure 2D). In other words, 22D1 treatment was not consuming C3 and not preventing direct fixation of C3 from serum by S. pneumoniae. However, the kinetics of C3 processing was much more rapid when S. pneumoniae was administered in vivo, being detectable within 5 min (Figure 2E), as opposed to adding organisms to serum in vitro, where 30 min were required for significant C3 catabolism (Figure 2D). Thus CPS and S. pneumoniae are both captured within minutes from the blood by SIGN-R1+ MZ macrophages, and the latter dominate the early processing of C3 in vivo. We next wanted to verify a role for SIGN-R1 during C3 fixation by S. pneumoniae directly in spleen sections in vivo. We injected i.v. 108 CFU (colony-forming units), mitomycin C-treated type 14 (Pn14) organisms that were also labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). 90 min later, to allow for uptake by MZ macrophages and deposition of CPS on FDCs, the spleens were sectioned and examined by deconvolution microscopy. The Pn14 were taken up in the MZ in control and C3 deficient (C3−/−) mice, but there were diminished numbers of organisms in the MZ of mice treated beforehand with anti-SIGN-R1 mAb 22D1, possibly because there were residual SIGN-R1 molecules and/or receptors for the organisms in addition to SIGN-R1 (Figure 3A). C3 was deposited on the organisms in control mice in the MZ when mice were given pneumococci but not in C3−/− mice, as expected (Figure 3A, yellow labeling). In 22D1 treated mice, when we selected regions of the MZ where there was some uptake of CFSE-labeled S. pneumoniae, C3 was minimally fixed to the individual organisms (Figure 3A). C3 likewise was clearly deposited on CD21/35+ FDCs in control mice given the CFSE labeled organisms, very little in the 22D1 treated animals, and not at all in C3−/− mice (Figure 3B). Likewise, when we injected either CPS (or dextran, data not shown) i.v. in control mice, the polysaccharides were taken up in MZ macrophages and C3 was deposited there, but again not in 22D1 treated animals (Supplemental Figure S2). Therefore when pneumococci or polysaccharides are injected i.v., SIGN-R1 dominates the fixation of C3 to MZ macrophages and subsequently, FDCs. Because we were using treatment with mAb 22D1 to selectively deplete SIGN-R1, we needed to verify that this approach was relevant to the resistance of mice to infection with S. pneumoniae, as described by Lanoue et al., who showed that genetic deletion of SIGN-R1 decreased innate resistance (Lanoue et al., 2004Lanoue A. Clatworthy M.R. Smith P. Green S. Townsend M.J. Jolin H.E. Smith K.G. Fallon P.G. McKenzie A.N. SIGN-R1 contributes to protection against lethal pneumococcal infection in mice.J. Exp. Med. 2004; 200: 1383-1393Crossref PubMed Scopus (118) Google Scholar). First, we repeated the experiments of J.S. Brown et al. (Brown et al., 2002Brown J.S. Hussell T. Gilliland S.M. Holden D.W. Paton J.C. Ehrenstein M.R. Walport M.J. Botto M. The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice.Proc. Natl. Acad. Sci. USA. 2002; 99: 16969-16974Crossref PubMed Scopus (276) Google Scholar) on the heightened susceptibility of C3−/− mice, as well as mice treated with cobra venom factor (CVF) to deplete C3 (Figure 3C). C4−/− mice were also very susceptible to infection, although somewhat less than the C3 deficient mice (Figure 3C), again confirming prior results that a classical C4 dependent pathway is the dominant pathway for C3 fixation during innate resistance (Brown et al., 2002Brown J.S. Hussell T. Gilliland S.M. Holden D.W. Paton J.C. Ehrenstein M.R. Walport M.J. Botto M. The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice.Proc. Natl. Acad. Sci. USA. 2002; 99: 16969-16974Crossref PubMed Scopus (276) Google Scholar). Splenectomized mice were likewise very sensitive to i.v. infection (Figure S3). 22D1 treated mice also showed a decrease in resistance to live S. pneumoniae in BALB/c and C57BL/6 strains (Figures 3D,E), comparable to genetic knockouts, but this pathway is only one of several that contribute to C3 fixation. For example, other contributions arise from the alternative pathway in serum (Figure 2D) (Brown et al., 2002Brown J.S. Hussell T. Gilliland S.M. Holden D.W. Paton J.C. Ehrenstein M.R. Walport M.J. Botto M. The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice.Proc. Natl. Acad. Sci. USA. 2002; 99: 16969-16974Crossref PubMed Scopus (276) Google Scholar), natural antibody (Koppel et al., 2005Koppel E.A. Wieland C.W. van den Berg V.C. Litjens M. Florquin S. van Kooyk Y. van der Poll T. Geijtenbeek T.B. Specific ICAM-3 grabbing nonintegrin-related 1 (SIGNR1) expressed by marginal zone macrophages is essential for defense against pulmonary Streptococcus pneumoniae infection.Eur. J. Immunol. 2005; 35: 2962-2969Crossref PubMed Scopus (65) Google Scholar), and perhaps other lectins in the SIGN-R family such as the related SIGN-R3 lectin (Takahara et al., 2004Takahara K. Yashima Y. Omatsu Y. Yoshida H. Kimura Y. Kang Y.S. Steinman R.M. Park C.G. Inaba K. Functional comparison of the mouse DC-SIGN, SIGNR1, SIGNR3 and Langerin, C-type lectins.Int. Immunol. 2004; 16: 819-829Crossref PubMed Scopus (123) Google Scholar). Thus SIGN-R1 is one pathway for resistance to S. pneumoniae, most likely contributing to the early phase of C3 fixation (Figure 2B,E). To understand the linkage between SIGN-R1 and C3 processing, we searched for SIGN-R1 binding proteins in the spleen. We biotinylated the soluble extracellular domain of SIGN-R1, which was expressed by CHO cells, to allow for binding to streptavidin beads for 2 hr at 4°C. Binding was verified by the reactivity of the SIGN-R1-coated beads with mAb 22D1. We then added 10 mg of spleen or lymph node extracts to the SIGN-R1 coated beads, or beads coated with transferrin as control, for 12 hr at 4°C. The bound proteins were eluted and separated on a 4%–15% gradient SDS page gel and developed with silver staining. Since the transmembrane lectin SIGN-R1 is a mannose binding lectin (Galustian et al., 2004Galustian C. Park C.G. Chai W. Kiso M. Bruening S.A. Kang Y.S. Steinman R.M. Feizi T. High and low affinity carbohydrate ligands revealed for murine SIGN-R1 by carbohydrate array and cell binding approaches, and differing specificities for SIGN-R3 and langerin.Int. Immunol. 2004; 16: 853-866Crossref PubMed Scopus (118) Google Scholar), we expected SIGN-R1 to retrieve proteases called MASPs, which are soluble, mannose lectin-associated, serine proteases (Fujita, 2002Fujita T. Evolution of the lectin-complement pathway and its role in innate immunity.Nat. Rev. Immunol. 2002; 2: 346-353Crossref PubMed Scopus (531) Google Scholar). Instead, the 30 kDa band that was selectively bound to SIGN-R1-coated beads was repeatedly isolated and identified" @default.
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• W2017479299 title "A Dominant Complement Fixation Pathway for Pneumococcal Polysaccharides Initiated by SIGN-R1 Interacting with C1q" @default.
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