Carbohydrate specificities of the murine DC-SIGN homologue mSIGNR1

C-type lectins are important receptors expressed by antigen presenting cells that are involved in cellular communications as well as in pathogen uptake. An important C-type lectin family is represented by DC-SIGN and its homologues in human and mouse. Here we have investigated the carbohydrate specificity of cellular mSIGNR1 and compared it with DC-SIGN and L-SIGN. mSIGNR1 has a similar specificity as human DC-SIGN for high mannose-containing ligands present on both cellular and pathogen ligands. However, the DC-SIGN molecules differ in their recognition of Lewis antigens; mSIGNR1 interacts not only with Le(x/y) and Le(a/b) antigens similar to DC-SIGN, but also with sialylated Lex, a ligand for selectins. The differential recognition of Lewis antigens suggests differences between mSIGNR1 and DC-SIGN in the recognition of cellular ligands and pathogens that express Lewis epitopes.     

Binding sites for Lewis antigens are expressed by human colon cancer cells and negatively affect their migration

In colon cancer, endothelial cell selectins can promote tumor cell attachment via interactions with sialylated Lewis antigens present at the surface of tumor cells, thereby facilitating tumor cell arrest and transmigration into the extravascular space. However, it is not known whether Lewis antigens interact with colon tumor cells and modify their migration. Our aim was to detect the presence of binding sites on human tumor cells for Lewis(a/x) antigens and their sialylated derivatives in vitro and in vivo and to analyze their influence on migration of colon cancer cells. The immunocytochemical and histochemical levels of expression of the four Lewis antigens were quantitatively determined in four human colon cancer cell lines and in in vivo nude mice xenografts. The levels of expression of specific binding sites for these sugar epitopes were determined by synthetic neoglycoconjugates. The influence of binding of these carbohydrate ligands on cancer cell migration was quantitatively evaluated by computer-assisted phase-contrast videomicroscopy performed on Matrigel culture supports either left uncoated or coated with neoglycoconjugate presenting synthetic Lewis(a), sialyl Lewis(a), Lewis(x), or sialyl Lewis(x) antigens. The influence of the calcium concentration in the culture medium on the Lewis antigen-mediated effects was checked. Human colon cancer cells expressed significant amounts of specific binding sites detected by the synthetic probes in addition to the oligosaccharide epitopes. The expression levels differed considerably between the four cell lines and between in vitro and in vivo specimens. Cell migration analysis revealed that the four Lewis antigens markedly decreased the levels of migration of the HCT-15 and LoVo cancer cells. This effect depends on the calcium concentration in the culture medium. Binding sites for Lewis epitopes are present on colon cancer cells. The functional relevance of these sites is indicated by the negative influence on cell migration of a matrix containing the oligosaccharides as ligand parts.     

Avidin-binding to peripheral blood B lymphocytes and monocytes.

Avidin-coated magnetic beads bind peripheral blood B lymphocytes and monocytes. This unwanted reactivity is not due to the membrane expression of avidin target molecules since beads coated with a biotin-binding analogue are non-reactive and binding occurs even when cellular carbohydrate-binding sites are not active, in the absence of Mg2+ and Ca2+ cations, or when they are blocked by a alpha-D-glucose or alpha-D-mannose in presence of Ca2+ and Mg2+. The non-polar residues of avidin appear not to be engaged in a hydrophobic bond with the membrane molecule since suroptimal quantities of serum albumin do not prevent the avidin binding. It is suggested that ionic interactions explain the binding of avidin-coated beads to B lymphocytes and monocytes and that these can be inhibited with high molecular weight serum molecules or with 0.4 M NaCl.     

Epitope mapping on the dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) pathogen-attachment factor

DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin) is a myeloid pathogen-attachment factor C-type lectin which recognizes mannose- and fucose-containing oligosaccharide ligands on clinically relevant pathogens. Intracellular signaling initiated upon ligand engagement of DC-SIGN interferes with TLR-initiated signals, and modulates the T cell activating and polarizing ability of antigen-presenting cells. The C-terminal carbohydrate-recognition domain (CRD) of DC-SIGN is preceded by a neck domain composed of eight 23-residue repeats which mediate molecule multimerization, and whose polymorphism correlates with altered susceptibility to SARS and HIV infection. Naturally occurring isoforms and chimaeric molecules, in combination with established recognition properties, were used to define seven structural and functional epitopes on DC-SIGN. Three epitopes mapped to the CRD, one of which is multimerization-dependent and only exposed on DC-SIGN monomers. Epitopes within the neck domain were conformation-independent and unaltered upon molecule multimerization, but were differentially affected by neck domain truncations. Although neck-specific antibodies exhibited lower function-blocking ability, they were more efficient at inducing molecule internalization. Moreover, crosslinking of the different epitopes resulted in distinct levels of microclustering on the cell surface. The identification of independent epitopes on the DC-SIGN molecule might facilitate the design of reagents that modulate the T cell activating and polarizing ability of DC-SIGN-expressing cells without preventing its antigen- and pathogen-recognition capacities.     

High glucose disrupts oligosaccharide recognition function via competitive inhibition: A potential mechanism for immune dysregulation in diabetes mellitus

Diabetic complications include infection and cardiovascular disease. Within the immune system, host–pathogen and regulatory host–host interactions operate through binding of oligosaccharides by C-type lectin. A number of C-type lectins recognise oligosaccharides rich in mannose and fucose – sugars with similar structures to glucose. This raises the possibility that high glucose conditions in diabetes affect protein–oligosaccharide interactions via competitive inhibition. Mannose-binding lectin, soluble DC-SIGN and DC-SIGNR, and surfactant protein D, were tested for carbohydrate binding in the presence of glucose concentrations typical of diabetes, via surface plasmon resonance and affinity chromatography. Complement activation assays were performed in high glucose. DC-SIGN and DC-SIGNR expression in adipose tissues was examined via immunohistochemistry. High glucose inhibited C-type lectin binding to high-mannose glycoprotein and binding of DC-SIGN to fucosylated ligand (blood group B) was abrogated in high glucose. Complement activation via the lectin pathway was inhibited in high glucose and also in high trehalose – a nonreducing sugar with glucoside stereochemistry. DC-SIGN staining was seen on cells with DC morphology within omental and subcutaneous adipose tissues. We conclude that high glucose disrupts C-type lectin function, potentially illuminating new perspectives on susceptibility to infectious and inflammatory disease in diabetes. Mechanisms involve competitive inhibition of carbohydrate binding within sets of defined proteins, in contrast to broadly indiscriminate, irreversible glycation of proteins.     

Surface molecules involved in the adherence of recombinant interferon-gamma (rIFN-gamma)-stimulated human monocytes to vascular endothelial cells.

During an inflammatory reaction, an increased number of circulating monocytes adhere to the endothelial cells (EC) of the vessel wall. This process is mediated by molecules located on the surface of monocytes and EC. Locally released inflammatory mediators can modulate monocyte-EC interaction. In an earlier study we reported that stimulation of monolayers of cultured venous EC with rIFN-gamma enhanced their adhesiveness for monocytes. The aim of the present study was to investigate the effect of rIFN-gamma on peripheral blood monocytes with regard to the expression of surface molecules and the binding to non-stimulated or cytokine-stimulated EC. Flow cytometric analysis demonstrated that monocytes stimulated with 500 U/ml rIFN-gamma for 24 h showed increased expression of CR3 (CD11b/CD18), p150,95 (CD11c/CD18) and Fc gamma RI (CD64); the expression of LFA-1 (CD11a/CD18), L-selectin, CD14 and VLA-4 (CD49d/CD29) did not change or was slightly reduced. Upon stimulation with rIFN-gamma monocytes showed an enhanced binding to both non-stimulated or rIFN-gamma-stimulated EC. This was even more pronounced when EC had been stimulated with rIL-1 alpha for 24 h. The increased binding of rIFN-gamma-stimulated monocytes to rIL-1 alpha-stimulated EC was further analysed. Studies with MoAbs against adhesion molecules on monocytes revealed that the binding of rIFN-gamma-stimulated monocytes, but not that of non-stimulated monocytes, to rIL-1 alpha-stimulated EC was inhibited by about 30-60% with MoAbs against CD11a, CD11b, CD18, L-selectin or CD14. MoAbs against CD11c or CD49d had little or no effect. From these results, we conclude that exposure of monocytes to rIFN-gamma enhances their adhesiveness to cytokine-stimulated EC by a mechanism which involves CD11a/CD18, CD11b/CD18, CD14 and L-selectin on monocytes.     

