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Having identified an oligonucleotide (ON) receptor in the HepG2 cell line, we have re-examined here the kinetics of ON uptake, subcellular distribution and intracellular localisation in these cells, at concentrations relevant for the study of a receptor-dependent process. Kinetic parameters of ON endocytosis were comparable with those of the receptor-mediated endocytosis tracer, transferrin (uptake equilibrium, saturation with concentration, specific competition and rapid efflux) and were clearly distinct from those of fluid-phase endocytosis. By analytical subcellular fractionation, particulate ON showed a bimodal distribution after 2 h of uptake, with a low-density peak superimposed on the distribution of endosomes, and a high-density peak overlapping lysosomes. After an overnight chase, only the high-density peak remained, but it could be dissociated from lysosomes, based on its refractoriness to displacement upon chloroquine-induced swelling. After 2 h of uptake at 300 nM ON-Alexa, a punctate pattern was resolved, by confocal microscopy, from those of transferrin, of a fluid-phase tracer, and of vital staining of lysosomes by LysoTracker. At 3 microM ON-Alexa, its pattern largely overlapped with the fluid-phase tracer and LysoTracker. Taken together, these data suggest that ON may be internalised at low concentrations by receptor-mediated endocytosis into unique endosomes, then to dense structures that are distinct from lysosomes. The nature of these two compartments and their significance for ON effect deserve further investigation.

The low and unpredictable uptake and cytosolic transfer of oligonucleotides (ODN) is a major reason for their limited benefit. Improving the ODN potential for therapy and research requires a better understanding of their receptor-mediated endocytosis. We have undertaken to identify a membrane ODN receptor on HepG2 cells by ligand blotting of cell extracts with [(125)I]ODN and by photolabelling of living cells with a [(125)I]ODN-benzophenone conjugate. A major band at 66 kDa was identified by the two methods. Its labelling was saturable and competed for by unlabelled ODN of various sequences and irrespective of the presence of a phosphodiester or phosphoro-thioate backbone. This protein remained sedimentable after carbonate extraction, indicating strong membrane association. About half of the total cell amount resisted extensive surface proteolysis, suggesting a dual localisation at the plasma membrane and cytoplasmic vesicles. The protein was purified using a biotinylated ODN-benzophenone conjugate by photocrosslinking followed by streptavidin affinity purification. A sequence obtained by Edman degradation showed no homology with known proteins. Using anti-peptide antisera, labelling by western blotting revealed at 66 kDa a band with comparable properties as found by ligand blotting. Thus, a new membrane protein acting as an ODN receptor has been demonstrated.

Galectin-3 and asialoglycoprotein receptor are lectins belonging to the classes of soluble lectins and of membrane C type lectins respectively. Conflicting results have been reported concerning their transcription level in the time course development of tumours. In the present study we investigated the abnormalities and the transcription levels of galectin-3 and asialoglycoprotein receptor genes in liver-targeted SV40 large T transgenic mice related to normal mice. In the strain expressing the highest level of large T, 100% of the male mice reproducibly developed an hepatocarcinoma. We provide evidence that the galectin-3 and asialoglycoprotein receptor genes are stable in such mice. The galectin-3 gene is weakly transcribed and its level is identical and constant in normal and transgenic mice, suggesting a lack of involvement in the development of large T-induced hepatocarcinoma. The asialoglycoprotein receptor gene is actively transcribed and its level remains high all along the development of the tumour; therefore, in such an hepatocarcinoma the asialoglycoprotein receptor could be used to take up drugs, genes or oligonucleotides associated with glycosylated carriers bearing galactose residues in a terminal non-reducing position.

