CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Immunity, Vol. 6, 187±197, February, 1997, Copyright 1997 by Cell Press The Mannose Receptor Delivers Lipoglycan Antigens to Endosomes for Presentation to T Cells by CD1b Molecules Theodore I. Prigozy,1 Peter A. Sieling,2 motif in their cytoplasmic tail that is similar to the endo- Daniel Clemens,3 Phoebe L. Stewart,5, 6, 7 somal targeting signals of lysosomal glycoprotein A and Samuel M. Behar,8 Steven A. Porcelli,8 CD63 (Sandoval and Bakke, 1994). Sugita et al. (1996) Michael B. Brenner,8 Robert L. Modlin,1, 2, 7 have established that the cytoplasmic tail of CD1b is a and Mitchell Kronenberg1, 4, 7 signal that targets CD1b to late endosomes and major 1 Department of Microbiology and Immunology histocompatibility complex (MHC) class II compart- 2 Division of Dermatology ments (MIICs), possibly via internalization from the 3 Division of Infectious Diseases plasma membrane. 4 Division of Digestive Diseases The macrophage mannose receptor (MR) is a C-type Department of Medicine lectin expressed primarily by macrophages and den- 5 Department of Molecular and Medical Pharmacology dritic cells, as well as endothelial cells (Ezekowitz et al., 6 Crump Institute for Biological Imaging 1990; Taylor et al., 1990). Recently, the MR and a related 7 Molecular Biology Institute mouse protein, DEC-205 (Jiang et al., 1995), have been University of California at Los Angeles implicated as potential antigen capture molecules with Los Angeles, California 90095 the capability of directing the transport of ligands into 8 Division of Rheumatology and Immunology endosomal compartments (Sallusto et al., 1995). Neither Department of Medicine cell surface protein, however, has been linked to the Brigham and Women's Hospital uptake of antigens that are ultimately presented to T Boston, Massachusetts 02115 cells during an immune response to an infectious agent. In addition, the mechanismby whichlipoglycan antigens are transported to antigen-presenting compartments has not been explored. Because of the ability of the MR Summary to bind LAM (Schlesinger et al., 1994; Venisse et al., 1995) and other pathogen-associated polymannose We have characterized the CD1b-mediated presenta- structures (O'Riordan et al., 1995; Cross and Bancroft, tion pathway for the mycobacterial lipoglycan lipoara- 1995), we considered it to be a candidate molecule for binomannan (LAM) in monocyte-derived antigen-pres- mediating the internalization of LAM into endosomal enting cells. The macrophage mannose receptor (MR) compartments for presentation to LAM-specific T cells. was responsible for uptake of LAM. Antagonism of MR The goal of this study, therefore, was to define the function inhibited both the internalization of LAM and CD1b-mediated antigen presentation pathway for LAM, the presentation of this antigen to LAM-reactive T including the mechanism of LAM uptake, the fate of cells. Intracellular MRs were most abundant in early LAM in the endocytic pathway, and the likely site of endosomes, but they also were located in the com- encounter of LAM with CD1b in antigen-processing partment for MHC class II antigen loading (MIIC). In- compartments. These data outline a novel nonpeptide ternalized LAM was transported to late endosomes, antigen presentation pathway that links pattern recogni- lysosomes, and MIICs. MRs colocalized with CD1b tion molecules involved in ªfirst line defenseº to adap- molecules, suggesting that the MR could deliver LAM tive, T cell±mediated immune responses. to late endosomes for loading onto CD1b. LAM and CD1b colocalized in organelles that may be sites of lipoglycan antigen loading. This pathway links recog- Results nition of microbial antigens by a receptor of the innate immune system to the induction of adaptive T cell MR Function Is Required for the Presentation responses. of LAM to T Cells The involvement of the MR in the uptake of LAM by CD11 antigen-presenting cells (APC) was established Introduction by testing the ability of the MR ligand a-mannan to inhibit the antigen-dependent proliferation of the LAM-specific The CD1 proteins are nonpolymorphic, b2m-associated T cell line LDN4 (Sieling et al., 1995). In this experimental cell surface glycoproteins that have been shown to have system, APC for the LDN4 T cells are derived by treat- antigen presentation function (Calabi and Bradbury, ment of peripheral blood mononuclear cells with granu- 1991; Porcelli, 1995; Porcelli and Modlin, 1995). Human locyte/macrophage colony-stimulating factor (GM-CSF) CD1b presents nonpeptide components of mycobact- and interleukin-4 (IL-4) (Porcelli et al., 1992). The re- eria, including the lipid mycolic acid and the lipoglycan sulting population of APC consists of dendritic cells and lipoarabinomannan (LAM), to double negative T cells. macrophages, of which greater than 50% are