Role of DC-SIGN in the activation of dendritic cells by HPV-16 L1 virus-like particle vaccine

Dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN), a specific C-type lectin expressed on DC, binds and transmits different pathogens to susceptible cells. In the present study, we examined the role of DC-SIGN in the capture of human papillomavirus (HPV) pseudovirions and activation of DC. We demonstrate that HPV virus-like particles (VLP) bind to DC-SIGN expressed on transfected Raji cells and that antibodies against DC-SIGN block this interaction. DC take up VLP, which activate expression of costimulatory markers and cytokines/chemokines. Although our results indicate that DC-SIGN is not the major receptor for VLP in DC, this interaction contributes to the activation of DC surface antigens (HLA class I) and of various cytokines/chemokines, particularly TNF-alpha, IL-6, and RANTES. Induction of these markers in DC by VLP was significantly abrogated when binding to DC-SIGN was blocked by anti-DC-SIGN antibodies. These results suggest that DC-SIGN has a functional role in DC activation induced by HPV-16 L1-VLP, and thus highlight new aspects of DC interactions with HPV VLP.     

Interactions of LSECtin and DC-SIGN/DC-SIGNR with viral ligands: Differential pH dependence, internalization and virion binding

The calcium-dependent lectins DC-SIGN and DC-SIGNR (collectively termed DC-SIGN/R) bind to high-mannose carbohydrates on a variety of viruses. In contrast, the related lectin LSECtin does not recognize mannose-rich glycans and interacts with a more restricted spectrum of viruses. Here, we analyzed whether these lectins differ in their mode of ligand engagement. LSECtin and DC-SIGNR, which we found to be co-expressed by liver, lymph node and bone marrow sinusoidal endothelial cells, bound to soluble Ebola virus glycoprotein (EBOV-GP) with comparable affinities. Similarly, LSECtin, DC-SIGN and the Langerhans cell-specific lectin Langerin readily bound to soluble human immunodeficiency virus type-1 (HIV-1) GP. However, only DC-SIGN captured HIV-1 particles, indicating that binding to soluble GP is not necessarily predictive of binding to virion-associated GP. Capture of EBOV-GP by LSECtin triggered ligand internalization, suggesting that LSECtin like DC-SIGN might function as an antigen uptake receptor. However, the intracellular fate of lectin-ligand complexes might differ. Thus, exposure to low-pH medium, which mimics the acidic luminal environment in endosomes/lysosomes, released ligand bound to DC-SIGN/R but had no effect on LSECtin interactions with ligand. Our results reveal important differences between pathogen capture by DC-SIGN/R and LSECtin and hint towards different biological functions of these lectins.     

Inhibition of DC-SIGN-mediated trans infection of T cells by mannose-binding lectin

Some dendritic cells (DC) express a cell-surface lectin called 'dendritic cell-specific intracellular adhesion molecule 3 (ICAM-3)-grabbing non-integrin' (DC-SIGN). DC-SIGN has been shown to mediate a type of infection called 'trans' infection, where DC bind human immunodeficiency virus (HIV) and efficiently transfer the virus to T cells. We investigated the possibility that mannose-binding lectin (MBL), a soluble lectin that functions as a recognition molecule in innate immunity and that binds to HIV, could block trans infection mediated by DC-SIGN. Binding studies with glycoprotein (gp)120/gp41-positive and -negative virus preparations suggested that DC-SIGN and MBL bind primarily to glycans on gp120/gp41, as opposed to glycans on host-cell-derived proteins, indicating a close overlap in the binding site of the two lectins and supporting the notion that MBL could prevent binding of HIV to DC-SIGN. Preincubation of X4, R5 or dual-tropic HIV strains with MBL prevented DC-SIGN-mediated trans infection of T cells. The mechanism of MBL blocking trans infection of T cells was at least partly caused by blocking of virus binding to DC-SIGN positive cells. This study shows that MBL prevents DC-SIGN-mediated trans infection of T cells in vitro and suggests that in infected persons, MBL may inhibit DC-SIGN-mediated uptake and spread of HIV.     