Dendritic cells (CD) are the most efficient antigen presenting cells for T lymphocytes. CD1a+ CD14- CD with high antigen-presenting capacities can now be obtained easily from adherent peripheral blood monocytes by culture in the presence of granulocyte/macrophage colony-stimulating factor and interleukin-4 (Sallusto et al., J. Exp. Med. 1994. 179: 1109). Human macrophages express a membrane lectin, or sugar-specific receptor, which specifically mediates the binding and endocytosis of mannose- and fucose-terminated glycoproteins and is involved in the phagocytosis of pathogens. A similar lectin activity was sought on cultured human DC using flow cytometry and confocal microscopy to detect binding and internalization of fluoresceinated neoglycoproteins [bovine serum albumin (BSA) substituted with sugar residues]. Several neoglycoproteins, especially alpha-L-fucosyl-, alpha-D-mannosyl-, N,N'-di-acetyl-beta-chitobiosyl- and beta-D-glucosyl-BSA, were endocytosed by cultured human CD1a+ DC as well as by CD1a- CD14- cells which were also obtained in the culture. Fuc-BSA and Man-BSA had the same number of binding sites (1.7 x 10(6)/cell) on CD1a+ DC, and bound with an affinity constant close to 10(7) 1/mol. Inhibition experiments indicated that these two neoglycoproteins bound to the same membrane lectin. CD1a+ and CD1a- cells were both labeled by an antiserum specific for the human macrophage mannose receptor. The membrane lectin specific for mannose and fucose that is evidenced in these experiments on cultured DC may be similar to the macrophage membrane lectin or may share functional and structural properties with it.

Mannose receptor is a differentiation marker of macrophages. Circulating monocytes isolated from plasma are devoid of this receptor; upon culture this receptor is rapidly expressed. Its expression is modulated by a variety of inflammatory and anti-inflammatory agents. In the present study, we investigated its transcription level during the differentiation process. Mannose receptor mRNA was monitored by quantitative RT-PCR on freshly harvested monocytes and on monocytes cultivated up to four days. No transcription was detected in freshly harvested cells, the transcription increased during the first 24 h upon adhesion and then decreased.

We have cloned the putative promoter of the human mannose receptor gene using the ligation-mediated polymerase chain reaction. This modified polymerase chain reaction method depends upon the ligation of restricted genomic DNA fragments to a sequence of DNA containing a generic primer site. Approximately 400 bp of genomic DNA sequence immediately upstream from the 5' end of the lectin gene was amplified with this strategy. Primer-extended reverse transcription identified several 5' ends of the mannose receptor mRNA corresponding to differential use of initiation transcription sites. DNA sequence analysis of the 5' flanking sequence of the mannose receptor gene indicated the presence of a TATA box and various putative binding sites for several transcription activators. The insertion of the sequence into a plasmid containing a promoterless luciferase reporter gene reveals a promoter activity with a high cell type specificity, efficient expression upon transfection into macrophage type cells, and lack of efficiency upon transfection into nonmyeloid cells. A series of deletion mutants reveals that this cell-type-specific promoter activity is mediated by a negative regulatory element located at the 5' end of the isolated promoter.

Myelomonocytic lineage cells express an M(r) 60,000 mannose specific lectin, MR60 (Pimpaneau et al. (1991), Carbohydr Res 213: 95-108). Under non-reducing conditions, this protein migrates as a 120,000 protein. MR60 does not contain any N-glycan moiety cleavable by the action of N-glycanase. MR60 induces a sugar selective aggregation of beads coated with glycosylated albumin: beads bearing alpha-D-mannosyl residues are aggregated while beads bearing alpha-D-glucosyl residues are not. A monoclonal antibody Lec101B, specific for MR60, recognizes a single M(r) 60,000 protein by Western blotting. This monoclonal antibody does not label the cell surface of cells expressing MR60, but decorates intracellular vesicles upon permeabilization of these cells.

