Interleukin-4-induced macrophage fusion is prevented by inhibitors of mannose receptor activity

A potential role for the macrophage mannose receptor in human monocyte-derived macrophage fusion was explored by testing the effects of previously described inhibitors of its activity on the formation of interleukin-4-induced foreign body giant cells in vitro Giant cell formation was prevented or reduced in the presence of alpha-man-nan and synthetic neoglycoprotein conjugates according to the following pattern of relative inhibition: mannose-bovine serum albumin (BSA) > N-acetylgucosamine-BSA congruent to glucose-BSA. Laminarin (beta-glucan) or galactose-BSA were not inhibitory. Swainsonine and castanospermine, inhibitors of glycoprotein processing that interfere with the arrival of newly synthesized mannose receptors at the cell surface, also attenuated macrophage fusion and the formation of giant cells, whereas another glycosidase inhibitor, deoxymannojirimycin, was without effect. Mannose receptors were confirmed to be specifically up-regulated by interleukin-4 in this culture system and also demonstrated to be present and concentrated at macrophage fusion interfaces. These data suggest that the macrophage mannose receptor may be an essential participant in the mechanism of interleukin-4-induced macrophage fusion and implicate a novel function for this endocytic/phagocytic receptor in mediating foreign body giant cell formation at sites of chronic inflammation.     

IL-10 prevents the differentiation of monocytes to dendritic cells but promotes their maturation to macrophages

Human monocytes cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-13 for 7 days differentiate into cells with the morphology and function of dendritic cells (DC). We have investigated the effect of IL-10 on this differentiation pathway. In the presence of IL-10 cells did not develop DC morphology, did not express CD1a and had lower levels of MHC class II. IL-10 promoted the differentiation of large cells with the morphology, cytochemistry and membrane phenotype of macrophages, including staining for nonspecific esterase and high levels of CD14, CD16 and CD68. The effect of IL-10 was dose dependent and was best appreciated when the cytokine was added at the initiation of the culture, as addition on day 3 was less inhibitory. When added to already differentiated DC on day 6, IL-10 caused only a modest reduction of MHC class II and CD1a expression, and no acquisition of the macrophage markers CD14, CD16 and CD68. Prolonged incubation up to 5 days with IL-10 did not induce a shift of differentiated DC to macrophages. On the other hand, the macrophages obtained by culturing for 7 days with GM-CSF+IL-13+IL-10 did not shift to DC upon removal of IL-10 for up to 3 days. Thus, the effect of IL-10 on monocyte differentiation, occurs only at the precursor level and confers an irreversible phenotype. From a functional point of view, cells cultured in the presence of IL-10 were poor stimulators of allogeneic cord blood T cells in mixed lymphocyte reaction (MLR) and presented tetanus toxin (TT) to specific T cell lines with much less efficiency than control DC. In contrast, IL-10-cultured DC showed 7 times greater endocytosis of FITC-dextran. This increased endocytosis was mostly mediated via the mannose receptor, as demonstrated by blocking with unlabeled mannose. In conclusion, IL-10 inhibits DC differentiation from monocytes and, in a substantial proportion of the cells, promotes the differentiation to mature macrophages. Intriguingly, IL-10 inhibits antigen presentation while it stimulates endocytic activity.     

Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products

We have previously demonstrated that human peripheral blood low density mononuclear cells cultured in granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 develop into dendritic cells (DCs) that are extremely efficient in presenting soluble antigens to T cells. To identify the mechanisms responsible for efficient antigen capture, we studied the endocytic capacity of DCs using fluorescein isothiocyanate-dextran, horseradish peroxidase, and lucifer yellow. We found that DCs use two distinct mechanisms for antigen capture. The first is a high level of fluid phase uptake via macropinocytosis. In contrast to what has been found with other cell types, macropinocytosis in DCs is constitutive and allows continuous internalization of large volumes of fluid. The second mechanism of capture is mediated via the mannose receptor (MR), which is expressed at high levels on DCs. At low ligand concentrations, the MR can deliver a large number of ligands to the cell in successive rounds. Thus, while macropinocytosis endows DCs with a high capacity, nonsaturable mechanism for capture of any soluble antigen, the MR gives an extra capacity for antigen capture with some degree of selectivity for non-self molecules. In addition to their high endocytic capacity, DCs from GM-CSF + IL-4-dependent cultures are characterized by the presence of a large intracellular compartment that contains high levels of class II molecules, cathepsin D, and lysosomal-associated membrane protein-1, and is rapidly accessible to endocytic markers. We investigated whether the capacity of DCs to capture and process antigen could be modulated by exogenous stimuli. We found that DCs respond to tumor necrosis factor alpha, CD40 ligand, IL-1, and lipopolysaccharide with a coordinate series of changes that include downregulation of macropinocytosis and Fc receptors, disappearance of the class II compartment, and upregulation of adhesion and costimulatory molecules. These changes occur within 1-2 d and are irreversible, since neither pinocytosis nor the class II compartment are recovered when the maturation-inducing stimulus is removed. The specificity of the MR and the capacity to respond to inflammatory stimuli maximize the capacity of DCs to present infectious non-self antigens to T cells.     

Endocytosis during postnatal differentiation in superficial cells of the mouse urinary bladder epithelium

This electron microscopical study was performed in order to follow the endocytic pathway of horseradish peroxidase and colloidal gold tracers and to determine the involvement of endocytosis in postnatal differentiation in superficial cells of the mouse urinary bladder epithelium. Morphometric analyses of late endosomes/multivesicular bodies from day of birth to day 25 were performed. The internalisation and intracellular transport of luminal plasmalemma to multivesicular bodies via endocytic vesicles, early endosomes and pleomorphic compartments was established. Dynamic changes in endocytic activity took place within the first few days of postnatal differentiation. During this period the number of multivesicular bodies changed in an inverse ratio to their size. After the third day endocytic activity gradually approached the low rate of adult urothelium.     

Paclitaxel and nocodazole differentially alter endocytosis in cultured cells

PURPOSE: Microtubule-based transport facilitates the endocytosis of exogenous macromolecules. We have determined how microtubule accumulation and disassembly alter endocytosis. METHODS: The effects of paclitaxel, which promotes microtubule assembly, and nocodazole, which promotes microtubule disassembly, on fluid-phase and receptor-mediated endocytosis were measured using uptake of horseradish peroxidase and 125I-transferrin, respectively. Changes in membrane and microtubule organization were examined by fluorescence microscopy. RESULTS: Neither paclitaxel (4 microM, 60 min pretreatment) nor nocodazole (1 microgram/ml, 60 min pretreatment) significantly inhibited fluid-phase endocytosis. However, paclitaxel caused a redistribution of fluorescent fluid-phase marker to the periphery. Both paclitaxel and nocodazole treatment significantly (p < or = 0.05) reduced the initial uptake of 125I-transferrin at 5 min to approximately 50% of control. Despite the similarity of the effects on initial endocytic uptake, the effects on steady state accumulation of 125I-transferrin were quite distinct. Exposure of CV-1 cells to paclitaxel for an additional 30, 60 or 90 min also showed reduced accumulation of 125I-transferrin up to a maximum significant (p < or = 0.05) inhibition of 48% +/- 10% of control at 90 min. In contrast, nocodazole caused an initial significant (p < or = 0.05) increase in 125I-transferrin accumulation after 30 min (159% +/- 13% of control), while by 90 min 125I-transferrin accumulation had returned to control levels. Microtubule content, particularly of stable microtubules, was increased in CV-1 cells by paclitaxel, but abolished by nocodazole treatment. CONCLUSIONS: Our data show that changes in the microtubule array can alter the dynamics of receptor movement through the endosomal pathway. However, microtubule assembly versus disassembly have different effects.     

