Claude Vincent

Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules

We have identified a type II Ca2+-dependent lectin displaying mannose-binding specificity, exclusively expressed by Langerhans cells (LC), and named Langerin. LC are uniquely characterized by Birbeck granules (BG), which are organelles consisting of superimposed and zippered membranes. Here, we have shown that Langerin is constitutively associated with BG and that antibody to Langerin is internalized into these structures. Remarkably, transfection of Langerin cDNA into fibroblasts created a compact network of membrane structures with typical features of BG. Langerin is thus a potent inducer of membrane superimposition and zippering leading to BG formation. Our data suggest that induction of BG is a consequence of the antigen-capture function of Langerin, allowing routing into these organelles and providing access to a nonclassical antigen-processing pathway.

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.

ANTIBODIES AGAINST FUNCTIONAL LEUKOCYTE SURFACE MOLECULES IN POLYCLONAL ANTILYMPHOCYTE AND ANTITHYMOCYTE GLOBULINS

Antilymphocyte or antithymocyte globulins were shown to be immunosuppressive when administered to recipients of organ transplants as prophylactic or rescue treatment of acute rejection or in patients with acute graft versus host reactions following bone marrow transplantation. Several monoclonal antibodies specific for activation or adhesion molecules of the T lymphocyte surface can also inhibit experimental or clinical allograft reactions. We have investigated the presence of some antibodies, of defined specificity and documented biological activity, in polyclonal antilymphocyte and antithymocyte globulins in order to get further insight into the mechanism of action of these polyclonal antibodies. Using a quantitative immunofluorescence assay by flow cytometry we could estimate the minimal amounts of antibodies to LFA-1 (CD11a and CD18), CD45, CD3, and CD5. Antibodies to HLA-DR, CD2, CD4, CD8, and CD25 were also demonstrated but could not be quantified. Antibodies to beta 2-microglobulin were determined by ELISA. These data suggest that interference with functional lymphocyte surface molecules may account at least in part for the immunosuppressive activity of antilymphocyte and antithymocyte globulins.

Lipoteichoic Acid Increases TLR and Functional Chemokine Expression while Reducing Dentin Formation in In Vitro Differentiated Human Odontoblasts

Gram-positive bacteria entering the dentinal tissue during the carious process are suspected to influence the immune response in human dental pulp. Odontoblasts situated at the pulp/dentin interface are the first cells encountered by these bacteria and therefore could play a crucial role in this response. In the present study, we found that in vitro-differentiated odontoblasts constitutively expressed the pattern recognition receptor TLR1–6 and 9 genes but not TLR7, 8, and 10. Furthermore, lipoteichoic acid (LTA), a wall component of Gram-positive bacteria, triggered the activation of the odontoblasts. LTA up-regulated the expression of its own receptor TLR2, as well as the production of several chemokines. In particular, an increased amount of CCL2 and CXCL10 was detected in supernatants from LTA-stimulated odontoblasts, and those supernatants augmented the migration of immature dendritic cells in vitro compared with controls. Clinical relevance of these observations came from immunohistochemical analysis showing that CCL2 was expressed in vivo by odontoblasts and blood vessels present under active carious lesions but not in healthy dental pulps. In contrast with this inflammatory response, gene expression of major dentin matrix components (type I collagen, dentin sialophosphoprotein) and TGF-β1 was sharply down-regulated in odontoblasts by LTA. Taken together, these data suggest that odontoblasts activated through TLR2 by Gram-positive bacteria LTA are able to initiate an innate immune response by secreting chemokines that recruit immature dendritic cells while down-regulating their specialized functions of dentin matrix synthesis and mineralization.

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.

In vitro evaluation of the sensitization potential of weak contact allergens using langerhans-like dendritic cells and autologous T cells

Contact hypersensitivity is a major public health concern in most industrial countries, which is why predictive tests which could identify potential allergens are needed. We have established an in vitro approach for the detection of primary immune response. This model uses Langerhans-like dendritic cells (LLDC) derived from cord blood progenitors and autologous T lymphocytes, isolated from the same blood sample. Treatment of day 12-14 LLDC, with strong haptens trinitrobenzene sulfonic acid (TNP), fluorescein isothiocyanate (FITC) or Bandrowskis base (BB), results in the proliferation of T lymphocytes, whereas weak allergens and irritants, such as sodium dodecyl sulfate (SDS) are ineffective. The use of immature (day 8) LLDC and the addition of a 48 h stage of incubation after hapten contact, result in phenotypic maturation of LLDC in addition to lymphocyte activation in all the cultures with strong haptens. The 48 h stage of incubation, results in sensitization and in some cases the induction of T cell proliferation to citronellal (1/8), coumarine (1/8) and to a prohapten p-phenylenediamine (pPDA; 2/8). The phenotype of DC after 48 h of contact with a strong hapten, becomes that of mature DC (CD83(+), CD86(+) and HLA-DR(++)). With fragrance molecules, weak haptens and prohaptens, a comparable phenotype is observed only when T lymphocytes are activated. These data suggest that the unresponsiveness observed with weak haptens, may be the consequence on an incomplete maturation of LLDC.