The culture of specialized high endothelial cells (HEC) from lymphoid organs (peripheral lymph nodes (PLN) and Peyer's patches (PP)) was undertaken in order to study and characterize the cell surface molecules which are involved in lymphocyte recognition and allow homing. Cells were stimulated in vivo by a graft versus host (GVH) type of reaction before isolation and culture. The resulting adherent and growing cells were characterized as endothelial cells because of their typical aspect and their ability to produce angiotensin-converting enzyme and factor VIII-related antigen. They possess tissue-specific endothelial addressins. MECA 79 antigen is present on cells isolated from PLN while MECA 367 antigen is detected on cells from PP. Surface receptors for glycans were studied cytochemically using neoglycoproteins and fluorescence microscopy and quantified by flow cytometry experiments which showed that the specificity of sugar receptors depends upon endothelial cell origin. Indeed, sugar receptors for alpha-L-fucosyl residues were specifically expressed by endothelial cells from PLN. These receptors were inducible upon action of activated lymphocyte-conditioned medium. Further characterization of endothelial cells from peripheral lymph nodes indicates that they indeed mediate adhesion of lymphocytes in vitro. The role of protein-sugar interactions in this process was assessed by inhibition experiments performed with the help of neoglycoproteins. Best inhibitory effects were obtained when endothelial cells had been preincubated with alpha-L-fucosyl-BSA and when lymphoid cells were preincubated with beta-D-galactosyl-BSA. Concomitant inhibition assays indicate the participation of sugar specific receptors--endogenous lectins--on the surface of both endothelial and lymphoid cells to achieve recognition and adhesion.

Most mammalian macrophages express D-mannose-specific receptor (membrane lectin, Mr 175,000) allowing endocytosis of their ligands, but cells of the monocytic lineage (HL60, U937, monocyte) lack this receptor. However, after permeabilization, promyelocytic, promonocytic cells and monocytes bound fluoresceinylated D-mannose-terminated neoglycoproteins as evidenced by flow cytometry. Under these conditions, confocal analysis confirmed the intracellular membrane localization of the labeling and the absence of nuclear binding. An intracellular D-mannose-specific receptor was isolated from the human promyelocytic cell line HL60, by affinity chromatography on 4-isothiocyanatophenyl alpha-D-mannopyranoside-substituted Affi-gel as a 60,000-Mr membrane protein requiring divalent cations for the ligand binding. Under the same conditions, mouse macrophages were shown to express a 175,000-Mr D-mannose-specific receptor but not the 60,000-Mr receptor.

Suppression of the immune response, which involves suppressor factors released from specialized T cells, is inhibited by alpha-L-rhamnose. In this paper, we show the presence of rhamnose-specific receptors on a human CD8+ T cell-rich population and describe a novel method to isolate cells which express a given sugar-binding protein on their surface. We describe the isolation of alpha-L-rhamnose-specific molecules (rhamnose-binding fractions: RBF) from a water-soluble extract from lymphocytes, their purification by affinity chromatography on immobilized neoglycoproteins containing rhamnose residues. RBF kept their ability to bind rhamnose, as shown by the binding of fluorescein-labeled RBF to rhamnosylated BSA-substituted beads. RBF efficiently suppresses DNA synthesis of mitogen-stimulated human lymphocytes as well as B cell immunoglobulin production. Therefore, these rhamnose-binding molecules appear to be antigen-independent suppressor factors.

The nuclear sugar-binding components (i.e., lectin-like molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 h of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation.

Using gold labelled neoglycoproteins containing either alpha-D-glucose, N-acetyl-beta-D-glucosamine, alpha-D-mannose, 6-phospho-alpha-D-mannose, and alpha-L-fucose (BSA), we investigated their intranuclear binding sites in the TG human cell line. Although gold-labelled BSA did not give any noticeable labelling, the presence of 1% free BSA in the medium containing the gold labelled neoglycoproteins was revealed to be a key factor of the labelling. During interphase in the presence of free BSA most of the labelling was detected in the nucleoplasm. The border of the condensed chromatin, known to be the site of hnRNA synthesis as well as the interchromatin areas enriched in RNPs were labelled. Condensed chromatin also contained binding-sites. The nucleolus was seen to present low labelling in comparison with the labelling observed over the nucleoplasm. These nucleolar binding sites were located both in the dense fibrillar and granular components. No labelling could be detected over the fibrillar centers which are very conspicuous in this cell line. During mitosis sugar-binding sites were observed over the chromosomes. Data reported here show for the first time that lectin-like proteins and chromatin components are colocalized both during interphase and mitosis. In addition, within the nucleolus the presence of sugar-binding proteins was seen to be restricted to the dense fibrillar and granular components.