Kaori Denda-Nagai

The Macrophage C-type Lectin Specific for Galactose/N-Acetylgalactosamine Is an Endocytic Receptor Expressed on Monocyte-derived Immature Dendritic Cells

Lectins on antigen presenting cells are potentially involved in the antigen uptake and the cellular recognition and trafficking. Serial analysis of gene expression in monocyte-derived dendritic cells (DCs), monocytes, and macrophages revealed that 7 of the 19 C-type lectin mRNA were present in immature DCs. Two of these, the macrophage mannose receptor and the macrophage lectin specific for galactose/N-acetylgalactosamine (MGL), were found only in immature DCs, as confirmed by reverse transcriptase-PCR and flow cytometric analysis. By subcloning and sequencing the amplified mRNA, we obtained nucleotide sequences encoding seven different human MGL (hMGL) subtypes, which were apparently derived from alternatively spliced mRNA. In addition, the hMGL gene locus on human chromosome 17p13 contains one gene. A single nucleotide polymorphism was identified at a position in exon 3 that corresponds to the cytoplasmic region proximal to the transmembrane domain. Of all the splicing variants, the hMGL variant 6C was expressed at the highest levels on immature DCs from all donors tested. Immature DCs could incorporate α-GalNAc-modified soluble acrylamide polymers, and this was significantly inhibited by pretreatment of the cells with an anti-hMGL monoclonal antibody that blocks the lectin-carbohydrate interaction. We propose that hMGL is a marker of imDCs and that it functions as an endocytic receptor for glycosylated antigens.

Tumor-Associated Tn-MUC1 Glycoform Is Internalized through the Macrophage Galactose-Type C-Type Lectin and Delivered to the HLA Class I and II Compartments in Dendritic Cells

The type of interaction between tumor-associated antigens and specialized antigen-presenting cells such as dendritic cells (DCs) is critical for the type of immunity that will be generated. MUC1, a highly O-glycosylated mucin, is overexpressed and aberrantly glycosylated in several tumor histotypes. This results in the expression of tumor-associated glycoforms and in MUC1 carrying the tumor-specific glycan Tn (GalNAcalpha1-O-Ser/Thr). Glycopeptides corresponding to three tandem repeats of MUC1, enzymatically glycosylated with 9 or 15 mol of GalNAc, were shown to specifically bind and to be internalized by immature monocyte-derived DCs (iDCs). Binding required calcium and the GalNAc residue and was competed out by GalNAc polymer and Tn-MUC1 or Tn-MUC2 glycopeptides. The macrophage galactose-type C-type lectin (MGL) receptor expressed on iDCs was shown to be responsible for the binding. Confocal analysis and ELISA done on subcellular fractions of iDCs showed that the Tn-MUC1 glycopeptides colocalized with HLA class I and II compartments after internalization. Importantly, although Tn-MUC1 recombinant protein was bound and internalized by MGL, the glycoprotein entered the HLA class II compartment, but not the HLA class I pathway. These data indicate that MGL expressed on iDCs is an optimal receptor for the internalization of short GalNAcs carrying immunogens to be delivered into HLA class I and II compartments. Such glycopeptides therefore represent a new way of targeting the HLA class I and II pathways of DCs. These results have possible implications in designing cancer vaccines.

The epitope recognized by the unique anti-MUC1 monoclonal antibody MY.1E12 involves sialylα2-3galactosylβ1-3N-acetylgalactosaminide linked to a distinct threonine residue in the MUC1 tandem repeat

The specificity of the MY.1E12 mAb that was generated by immunizing mice with human milk fat globule (HMFG) was investigated. Fluorescein isothiocyanate (FITC)-conjugated peptides corresponding to a portion of the MUC1 tandem repeat were enzymatically glycosylated with N-acetylgalactosamine, galactose, and then sialic acid. The MY.1E12 mAb was examined for its affinity to the resulting glycopeptides by fluorescence polarization. Its affinity for the peptide whose Thr within the VTS sequence bears a Neu5Ac alpha 2-3Gal beta 1-3GalNAc trisaccharide (K(d)=1.4 x 10(-7) M) was significantly higher than for the same peptide whose Thr bears an unsialylated disaccharide (K(d)=3.9 x 10(-6) M). The MY.1E12 mAb also bound strongly to a purified recombinant MUC1 fusion protein with six tandem repeats that was expressed by transfected MCF-7 breast cancer cells. The removal of sialic acids from the fusion protein significantly decreased MY.1E12 mAb reactivity, much more so than the MUC1-specific 115D8 antibody, whose epitope is known to be destroyed by desialylation. Thus, the attachment of the sialyl alpha 2-3Gal beta 1-3 beta 1-3GalNAc trisaccharide onto the Thr within the VTS motif significantly increases the binding of the MY.1E12 antibody to the MUC1 repeat sequence.