Human Schistosomiasis: Modulation of In Vitro Granulomatous Hypersensitivity and Lymphocyte Proliferative Response by Macrophages Undergoing Differentiation

The mechanisms associated with the modulation of immune response in the chronic phase of human schistosomiasis mansoni infection are complex and involve many cell types. In the present paper the authors demonstrate that antigenic stimulation of peripheral blood mononuclear cells (PBMC) from chronic-intestinal schistosomiasis mansoni patients with polyacrylamide beads (PB) conjugated to Schistosoma mansoni soluble egg antigens (PB-SEA) or adult worm antigen preparation (PB-SWAP) were able to induce a statistically significant increase on the in vitro multinucleated giant cell (MGC) formation after the 15th day in culture. A correlation between an increase in the number of MGC and a decrease in in vitro granuloma formation index to PB-SEA and PB-SWAP was observed. Moreover, the authors demonstrated a down-regulation of lymphocyte proliferative responses to S. mansoni antigens, during the differentiation pathway of monocytes towards MGC formation, due to a decrease in the antigen-presenting capacity of these cells. These phenomena also correlate with a concomitant decrease in the expression of HLA-DR and CD54 adhesion molecules on the surface of MGC. The results suggest that differentiation of monocytes to MGC may be one of the immunoregulatory mechanisms involved in the down-regulation of the granuloma reaction against S. mansoni eggs.     

Relevance of DC-SIGN in DC-induced T cell proliferation

The role of dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in DC-T cell communication was assessed by analyzing the effect of DC-SIGN-blocking mAb in MLR. The results show that the degree of inhibition by DC-SIGN and LFA-1 mAb depends on the magnitude of the MLR and the maturation status of the DC. Addition of DC-SIGN mAb at several time-points during MLR showed that DC-SIGN is involved early on in DC-T cell contacts. This initial role is masked by strong adhesive and costimulatory mechanisms, indicating a short-lived effect of DC-SIGN in DC-T cell interactions. To examine this concept in more detail, the percentage of PBL capable of binding DC-SIGN was determined. Analysis of several donors revealed that 1-20% PBL bind to beads coated with recombinant DC-SIGN, and the DC-SIGN-binding cells comprised all major cell subsets found in blood. PBL isolated from a donor with high DC-SIGN-binding capacity were more prone to blocking by DC-SIGN mAb in MLR than PBL from a donor with low DC-SIGN-binding capacity. This study indicates an initial and transient role for DC-SIGN in T cell proliferation, which becomes apparent when T cell proliferation is low and when the percentage of DC-SIGN binding PBL is high.     

Membrane expression and shedding of tumour necrosis factor receptors during activation of human blood monocytes: regulation by desferrioxamine.

Previous studies have shown that desferrioxamine (DFX), an iron chelator preventing the synthesis of hydroxyl radical (OH.), up-regulates the cell-surface expression of tumour necrosis factor-alpha (TNF-alpha) receptors on unactivated human blood monocytes. In the present study, we investigated the regulatory action of DFX on 125I-TNF-alpha binding to monocytes upon exposure to bacterial lipopolysaccharide (LPS). Exposure to LPS (1 microgram/ml) resulted in almost complete loss of 125I-TNF-alpha binding to the surface of monocytes. This down-regulation was reversible and the recovery observed after 18 hr was enhanced by addition of DFX (5 mM). However, binding studies on monocytes pre-exposed to low pH suggested that the DFX-induced increase of 125I-TNF-alpha binding was not due to differences in the number of receptors available but was probably due to a reduction of receptor occupancy by endogenously generated TNF-alpha. Time-course studies of TNF-alpha release from monocytes confirmed the ability of DFX to reduce the extracellular concentration of bioactive TNF-alpha through a decrease of its synthesis and an increase of its inactivation. The latter process was associated with an increased expression of the soluble form of TNF-alpha receptor type II. These results indicate that, in the presence of LPS, DFX increases the release of soluble TNF-alpha receptors from monocytes. Thus, conversely, OH. generated in situ could reduce the shedding of soluble TNF-alpha receptors and, hence, increase the widespread release of bioactive TNF-alpha.     