A novel macrophage receptor enhances MHC II-peptide loading and surface expression of a hapten-protein conjugate

A receptor possessing specificity for fluorescein was previously identified on murine macrophage. The goal of the present study was to determine if this receptor influenced MHC II-peptide loading and surface expression of a hapten-protein conjugate within murine macrophage. Although inhibition of fluid-phase pinocytosis had no detectable effect, lower levels of intracellular MHC II-peptide complexes were observed upon inhibition of receptor-mediated endocytosis. Moreover, lower levels of surface expressed MHC II-fluoresceinated peptide complexes were also detected. Following subcellular fractionation experiments, it was revealed that the receptor altered the endocytic trafficking of the antigen within the cell. Namely, degraded antigen and MHC II-peptide complexes were not observed in dense transferrin receptor positive, cathepsin D positive, LAMP-1 positive organelles upon inhibition of the receptor. Previous studies also suggested that this receptor enhanced MHC II-peptide loading by concentrating high levels of antigen to endocytic organelles. The implications of these findings on subsequent development of the immune response were also discussed.     

Further studies on the endocytic activity of Tritrichomonas foetus

The endocytic activity of Tritrichomonas foetus was studied at the ultrastructural level using gold-labeled macromolecules (bovine lactoferrin, human and bovine transferrin, bovine albumin, human low-density lipoprotein, horseradish peroxidase, and protein A). All macromolecules were ingested by the protozoan. Binding experiments showed that only bovine lactoferrin bound to the parasite surface in a process that could be inhibited by the unlabeled protein, suggesting that it binds and is internalized via receptors. Label-fracture experiments showed that the receptors were distributed in clusters that did not colocalize with intramembranous particles. Kinetics analysis of the internalization of bovine lactoferrin and horseradish peroxidase, associated with the cytochemical detection of acid phosphatase, revealed that proteins were rapidly ingested through small uncoated vesicles and delivered to acid phosphatase-containing compartments. The colocalization of gold-labeled proteins and reaction product indicative of enzyme activity was confirmed by electron spectroscopic imaging. Simultaneous incubation of cells in the presence of two proteins labeled with gold particles of different diameters showed that they were ingested through the same pathway and were concentrated into cytoplasmic vacuoles corresponding to lysosome-like organelles. These data suggest that the endocytic process in T. foetus is very rapid and that the intracellular pathway for receptor-mediated and fluid-phase endocytosis seems to be the same.     

Intracellular routes and selective retention of antigens in mildly acidic cathepsin D/lysosome-associated membrane protein-1/MHC class II-positive vesicles in immature dendritic cells

Immature dendritic cells (DC) use both macropinocytosis and mannose receptor-mediated endocytosis to internalize soluble Ags efficiently. These Ags are ultimately presented to T cells after DC maturation and migration into the lymph nodes. We have previously described the immortalized myeloid cell line FSDC as displaying the characteristics of early DC precursors that efficiently internalize soluble Ags. To describe the different routes of Ag uptake and to identify the Ag retention compartments in FSDC, we followed the intracellular fate of FITC-coupled OVA, dextran (DX), transferrin, and Lucifer Yellow using flow cytometry, confocal microscopy, and immunoelectron microscopy. OVA and DX gained access into macropinosomes and early endosomes. DX was preferentially sorted into endosomal compartments, while most of the OVA entered macropinosomes via fluid phase uptake. We found a long-lasting retention of DX and OVA of up to 24 h. After 6 h of chase, these two molecules were concentrated in common vesicular compartments. These retention compartments were distinct from endosomes and lysosomes; they were much larger, only mildly acidic, and lacked the small GTP binding protein rab7. However, they were positive for lysosome-associated membrane protein-1, the protease cathepsin D, and MHC class II molecules, thus representing matured macropinosomes. These data suggest that the activity of vacuolar proteases is reduced at the mildly acidic pH of these vesicles, which explains their specific retention of an Ag. The retention compartments might be used by nonlymphoid tissue DC to store peripheral Ags during their migration to the lymph node.     