The dermal microenvironment induces the expression of the alternative activation marker CD301/mMGL in mononuclear phagocytes, independent of IL-4/IL-13 signaling

Recently, we have shown that mononuclear phagocytes comprise the majority of interstitial cells in the mouse dermis, as indicated by their phenotypic and functional characteristics. In particular, these cells express the mouse macrophage galactose‐/N‐acetylgalactosamine‐specificlectin (mMGL)/CD301, identified by the monoclonal antibody ER‐MP23, as well as other macrophage markers. As expression of mMGL is induced by IL‐4 or IL‐13 and is therefore a marker of alternatively activated macrophages, we asked whether dermal mononuclear phagocytes are genuinely alternatively activated. We observed that these cells expressed, next to mMGL, two other alternative activation markers, namely, the mannose receptor/CD206 and Dectin‐1. Yet, as this expression profile was similar in IL‐4 receptor α knockout mice, neither IL‐4 nor IL‐13 signaling appeared to be required for this phenotype. We also found that Langerhans cells (LC), which showed only a low level of mMGL in the epidermis, up‐regulated mMGL expression upon migration through the dermis, allowing these cells to internalize limited amounts of mMGL ligands. LC isolated from epidermal preparations did not show this up‐regulation when cultured in standard medium, but whole skin‐conditioned medium did stimulate mMGL expression by LC. The vast majority of mMGL molecules was present in the cytoplasm, however. LC, which arrived in skin‐draining lymph nodes, quickly down‐regulated mMGL expression, and dermally derived cells retained significant mMGL levels. Taken together, these data suggest that the dermal microenvironment induces mononuclear phagocyte subpopulations to express mMGL and possibly other markers of alternatively activated macrophages, independent of IL‐4/IL‐13 signaling.

A Unique Dermal Dendritic Cell Subset That Skews the Immune Response toward Th2

Dendritic cell (DC) subsets in the skin and draining lymph nodes (LNs) are likely to elicit distinct immune response types. In skin and skin-draining LNs, a dermal DC subset expressing macrophage galactose-type C-type lectin 2 (MGL2/CD301b) was found distinct from migratory Langerhans cells (LCs) or CD103+ dermal DCs (dDCs). Lower expression levels of Th1-promoting and/or cross-presentation-related molecules were suggested by the transcriptome analysis and verified by the quantitative real-time PCR analysis in MGL2+ dDCs than in CD103+ dDCs. Transfer of MGL2+ dDCs but not CD103+ dDCs from FITC-sensitized mice induced a Th2-type immune response in vivo in a model of contact hypersensitivity. Targeting MGL2+ dDCs with a rat monoclonal antibody against MGL2 efficiently induced a humoral immune response with Th2-type properties, as determined by the antibody subclass. We propose that the properties of MGL2+ dDCs, are complementary to those of CD103+ dDCs and skew the immune response toward a Th2-type response.

Distribution and Function of Macrophage Galactose-type C-type Lectin 2 (MGL2/CD301b): EFFICIENT UPTAKE AND PRESENTATION OF GLYCOSYLATED ANTIGENS BY DENDRITIC CELLS*

Dendritic cells (DCs) express cell surface lectins that are potentially involved in the recognition, uptake, and presentation of glycosylated foreign substances. A unique calcium-type (C-type) lectin, the macrophage galactose (Gal)-type C-type lectin (MGL/CD301) expressed on DCs, is thought to participate in the recognition of molecules from both altered self and pathogens due to its monosaccharide specificity for Gal and N-acetylgalactosamine (GalNAc). Although mice have two MGL genes, Mgl1 and Mgl2, their distinct roles have not been previously explored. The present report characterizes the properties of MGL2 by examining its distribution and its role in antigen presentation by DCs. We generated an MGL2-specific monoclonal antibody and examined MGL2 expression in tissues by immunohistochemistry and in isolated cells by flow cytometry. The cells reactive with this antibody were shown to be a portion of MGL1-expressing cells, mostly conventional DCs. Internalization of soluble polyacrylamide polymers (PAA) with α-GalNAc residues (GalNAc-PAA) by bone marrow-derived DCs (BM-DCs) was mediated by MGL2, as revealed by a comparison of Mgl1−/− and Mgl2−/− BM-DCs with wild-type BM-DCs. Biotinylated GalNAc-PAA conjugated to streptavidin (SAv) was more efficiently presented to SAv-primed T cells by BM-DCs than β-N-acetylglucosamine-PAA conjugated to SAv or SAv alone as shown by thymidine uptake and cytokine production. This is the first report that demonstrates the involvement of GalNAc residues in antigen uptake and presentation by DCs that lead to CD4+ T cell activation.

MGL2 Dermal dendritic cells are sufficient to initiate contact hypersensitivity in vivo.