Redistribution of selectin counter-ligands induced by cytokines

Soluble recombinant (r) P-selectin and rP-selectin immobilized on plastic surfaces were tested for their capacity to activate neutrophils to produce superoxide anion. Soluble rP-selectin was incapable of activating leukocytes, whereas immobilized rP-selectin was able to induce leukocyte activation. When neutrophils were pretreated with a low dose of IL-8, granulocyte colony stimulating factor or granulocyte macrophage colony stimulating factor, soluble rP-selectin was no longer inert. These cytokine-primed leukocytes produced superoxide anion in the presence of soluble rP-selectin. During this priming period, sialyl Lewis X (sLe(X)) epitopes redistributed to one end of the leukocytes. Similar polarization of sLe(X) epitopes was observed at the attachment site of cells that adhered to immobilized rP-selectin. Cap formation and superoxide anion production induced by solid-phase P-selectin or by IL-8 and soluble rP-selectin treatment were inhibited by treatment of the leukocytes with cytochalasin B. These observations suggest that the redistribution of the carbohydrate ligands and the polarization of the leukocyte surface through an active process is a prerequisite but not sufficient to leukocyte superoxide production through P-selectin.     

Potency of HIV-1 envelope glycoprotein gp120 antibodies to inhibit the interaction of DC-SIGN with HIV-1 gp120

The interaction of DC-SIGN with gp120 provides an attractive target for intervention of HIV-1 transmission. Here, we have investigated the potency of gp120 antibodies to inhibit the DC-SIGN-gp120 interaction. We demonstrate that although the V3 loop is not essential for DC-SIGN binding, antibodies against the V3 loop partially inhibit DC-SIGN binding, suggesting that these antibodies sterically hinder DC-SIGN binding to gp120. Polyclonal antibodies raised against non-glycosylated gp120 inhibited both low and high avidity DC-SIGN-gp120 interactions in contrast to polyclonal antibodies raised against glycosylated gp120. Thus, glycans present on gp120 may prevent the generation of antibodies that block the DC-SIGN-gp120 interactions. Moreover, the polyclonal antibodies against non-glycosylated gp120 efficiently inhibited HIV-1 capture by both DC-SIGN transfectants and immature dendritic cells. Therefore, non-glycosylated gp120 may be an attractive immunogen to elicit gp120 antibodies that block the binding to DC-SIGN. Furthermore, we demonstrate that DC-SIGN binding to gp120 enhanced CD4 binding, suggesting that DC-SIGN induces conformational changes in gp120, which may provide new targets for neutralizing antibodies.     

Molecular mechanisms that set the stage for DC-T cell engagement

The unsurpassed capacity of dendritic cells (DC) to prime naive T cells is thought to depend on the formation of an immunological synapse. DC-SIGN, a C-type lectin exclusively expressed at the cell surface of DC, functions as an adhesion receptor facilitating T cell binding and priming through recognition of glycosylated ICAM-3 on naive T cells. Yet, DC-SIGN also mediates binding to pathogens such as HIV by recognizing glycosylated gp120. The scope of the present study was to investigate whether DC-SIGN upon recognition of its cellular ligand and pathogenic ligand affects DC synapse formation and activation/mobilization of other adhesion receptors such as LFA-1 to the cell contact site. Using a DC-SIGN deletion mutant, we show that DC-SIGN is a constitutively active receptor that mediates ligand binding independent of signaling through the cytoplasmic domain. Surprisingly, initial binding of gp120 to DC-SIGN did not result in increased adhesion levels of LFA-1 to its ligand ICAM-1 in both immature DC and Raji-DC-SIGN cells. However, ligand binding to DC-SIGN induced recruitment of LFA-1 to the adhesion site. Moreover, we could demonstrate that activation of LFA-1 results in DC-SIGN-LFA-1 co-clustering in the cell membrane. This triggers binding of ligands to LFA-1 that are shared with DC-SIGN, such as ICAM-3, but not of ligands that are not shared with DC-SIGN, such as ICAM-1. Thus, we propose that upon ligand binding DC-SIGN recruits LFA-1 to the contact site, resulting in the formation of DC-SIGN-LFA-1 co-clusters, in which the initial DC-SIGN-mediated interactions with ligand are transient and eventually shift to more stable LFA-1-dependent interactions.     