Expression of maturation-/migration-related molecules on human dendritic cells from blood and skin

Progress in dendritic cell research has been overwhelming in the past few years. This was made possible by the recent development of simple methods to generate large numbers of dendritic cells. These methods use as starting populations for culture either CD34+ progenitor cells from cord blood or bone marrow, or monocytes from peripheral blood. The latter approach is critically dependent on the combination of GM-CSF and interleukin 4. Such "priming cultures" yield populations of immature dendritic cells (CD83-/CD86 +/- /CD115+/antigen uptake high/antigen processing high/T cell sensitization low). In order to generate mature dendritic cells a subsequent "differentiation culture" has to be added whereby monocyte-conditioned medium appears to be the optimal stimulus for maturation. This results in terminally mature dendritic cells (CD83+/CD86++/CD115-/antigen uptake low/antigen processing low/T cell sensitization high). We investigated the expression of some molecules involved in maturation and migration on human monocyte-derived dendritic cells from blood in comparison with dermal dendritic cells and epidermal Langerhans cells. We present a method to highly enrich epidermal Langerhans cells. Survival of purified Langerhans cells in culture is dependent on the presence of GM-CSF and TNF-alpha. During maturation a substantial part of the Langerhans cells loses expression of the cutaneous lymphocyte antigen (CLA); mature dendritic cells from the dermis are completely devoid of CLA. Similarly, CLA as well as CD15s (Sialyl Lewis x) and CD31 (PECAM-1) that can be readily detected on immature monocyte-derived dendritic cells are down-regulated upon maturation. CD68 expression is very low in cutaneous dendritic cells; in monocyte-derived dendritic cells this molecule is abundantly present. Subsets of monocyte-derived dendritic cells express E-cadherin; CD87 (urokinase plasminogen activator receptor) is weakly expressed on both immature and mature monocyte-derived dendritic cells. Taken together, these data suggest that the phenotype of monocyte-derived dendritic cells (E-cadherin low to negative, CD68++) is not indicative for a cutaneous destiny. Furthermore, the downregulation upon maturation of molecules involved in migration through vessel walls (CD31, CLA, CD15s) indicates that the entry of mature dendritic cells into lymphatic vessels may not be as rigidly regulated by adhesion molecules as the process of extravasation from blood vessels.     

Interleukin-10 in non-Hodgkin's lymphoma

Interleukin-10 (IL-10) is a pleiotropic cytokine produced by type 2 helper cells (Th2), as well as by monocytes and macrophages, and normal and neoplastic B lymphocytes. It is highly homologous to an open reading frame of Epstein-Barr virus (EBV) called BCRF1, and EBV infection of B-cells up-regulates IL-10. IL-10 production has strong immunosuppressive effects via inhibition of Th1 type cytokines, including interferon gamma and interleukin-2. On B-cells, IL-10 has a potent stimulating effect, inducing proliferation and differentiation. Interestingly, in cell lines derived from B-cell lymphomas, IL-10 production has been found to be up-regulated, and it serves as an autocrine growth factor. In patients with non-Hodgkin's lymphoma (NHL), serum IL-10 levels are significantly increased when compared to normal individuals and NHL patients in remission. The prognostic significance of these increased levels vary according to the assay used. Both human IL-10 and viral IL-10 are increased, and when specific assays for human IL-10 are used, there seems to be no prognostic significance, whereas when the assay cross-reacts with viral IL-10, high levels correlate with poor prognosis. These results suggest that viral IL-10 might have some pathogenic role in NHL.     

The monomeric guanosine triphosphatase rab4 controls an essential step on the pathway of receptor-mediated antigen processing in B cells

Each member of the rab guanosine triphosphatase protein family assists in the regulation of a specific step within the biosynthetic or endocytic pathways. We have found that the early endosome-associated rab4 protein controls a step critical for receptor-mediated antigen processing in a murine A20 B cell line. Expression of the dominant negative rab4N121I mutant dramatically inhibited the processing and presentation of ovalbumin, lambda cI repressor, or rabbit immunoglobulin G internalized as antigens by B cell antigen receptors or transfected Fc receptors. This defect did not reflect a block in antigen endocytosis or degradation, and transfected cells remained completely capable of presenting exogenously added ovalbumin and lambda repressor peptides. Most remarkably, rab4N121I-expressing cells were undiminished in their ability to present each of these antigens when whole proteins were internalized at high concentration by fluid-phase endocytosis. Thus, expression of the rab4N121I selectively inactivated a portion of the endocytic pathway required for the processing of receptor-bound, but not nonspecifically internalized, antigens. These results suggest that elements of the early endosome-recycling pathway play an important and selective role in physiologically relevant forms of antigen processing in B cells.     

