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.

SIGN-R1, a recently discovered C-type lectin expressed at high levels on macrophages within the marginal zone of the spleen, mediates the uptake of dextran polysaccharides by these phagocytes. We now find that encapsulated Streptococcus pneumoniae are rapidly cleared by these macrophages from the bloodstream, and that capture also takes place when different cell lines express SIGN-R1 after transfection. To assess the role of the capsular polysaccharide of S. pneumoniae (CPS) in the interaction of SIGN-R1 with pneumococci, we first studied binding and uptake of serotype 14 CPS in transfected cells. Binding was observed and was of a much higher avidity (3,000-fold) for CPS 14 than dextran. The CPSs from four different serotypes were also cleared by marginal zone macrophages in vivo. To establish a role for SIGN-R1 in this uptake, we selectively down-regulated expression of the lectin by pretreatment of the mice with SIGN-R1 antibodies, including a newly generated hamster monoclonal called 22D1. For several days after this transient knockout, the marginal zone macrophages were unable to take up either CPSs or dextrans. Therefore, marginal zone macrophages in mice have a receptor that interacts with capsular pneumococcal polysaccharides, setting the stage for further studies of the functional consequences of this interaction.T he spleen functions at several points in innate and adaptive immunity. A major innate function is exerted by macrophages that are abundant in vascular regions termed the splenic red pulp, whereas adaptive functions are carried out by B and T lymphocytes, typically located in white pulp nodules. At the junction of each white pulp nodule with the red pulp is a specialized region called the marginal zone, which is composed of several concentric regions (1). Innermost is a ring of macrophages termed marginal metallophils, expressing a hemagglutinin termed sialoadhesin or CD169 (2, 3). Then there is a vascular sinus that receives blood via penetrating small arterial vessels from the white pulp. Surrounding the marginal sinus is a zone composed of large macrophages as well as specialized B lymphocytes (4). Within and surrounding the marginal zone are also dendritic cells (5, 6), possibly in the process of migrating from the blood to the T cell regions of the white pulp.With respect to host defense, the spleen plays a special role during blood-borne infection with encapsulated microorganisms, particularly Streptococcus pneumoniae bacteria (7-12). A critical role of the spleen is the formation of antibodies by marginal zone B cells (13-15), particularly complement-fixing antibodies (16)(17)(18)(19)(20). The role of macrophages in the processes of microbial clearance and resistance and antibody formation to S. pneumoniae needs to be considered (21), particularly given recent data that marginal zone macrophages interact and retain B cells in this region (22). Here we show that marginal zone macrophages express a receptor called SIGN-R1 that is able to bind and internalize the capsular pneumococcal polys...

The marginal zone macrophages of the spleen are implicated in the clearance of polysaccharides, but underlying mechanisms need to be pinpointed. SIGN-R1 is one of five recently identified mouse genes that are homologous to human DC-SIGN and encode a single, external, C-terminal C-type lectin domain. We find that a polyclonal antibody to a specific SIGN-R1 peptide reacts primarily and strongly with a subset of macrophages in the marginal zone of spleen and lymph node medulla. In both sites, SIGN-R1 exists primarily in an aggregated form, resistant to dissociation into monomers upon boiling in SDS under reducing conditions. Upon transfection into three different cell lines, high-mol.-wt forms bearing SIGN-R1 are expressed, as well as reactivity with ER-TR9, a mAb previously described to react selectively with marginal zone macrophages. SIGN-R1-expressing macrophages preferentially sequester dextrans following i.v. injection. Likewise, when phagocytic cells are enriched from spleen and tested in culture, dextran is selectively endocytosed by a subset of very large SIGN-R1(+) cells representing approximately 5% of total released macrophages. Uptake of FITC-dextran by these macrophages in vivo and in vitro is blocked by ER-TR9 and polyclonal anti-SIGN-R1 antibodies. Following transfection with SIGN-R1, cell lines become competent to endocytose dextrans. The dextran localizes primarily to compartments lacking transferrin receptor and the LAMP-1 CD107a panlysosomal antigen. Therefore, SIGN-R1 mediates the uptake of dextran polysaccharides, and it is predominantly expressed in the macrophages of the splenic marginal zone and lymph node medulla.

The mouse (m) DC-SIGN family consists of several homologous type II transmembrane proteins located in close proximity on chromosome 8 and having a single carboxyl terminal carbohydrate recognition domain. We first used transfected non-macrophage cell lines to compare the polysaccharide and microbial uptake capacities of three of these lectins--DC-SIGN, SIGNR1 and SIGNR3--to another homologue mLangerin. Each molecule shares a potential mannose-recognition EPN-motif in its carbohydrate recognition domain. Using an anti-Tag antibody to follow Tag-labeled transfectants, we found that each molecule could be internalized, although the rates differed. However, mDC-SIGN was unable to take up FITC-dextran, FITC-ovalbumin, zymosan or heat-killed Candida albicans. The other three lectins showed distinct carbohydrate recognition properties, assessed by blocking FITC-dextran uptake at 37 degrees C and by mannan binding activity at 4 degrees C. Furthermore, only SIGNR1 was efficient in mediating the capture by transfected cells of Gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium, while none of the lectins tested were competent to capture Gram-positive bacteria, Staphylococcus aureus. Interestingly, transfectants with SIGNR1 lacking the cytoplasmic domain were capable of binding FITC-zymosan in a manner that was abolished by EDTA or mannan, but not laminarin. In addition, resident peritoneal CD11b+ cells expressing SIGNR1 bound zymosan at 4 degrees C in concert with a laminarin-sensitive receptor. Therefore these homologous C-type lectins have distinct recognition patters for microbes despite similarities in the carbohydrate recognition domains.