Background Dendritic cells (DCs) are the most potent antigen-presenting cells in the mammalian immune system. In the skin, epidermal Langerhans cells (LCs) and dermal dendritic cells (DDCs) survey for invasive pathogens and present antigens to T cells after migration to the cutaneous lymph nodes (LNs). So far, functional and phenotypic differences between these two DC subsets remain unclear due to lack of markers to identify DDCs. Methodology/Principal Findings In the present report, we demonstrated that macrophage galactose-type C-type lectin (MGL) 2 was exclusively expressed in the DDC subset in the skin-to-LN immune system. In the skin, MGL2 was expressed on the majority (about 88%) of MHCII+CD11c+ cells in the dermis. In the cutaneous LN, MGL2 expression was restricted to B220−CD8αloCD11b+CD11c+MHCIIhi tissue-derived DC. MGL2+DDC migrated from the dermis into the draining LNs within 24 h after skin sensitization with FITC. Distinct from LCs, MGL2+DDCs localized near the high endothelial venules in the outer T cell cortex. In FITC-induced contact hypersensitivity (CHS), adoptive transfer of FITC+MGL2+DDCs, but not FITC+MGL2−DCs into naive mice resulted in the induction of FITC-specific ear swelling, indicating that DDCs played a key role in initiation of immune responses in the skin. Conclusions/Significance These results demonstrated the availability of MGL2 as a novel marker for DDCs and suggested the contribution of MGL2+ DDCs for initiating CHS.

A C-Type Lectin MGL1/CD301a Plays an Anti-Inflammatory Role in Murine Experimental Colitis

Inflammatory bowel disease is caused by abnormal inflammatory and immune responses to harmless substances, such as commensal bacteria, in the large bowel. Such responses appear to be suppressed under healthy conditions, although the mechanism of such suppression is currently unclear. The present study aimed to reveal whether the recognition of bacterial surface carbohydrates by the macrophage galactose-type C-type lectin-1, MGL1/CD301a, induces both the production and secretion of interleukin (IL)-10. Dextran sulfate sodium salt (DSS) was orally administrated to mice that lacked MGL1/CD301a (Mgl1(-/-) mice) and their wild-type littermates. Mgl1(-/-) mice showed significantly more severe inflammation than wild-type mice after administration of DSS. MGL1-positive cells in the colonic lamina propria corresponded to macrophage-like cells with F4/80-high, CD11b-positive, and CD11c-intermediate expression. These cells in Mgl1(-/-) mice produced a lower level of IL-10 mRNA compared with wild-type mice after the administration of DSS for 2 days. Recombinant MGL1 was found to bind both Streptococcus sp. and Lactobacillus sp. among commensal bacteria isolated from mesenteric lymph nodes of DSS-treated mice. Heat-killed Streptococcus sp. induced an increase in IL-10 secretion by MGL1-positive colonic lamina propria macrophages, but not the macrophage population from Mgl1(-/-) mice. These results strongly suggest that MGL1/CD301a plays a protective role against colitis by effectively inducing IL-10 production by colonic lamina propria macrophages in response to invading commensal bacteria.

MUC1 in carcinoma-host interactions.

Many carcinoma-associated markers are glycoconjugates whose expression undergoes temporal or spatial regulation. Mucin-1 (MUC1), discovered through monoclonal antibody technology, is a well-documented example of such a molecule and influences numerous pathophysiological behaviors, such as the invasion and metastasis of carcinoma cells. Levels of MUC1 expression in carcinomas correlate with the clinical stage of the cancer and inversely correlate with the survival prospects of patients. The MUC1 immune response is known to provide a protective host defense mechanism against cancer. The multiple functions of MUC1 in carcinoma-host interactions are believed to be dependent on the polymorphic nature of MUC1, particularly its glycosylation status.

Vaccination of mice with MUC1 cDNA suppresses the development of lung metastases

C57BL/6 mice were immunized intradermally with various doses of purified pCEP4 plasmid DNA containing full-length MUC1 cDNA (22 tandem repeats). Mice immunized with MUC1 DNA three times at weekly intervals had serum antibodies to a synthetic peptide corresponding to the tandem repeats of MUC1. The antibody titer correlated with the plasmid DNA dose. After the third immunization mice were injected intravenously with 5×105 B16-F10 melanoma cells that had been stably transfected with MUC1 cDNA (F10-MUC1-C8 clone cells). The number of lung metastatic nodules three weeks after inoculation of F10-MUC1-C8 cells was significantly lower in mice immunized with MUC1 plasmid DNA than in mice immunized with the vector DNA alone. Thus, the suppression of lung metastasis was antigen-specific. In vivo depletion of lymphocyte subpopulations by specific antibodies revealed that natural killer cells are the major effector cells responsible for the suppression of lung metastasis. CD4+ cells and CD8+ cells apparently played some roles too.