Jean-Jacques Pin

Up-Regulation of Macrophage Inflammatory Protein-3α/CCL20 and CC Chemokine Receptor 6 in Psoriasis

Autoimmunity plays a key role in the immunopathogenesis of psoriasis; however, little is known about the recruitment of pathogenic cells to skin lesions. We report here that the CC chemokine, macrophage inflammatory protein-3α, recently renamed CCL20, and its receptor CCR6 are markedly up-regulated in psoriasis. CCL20-expressing keratinocytes colocalize with skin-infiltrating T cells in lesional psoriatic skin. PBMCs derived from psoriatic patients show significantly increased CCR6 mRNA levels. Moreover, skin-homing CLA+ memory T cells express high levels of surface CCR6. Furthermore, the expression of CCR6 mRNA is 100- to 1000-fold higher on sorted CLA+ memory T cells than other chemokine receptors, including CXCR1, CXCR2, CXCR3, CCR2, CCR3, and CCR5. In vitro, CCL20 attracted skin-homing CLA+ T cells of both normal and psoriatic donors; however, psoriatic lymphocytes responded to lower concentrations of chemokine and showed higher chemotactic responses. Using ELISA as well as real-time quantitative PCR, we show that cultured primary keratinocytes, dermal fibroblasts, and dermal microvascular endothelial and dendritic cells are major sources of CCL20, and that the expression of this chemokine can be induced by proinflammatory mediators such as TNF-α/IL-1β, CD40 ligand, IFN-γ, or IL-17. Taken together, these findings strongly suggest that CCL20/CCR6 may play a role in the recruitment of T cells to lesional psoriatic skin.

Mouse Strain Differences in Plasmacytoid Dendritic Cell Frequency and Function Revealed by a Novel Monoclonal Antibody

We report in this study the generation of a novel rat mAb that recognizes mouse plasmacytoid dendritic cells (pDC). This Ab, named 120G8, stains a small subset of CD11clow spleen cell with high specificity. This population produces high amounts of IFN-α upon in vitro viral stimulation. Both ex vivo- and in vitro-derived 120G8+ cells display a phenotype identical with that of the previously described mouse pDC (B220highLy6ChighGr1lowCD11b−CD11clow). Mice treated with 120G8 mAb are depleted of B220highLy6ChighCD11clow cells and have a much-reduced ability to produce IFN-α in response to in vivo CpG stimulation. The mAb 120G8 stains all and only B220highLy6ChighCD11clow pDC in all lymphoid organs. Immunohistochemical studies performed with this mAb indicate that pDC are located in the T cell area of spleen, lymph nodes, and Peyer’s patches. Although the Ag recognized by 120G8 is not yet known, we show that its expression is up-regulated by type I IFN on B cells and DC. Using this mAb in immunofluorescence studies demonstrates strain- and organ-specific differences in the frequency of pDC and other DC subsets. 129Sv mice have a much higher frequency of pDC, together with a lower frequency of conventional CD8α+CD11chigh DC, compared with C57BL/6 mice, both in spleen and blood. The higher ability of 129Sv mice to produce IFN-α in vivo is related to a higher number of pDC, but also to a higher ability of pDC from 129Sv mice to produce IFN-α in vitro in response to viral stimulation.

A Novel Lysosome-Associated Membrane Glycoprotein, DC-LAMP, Induced upon DC Maturation, Is Transiently Expressed in MHC Class II Compartment

We have identified a novel lysosome-associated membrane glycoprotein localized on chromosome 3q26.3-q27, DC-LAMP, which is homologous to CD68. DC-LAMP mRNA is present only in lymphoid organs and DC. A specific MAb detects the protein exclusively in interdigitating dendritic cells. Expression of DC-LAMP increases progressively during in vitro DC differentiation, but sharply upon activation with LPS, TNFalpha, or CD40L. Confocal microscopy confirmed the lysosomal distribution of the protein. Furthermore, DC-LAMP was found in the MHC class II compartment immediately before the translocation of MHC class II molecules to the cell surface, after which it concentrates into perinuclear lysosomes. This suggests that DC-LAMP might change the lysosome function after the transfer of peptide-MHC class II molecules to the surface of DC.