The inhibition of MAPK pathway is correlated with down-regulation of MMP-9 secretion induced by TNF-α in human keratinocytes

MMP-9 (92 kDa) is the major gelatinase able to degrade collagen IV, secreted by keratinocytes that are actively involved in wound-healing or tumorigenesis. Since the invasive phenotype of cancers is dependent on MMP-9 expression, it appeared of interest to precisely characterize which signal transduction pathways activated by TNF-alpha are involved in MMP-9 up-regulation induced by TNF-alpha. In HaCaT cells, activation of MMP-9 occurs at the transcriptional level. Inhibition of the MAPK pathway using specific inhibitors of the Ras, Raf, MEK1/2, and Erk1/2 cascade was correlated with a marked inhibition of MMP-9 activity, as determined by gene and protein expression. MAPK pathway activation via TNF-alpha was confirmed by marked AP-1 activation detected in EMSA. Under our experimental conditions, p38 MAPK and SAPK/JNK pathways were not activated. Gene and protein expression of other MMPs that regulate MMP-9, such as MMP-1 and MMP-13, were also up-regulated by TNF-alpha and inhibited by UO126, providing evidence that the MAPK pathway plays a fundamental role in the regulation of MMP-9 secretion by keratinocytes. As TNF-alpha is known to be a main activator of NF-kappaB pathway, the effects of campthothecin and caffeic acid were investigated, such as, TNF-alpha campthothecin up-regulated MMP-9 activity but caffeic acid only weakly inhibited MMP-9 activation induced by TNF-alpha. However, NF-kappaB is activated as shown from immunostaining data, a nuclear staining and higher Western blotting expression of p50 and p65 NF-kappaB subunits were detected after TNF-alpha treatment. A higher specific signal was also detected in EMSA for TNF-alpha-treated cells.

Anti-CD4 monoclonal antibody administration in renal transplanted patients

Administration of anti-CD4 antibodies in rodents was shown to prevent or to reverse spontaneous or experimentally induced autoimmune diseases and to delay organ or skin allograft rejection. Some anti-human CD4 antibodies were shown to be immunosuppressive when injected in monkeys. BL4, and IgG2a anti-human CD4 murine monoclonal antibody, which binds to an epitope located between the two N-terminal domains of the CD4 molecule, was administered to 12 recipients of a renal cadaver allograft, in association with azathioprine (2.5 mg/kg/day) and prednisolone (1 mg/kg/day). Treatment was started 1 day after transplantation and was discontinued after 3 to 14 days (median 5 days). Infusion of 10 or 15 mg of BL4 over 1 hr induced a selective but transient CD4+ lymphocytopenia. The lack of clinical side effect was remarkable. Acute rejection occurred in 4 out of 12 treated patients. Antibody response to BL4 3 weeks after completion of the treatment was demonstrated in only one patient. Residual antibody concentrations in serum, 24 hr after infusion, ranged from 0.1 to 0.5 microgram/ml, that is below the concentration required to achieve 50% inhibition of allogenic mixed lymphocyte reaction in vitro (1-10 micrograms/ml) or to saturate CD4 binding sites (5-10 micrograms/ml). Rapid degradation and dissociation of cell bound BL4 contributed to the failure to achieve high residual serum levels of the antibody.

Relationship between expression of matrix metalloproteinases and migration of epidermal and in vitro generated Langerhans cells

Langerhans cells (LC) are dendritic cells that capture foreign antigens and migrate with them to the regional lymph nodes where they are presented to naive T cells. The possible role of matrix metalloproteinase-9 (MMP-9) in migration was suggested following experiments in a mouse model and in human skin explants. Using in vitro generated LC (iLC) derived from CD34+ cord blood cells and epidermal LC (eLC), we investigated the correlation between MMP-9 and other MMPs production and cell migration. Cells were activated by Bandrowskis base (BB), a chemical allergen, or by recombinant birch pollen allergen 1 (rBetv 1). Contact with allergens triggered migration of these cells, with a maximum rate being reached after 24 h. Migration was preceded by production of MMP-2 and MMP-9; part of the molecules were recovered as pro-MMPs in cell culture supernatant and part were associated with cell membrane proteins. At the cellular level, membrane-type 1 (MT1) and MT3-MMP were also identified. Addition of tumor necrosis factor-alpha (TNF-alpha) initiated pro-MMP-2 and pro-MMP-9 production followed by cell migration in a dose-dependent manner. These data imply that TNF-alpha is a key molecule for MMP production and cell migration. Furthermore, activation of iLC with BB or rBet v 1 induced synthesis of TNF-a and expression of TNF RII on the cell membrane, suggesting an autocrine loop. In conclusion, membrane-associated MMP-2 and-9 rather than soluble MMPs appear to be involved in cell migration.

Malignant Langerhans cell tumor: A case with a favorable outcome associated with the absence of blood dendritic cell proliferation

Malignant Langerhans cell tumor is a rare malignant proliferation of Langerhans cells, with a negative prognosis due to its dissemination throughout the body, leading to death within 1 year. This disease has to be distinguished from Langerhans cell histiocytosis. The favorable evolution of a case of Langerhans cell tumor, characterized by the absence of metastasis 18 months after its occurrence, may be due to the initial treatment, which consisted of complete and large resection of the tumor. The authors searched for abnormal dendritic cells or progenitors in the blood but found no large amounts or proliferation of CD34(+) or CD1a(+) cells at the diagnosis and 1 year later. This case report shows that malignant Langerhans cell tumor is not always a lethal disease. The condition may be related to surgical treatment and the absence of malignant cells in the blood when the diagnosis was performed.