Expression of a mannose/fucose membrane lectin on human dendritic cells

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.     

Effect of bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: implications for viral uncoating and infection

Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used 35S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713-3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.     

Apoptosis of cardiac myocytes in Gsalpha transgenic mice

We examined the potential involvement of two CC chemokine receptors (CCRs), CCR-1 and CCR-3, in the functional activation of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4)-generated human peripheral blood monocyte-derived immature dendritic cells (DCs). Flow cytometric analysis showed that CCR-1, CCR-3, CCR-5, and CXC chemokine receptor (CXCR)-4 were expressed on the cell surface of monocyte-derived DCs. Treatment with a monoclonal antibody (MoAb) to either CCR-1 or CCR-3 but not MoAbs to CCR-5 and CXCR-4 abolished chemotactic migration of monocyte-derived DCs. The DCs treated with either the anti-CCR-1 MoAb or anti-CCR-3 MoAb were less efficient than untreated DCs in proliferation of allogeneic T cells (TCs) and TC-derived secretion of interferon-gamma (IFN-gamma). The homotypic aggregation of DCs and heterotypic aggregation of DCs with TCs were suppressed by the anti-CCR-1 MoAb or anti-CCR-3 MoAb. These results indicate that CCR-1 and CCR-3 specifically regulate interaction of TCs and DCs in the process of antigen presentation.     

Effective treatment of mice with type II collagen induced arthritis with lethal irradiation and bone marrow transplantation

Ricin A-chain is delivered into macrophages via receptor-mediated endocytosis. We have found that following uptake via the mannose receptor, ricin A-chain is rapidly cleaved by endosomal proteases. Inhibition of endosomal proteases such as cathepsin D and B leads to the accumulation of toxin inside the cell. Inhibition of cathepsin D reduces ricin A-chain cytotoxicity, while blocking cathepsin B enhances cytotoxicity. Similar results were obtained using fibroblasts transfected with the mannose receptor. Our data strongly suggest that the activation or membrane translocation of ricin A-chain is dependent upon the action of specific proteases.     

Reduced binding and phagocytosis of Pneumocystis carinii by alveolar macrophages from persons infected with HIV-1 correlates with mannose receptor downregulation

The macrophage mannose receptor, a pattern recognition molecule and component of innate immunity, mediates binding and phagocytosis of Pneumocystis carinii and likely represents an important clearance mechanism in the lungs of immunocompetent hosts. The purpose of this study was to examine the ability of alveolar macrophages from HIV-infected individuals to bind and phagocytose P. carinii, and to investigate the role of the macrophage mannose receptor in mediating this interaction. Compared with healthy individuals, alveolar macrophage phagocytosis of P. carinii from HIV+ persons was reduced up to 74% (P = 0.02), primarily reflecting a reduction in the number of organisms associated with each macrophage (P = 0.019). Furthermore, macrophages from HIV+ individuals demonstrated up to an 80% (P < 0.05) reduction in mannose receptor surface expression and endocytosis. Mannose receptor affinity was unaltered, and mRNA levels were modestly reduced (P < 0.05). Cells from HIV+ individuals with CD4(+) counts < 200 cells/mm3 (representing individuals at high clinical risk for P. carinii pneumonia) demonstrated the lowest levels of P. carinii phagocytosis and mannose receptor endocytosis. In vitro HIV infection of alveolar macrophages from healthy individuals reduced mannose receptor endocytosis to 53.2% (P < 0.05) and P. carinii binding and phagocytosis to 67.4% (P < 0.05) of control. Our studies suggest that HIV infection may alter innate immunity in the lungs, and that impaired alveolar macrophage mannose receptor-mediated binding and phagocytosis of P. carinii may contribute to the susceptibility of HIV-infected individuals to this opportunistic pulmonary pathogen.     