Dendritic Cell Infiltration and Prognosis of Early Stage Breast Cancer

Purpose: Although dendritic cells (DC) and T cells can infiltrate primary breast carcinoma, it remains unclear whether the immune response influences the clinical outcome. Experimental Design: T lymphocytes and DC infiltration within primary tumors was investigated in 152 patients with invasive nonmetastatic breast cancer. CD1a, CD3, CD68, CD123, CD207/Langerin, and CD208/DC-LAMP expression was assessed with semiquantitative immunohistochemical analysis. Expression of chemokines involved in DC migration (MIP-3a/CCL20, MIP-3b/CCL19, and 6Ckine/CCL21) was also examined. The correlation between these markers and the characteristics of the tumors, as well as relapse-free and overall survival was analyzed. Significant prognostic parameters were then tested in a validation series. Results: Infiltration by immature CD207/Langerin+ DC was found in a third of the cancers and did not correlate with clinicopathological data. Presence of mature CD208/DC-LAMP+ DC (56%) and CD3+ T cells (82%) strongly correlated with lymph node involvement and tumor grade. Among the chemokines analyzed, only the presence of MIP-3b/CCL19 in 57% of the tumors correlated with prolonged overall survival. CD123+ plasmacytoid DC (pDC) infiltrated 13% of the primary tumors. Their presence was strongly associated with shorter overall survival (93% versus 58% at 60 months) and relapse-free survival (90% versus 37% at 60 months) and was found to be an independent prognostic factor for overall survival and relapse-free survival and confirmed in an independent validation series of 103 patients. Conclusions: Infiltration by pDC of primary localized breast tumor correlates with an adverse outcome, suggesting their contribution in the progression of breast cancer.

The monoclonal antibody DCGM4 recognizes Langerin, a protein specific of Langerhans cells, and is rapidly internalized from the cell surface

We generated monoclonal antibody (mAb) DCGM4 by immunization with human dendritic cells (DC) from CD34+ progenitors cultured with granulocyte‐macrophage colony‐stimulating factor and TNF‐α. mAb DCGM4 was selected for its reactivity with a cell surface epitope present only on a subset of DC. Reactivity was strongly enhanced by the Langerhans cell (LC) differentiation factor TGF‐β and down‐regulated by CD40 ligation. mAb DCGM4 selectively stained LC, hence we propose that the antigen be termed Langerin. mAb DCGM4 also stained intracytoplasmically, but neither colocalized with MHC class II nor with lysosomal LAMP‐1 markers. Notably, mAb DCGM4 was rapidly internalized at 37 °C, but did not gain access to MHC class II compartments. Finally, Langerin was immunoprecipitated as a 40‐kDa protein with a pI of 5.2 – 5.5. mAb DCGM4 will be useful to further characterize Langerin, an LC‐restricted molecule involved in routing of cell surface material in immature DC.

Identification of Mouse Langerin/CD207 in Langerhans Cells and Some Dendritic Cells of Lymphoid Tissues

Human (h)Langerin/CD207 is a C-type lectin of Langerhans cells (LC) that induces the formation of Birbeck granules (BG). In this study, we have cloned a cDNA-encoding mouse (m)Langerin. The predicted protein is 66% homologous to hLangerin with conservation of its particular features. The organization of human and mouse Langerin genes are similar, consisting of six exons, three of which encode the carbohydrate recognition domain. The mLangerin gene maps to chromosome 6D, syntenic to the human gene on chromosome 2p13. mLangerin protein, detected by a mAb as a 48-kDa species, is abundant in epidermal LC in situ and is down-regulated upon culture. A subset of cells also expresses mLangerin in bone marrow cultures supplemented with TGF-β. Notably, dendritic cells in thymic medulla are mLangerin-positive. By contrast, only scattered cells express mLangerin in lymph nodes and spleen. mLangerin mRNA is also detected in some nonlymphoid tissues (e.g., lung, liver, and heart). Similarly to hLangerin, a network of BG form upon transfection of mLangerin cDNA into fibroblasts. Interestingly, substitution of a conserved residue (Phe244 to Leu) within the carbohydrate recognition domain transforms the BG in transfectant cells into structures resembling cored tubules, previously described in mouse LC. Our findings should facilitate further characterization of mouse LC, and provide insight into a plasticity of dendritic cell organelles which may have important functional consequences.