CD1b/MR The sensitivity of the antigen presentation of both my- double positive (data not shown). For consistency, the colic acid (Porcelli et al., 1992; Beckman et al., 1994) cells from this primary culture are referred to simply as and LAM (Sieling et al., 1995) to agents that inhibit endo- APC throughout. The APC were incubated with LAM in somal acidification suggests a CD1-mediated presenta- the presence of LDN4 cells and increasing concentra- tion pathway for nonpeptide antigens that involves en- tions of either mannan or mannose, which does not bind dosomes. Human CD1b molecules contain a sequence the MR, as a control ligand. T cell proliferation was Immunity 188 1B). The inhibition of antigen presentation by the anti- MR antiserum was observed only when the antiserum was added prior to the addition of LAM. In contrast, when the APC were pulsed with LAM for 2 hr prior to the addition of the blocking antiserum, no inhibition was observed. Because the MR antiserum must be added prior to LAM to inhibit the T cell response, we conclude that the antibodies most likely are blocking LAM uptake. Blockade of MR Function Inhibits the Internalization of LAM To test the hypothesis that the MR was indeed function- ing as the receptor for the uptake of LAM in these APC, we used quantitative immunoelectron microscopy to measure the amount of LAM that was internalized in the presence and absence of mannan. APC were pulsed with LAM for 3 hr with and without mannan. After fixation, sections were stained for both LAM and the late endoso- mal marker CD63. The amount of LAM-specific immuno- gold was quantitated by counting gold particles per square micron of cytoplasm in electron micrographs of 20 consecutive cells. LAM uptake was inhibited approxi- mately 4-fold by mannan (0.14 gold particles/mm2) rela- tive to the untreated control cells (0.62 gold particles/ mm2). The levels and distribution of CD63 in late endo- somes were not altered by the mannan treatment (data not shown). This established the MR as the principal receptor for LAM uptake into endocytic vesicles. The MR Is Present Predominantly in Early Endosomes We investigated the distribution of MR in APC to deter- mine whether the MR might transport LAM to late endo- somal compartments known to contain CD1b (Sugita et Figure 1. The MR Is Required for Presentation of LAM al., 1996). As the cytoplasmic tail of the MR contains (A) Inhibition of LDN4 proliferation by the MR ligand mannan. CD11 signals required for its internalization from the plasma APC were incubated with Mycobacterium leprae LAM (1 mg/ml) and membrane (Ezekowitz et al., 1990), we therefore ex- increasing concentrations of mannose (open squares) or mannan pected to find the MR in early endosomes. Cells were (closed squares), followed by the addition of LDN4 (1 3 104) T cells fluorescently labeled for various times with dextran± for 3 days. The resulting T cell proliferation data were plotted as Texas red to trace the trafficking of an internalized anti- percent inhibition versus the concentration of the ligand. Data from gen along the endocytic pathway. The majority of the three independent experiments gave similar results. (B) Anti-MR antiserum inhibits IFNg production by LDN4. CD11 APC MR colocalized with dextran after 15 min (Figure 2A), were subjected to the following protocols, as indicated in the figure indicating that the MR was most abundant in early endo- and described in Experimental Procedures: LAM, APC incubated somes. The MR colocalized only to a limited extent with with LAM for 2.5 hr followed by two washes with media; no antigen, dextran in perinuclear vesicles after 2 hr (Figure 2B), APC incubated for 2.5 hr without LAM followed by two washes with establishing that a minority of the MR molecules are media; anti-MR followed by LAM, APC were incubated with an anti- resident in late endosomes. APC were incubated for 1 hr MR antiserum (1:300) for 60 min, and after one wash with media, LAM was added for 2.5 hr followed by two washes with media; with dextran followed by an overnight chase to label preimmune followed by LAM, same as above except for the addition mature late endosomal compartments such as lyso- of preimmune goat serum rather than the anti-MR antiserum; LAM somes and MIICs. There was a moderate amount of followed by anti-MR, LAM was added to the APC for 2 hr followed colocalization of the MR and dextran (Figure 2C), which by one wash with media. The anti-MR antiserum was added for 1.5 was in general greater than that observed in the 2 hr 4 hr followed by two washes with media. LDN4 cells (5 3 10 ) were labeling. These data suggest that, although a significant added to the washed APC, and IFNg production was measured after 36 hr by ELISA. Data from three different experiments gave similar fraction of the MR is not resident in classical late endo- results.