Regulated endocytosis of G-protein-coupled receptors by a biochemically and functionally distinct subpopulation of clathrin-coated pits

beta-2 Adrenergic receptors (B2ARs) are endocytosed by clathrin-coated pits. This process serves specialized functions in signal transduction and receptor regulation, raising the question of whether B2ARs are associated with biochemically specialized membrane vesicles during their endocytic trafficking. Here we show that B2ARs are endocytosed by a distinct subpopulation of clathrin-coated pits, which represent a limited subset of coated pits present in the plasma membrane, even in cells overexpressing both B2ARs and beta-arrestin. Coated pits mediating agonist-induced endocytosis of B2ARs differ from other coated pits mediating constitutive endocytosis of transferrin receptors in their temperature dependence for fission from the plasma membrane and in the association of their membrane coats with beta-arrestin. Endocytosis of these coated pits generates endocytic vesicles selectively enriched in B2ARs, which fuse within approximately 10 min after their formation with a common population of endosomes containing both B2ARs and transferrin receptors. These observations demonstrate, for the first time, the existence of a functionally and biochemically distinct subpopulation of clathrin-coated pits that mediate the agonist-regulated endocytosis of G-protein-coupled receptors, and they suggest a new model for the formation of compositionally specialized membrane vesicles at the earliest stage of the endocytic pathway.     

Coronary arteries: breath-hold, gadolinium-enhanced, three-dimensional MR angiography

Cellular retinoic acid binding proteins (CRABP) are low molecular weight proteins whose precise function remains unknown. They bind retinoids and may thereby modulate the intracellular steady-state concentration of retinoids. Whereas CRABP I is ubiquitously expressed, CRABP II is mainly detected in various cell types of the skin. By representative difference analysis we found that CRABP II is also strongly expressed in human monocyte-derived macrophages (MAC) but not in freshly isolated monocytes (MO). The CRABP II mRNA was gradually upregulated during differentiation from MO to MAC in the presence of 2% serum. Adherence, which is important for MO differentiation, induced CRABP II expression, but the addition of 10(-7) M retinoic acid inhibited the upregulation of CRABP II expression during MO/MAC differentiation. As MO can differentiate along the classical pathway not only to MAC but also to dendritic cells we analyzed the expression of CRABP II in MO-derived dendritic cells cultured with 10% FCS, IL-4, and GM-CSF. In contrast to MAC, MO-derived dendritic cells showed an extremely low expression of CRABP II. From these results we conclude (1) that the availability and the metabolism of retinoids may be different in MAC compared to MO and dendritic cells and (2) that this may influence differentiation and activation of those cells.     

The gp200-MR6 molecule which is functionally associated with the IL-4 receptor modulates B cell phenotype and is a novel member of the human macrophage mannose receptor family

The human gp200-MR6 molecule has previously been shown to have either an antagonistic or agonistic effect on IL-4 function, demonstrated by inhibition of IL-4-induced proliferation of T cells or mimicking of IL-4-induced maturation of epithelium, respectively. We now show that gp200-MR6 ligation can also mimic IL-4 and have an anti-proliferative pro-maturational influence within the immune system, causing up-regulation of co-stimulatory molecules on B lymphocytes. Biochemical analysis and cDNA cloning reveal that gp200-MR6 belongs to the human macrophage mannose receptor family of multidomain molecules. It comprises 1722 amino acids in toto (mature protein, 1695 amino acids; signal sequence, 27 amino acids) organized into 12 external domains (an N-terminal cysteine-rich domain, a fibronectin type II domain and 10 C-type carbohydrate recognition domains), a transmembrane region and a small cytoplasmic C terminus (31 amino acids) containing a single tyrosine residue (Y1679), but no obvious kinase domain. Strong amino acid sequence identity (77%) suggests that gp200-MR6 is the human homologue of the murine DEC-205, indicating that this molecule has much wider functional activity than its classical endocytic role. We also show that the gp200-MR6 molecule is closely associated with tyrosine kinase activity; the link between gp200-MR6 and the IL-4 receptor may therefore be via intracellular signaling pathways, with multifunctionality residing in its extracellular multidomain structure.