Immature Human Dendritic Cells Express Asialoglycoprotein Receptor Isoforms for Efficient Receptor-Mediated Endocytosis

In a search for genes expressed by dendritic cells (DC), we have cloned cDNAs encoding different forms of an asialoglycoprotein receptor (ASGPR). The DC-ASGPR represents long and short isoforms of human macrophage lectin, a Ca2+-dependent type II transmembrane lectin displaying considerable homology with the H1 and H2 subunits of the hepatic ASGPR. Immunoprecipitation from DC using an anti-DC-ASGPR mAb yielded a major 40-kDa protein with an isoelectric point of 8.2. DC-ASGPR mRNA was observed predominantly in immune tissues. Both isoforms were detected in DC and granulocytes, but not in T, B, or NK cells, or monocytes. DC-ASGPR species were restricted to the CD14-derived DC obtained from CD34+ progenitors, while absent from the CD1a-derived subset. Accordingly, both monocyte-derived DC and tonsillar interstitial-type DC expressed DC-ASGPR protein, while Langerhans-type cells did not. Furthermore, DC-ASGPR is a feature of immaturity, as expression was lost upon CD40 activation. In agreement with the presence of tyrosine-based and dileucine motifs in the intracytoplasmic domain, mAb against DC-ASGPR was rapidly internalized by DC at 37°C. Finally, intracellular DC-ASGPR was localized to early endosomes, suggesting that the receptor recycles to the cell surface following internalization of ligand. Our findings identify DC-ASGPR/human macrophage lectin as a feature of immature DC, and as another lectin important for the specialized Ag-capture function of DC.

FDF03, a Novel Inhibitory Receptor of the Immunoglobulin Superfamily, Is Expressed by Human Dendritic and Myeloid Cells

In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14+/CD1a− DC derived from CD34+ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c+ blood and tonsil DC, but not by CD11c− DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcγRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.

CD208/Dendritic Cell-Lysosomal Associated Membrane Protein Is a Marker of Normal and Transformed Type II Pneumocytes

Dendritic cell-lysosomal associated membrane protein (DC-LAMP)/CD208, a member of the lysosomal associated membrane protein (LAMP) family, is specifically expressed by human DCs on activation. However, its mouse counterpart could not be detected in mature DCs. The present study demonstrates that DC-LAMP is constitutively expressed by mouse, sheep, and human type II pneumocytes. Confocal and immunoelectron microscopy showed that mouse DC-LAMP protein co-localizes with lbm180, a specific marker for the limiting membrane of lamellar bodies that contain surfactant protein B, as well as with intracellular MHC class II molecules that accumulate in the same organelles. Expression of DC-LAMP was also occasionally detected at the cell surface of type II pneumocytes. Interestingly, human bronchioloalveolar carcinoma tumor cells, which correspond to transformed type II pneumocytes, express DC-LAMP. Similar observations were made in the Jaagsiekte sheep retrovirus-associated ovine pulmonary adenocarcinoma, a model of human bronchioloalveolar carcinoma. This study establishes that DC-LAMP is constitutively expressed in normal type II pneumocytes. Furthermore, DC-LAMP appears to be a marker of transformed type II pneumocytes as well, an observation that may help the study and the classification of human lung adenocarcinomas.

Cleaved/Associated TLR3 Represents the Primary Form of the Signaling Receptor

TLR3 belongs to the family of intracellular TLRs that recognize nucleic acids. Endolysosomal localization and cleavage of intracellular TLRs play pivotal roles in signaling and represent fail-safe mechanisms to prevent self-nucleic acid recognition. Indeed, cleavage by cathepsins is required for native TLR3 to signal in response to dsRNA. Using novel Abs generated against TLR3, we show that the conserved loop exposed in LRR12 is the single cleavage site that lies between the two dsRNA binding sites required for TLR3 dimerization and signaling. Accordingly, we found that the cleavage does not dissociate the C- and N-terminal fragments, but it generates a very stable “cleaved/associated” TLR3 present in endolysosomes that recognizes dsRNA and signals. Moreover, comparison of wild-type, noncleavable, and C-terminal–only mutants of TLR3 demonstrates that efficient signaling requires cleavage of the LRR12 loop but not dissociation of the fragments. Thus, the proteolytic cleavage of TLR3 appears to fulfill function(s) other than separating the two fragments to generate a functional receptor.