The carbohydrate recognition domain of Langerin reveals high structural similarity with the one of DC-SIGN but an additional, calcium-independent sugar-binding site

Langerin is a type II transmembrane oligosaccharide receptor on Langerhans cells (LCs), a prominent subclass of dendritic cells (DCs) that mediate immune responses in epithelia and play a role in HIV degradation. Its extracellular moiety comprises a neck region with several heptad repeats and an exposed carboxy-terminal calcium-type carbohydrate-recognition domain (CRD). The CRD of human Langerin, which was expressed as a soluble protein in the periplasm of E. coli, was crystallized both alone and in the presence of two sugars, followed by X-ray analyses to resolutions of 2.5A for apo-Langerin and to 1.6A and 2.1A for the complexes with mannose and maltose, respectively. The fold of the Langerin CRD (dubbed LangA) resembles that of other typical C-type lectins such as DC-SIGN. However, especially in the long loop region (LLR), which is responsible for carbohydrate-binding, two additional secondary structure elements are present: a 3(10) helix and a small beta-sheet arising from the extended beta-strand 2, which enters into a hairpin and a new strand beta2'. Unexpectedly, the crystal structures in the presence of maltose and mannose reveal two sugar-binding sites. One is calcium-dependent and structurally conserved in the C-type lectin family whereas the second one represents a novel, calcium-independent type. Based on these data, a model for the binding of mannan, a component of many endogenous as well as viral glycoproteins, is proposed and the differences in binding behavior between Langerin and DC-SIGN with respect to the Lewis X carbohydrate antigen and its derivatives can be explained. Therefore, the crystal structure of LangA should be helpful for the development of new marker reagents selective for LCs and also of therapeutic compounds that may enhance the inhibitory role of Langerin towards HIV infection.     

Epitopes on the S1 subunit of pertussis toxin recognized by monoclonal antibodies.

To identify the neutralizing epitopes on the S1 subunit (A promoter) of pertussis toxin, we characterized anti-S1 monoclonal antibodies (MAbs) X2X5, 3CX4, and 6FX1. We confirmed by immunoblot analysis that these MAbs bind to the S1 subunit and not to the B oligomer of pertussis toxin and that they recognize different epitopes by a competitive binding enzyme-linked immunosorbent assay. These MAbs had differential abilities to neutralize the lymphocytosis-promoting factor activity of pertussis toxin in mice: 3CX4 and 6FX1 had partial neutralizing abilities, while MAb X2X5 had none. With these MAbs, the epitopes on the S1 subunit were examined by using trypsinized S1 peptides, recombinant truncated S1 molecules, and synthetic peptides. The non-neutralizing MAb X2X5 bound in immunoblots to tryptic peptides of various sizes as small as 1.5 kilodaltons; the neutralizing MAbs 3CX4 and 6FX1 bound only to a 24-kilodalton tryptic peptide band. Immunoblot studies with recombinant truncated S1 molecules demonstrated that amino acid residues 7 to 14 and 15 to 26 play an important role in the binding of neutralizing MAbs and the non-neutralizing MAb, respectively. The binding of these MAbs was not dependent upon the presence of C-terminal amino acid residues 188 to 234. To further define B-cell epitopes, the binding of the MAbs we tested to synthetic peptides representing the entire S1 subunit were examined. Neutralizing MAbs 3CX4 and 6FX1 bound to none of these peptides, further suggesting that these MAbs recognize conformational epitopes. The non-neutralizing MAb X2X5 bound to peptides 11 to 26 and 16 to 30, demonstrating that the major antigenic determinant recognized by this MAb is a linear epitope located within residues 16 to 26.     

Mycobacterium bovis Bacillus Calmette-Guerin infects DC-SIGN- dendritic cell and causes the inhibition of IL-12 and the enhancement of IL-10 production

The only available vaccine against tuberculosis is Mycobacterium bovis Bacillus Calmette Guérin (BCG), although its efficacy in preventing pulmonary tuberculosis is controversial. Early interactions between dendritic cells (DC) and BCG or Mycobacterium tuberculosis (Mtb) are thought to be critical for mounting a protective antimycobacterial immune response. Recent studies have shown that BCG and Mtb target the DC-specific C-type lectin intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) to infect DC and inhibit their immunostimulatory function. This would occur through the interaction of the mycobacterial mannosylated lipoarabinomannan to DC-SIGN, which would prevent DC maturation and induce the immunosuppressive cytokine interleukin (IL)-10 synthesis. Here, we confirm that DC-SIGN is expressed in DC derived from monocytes cultured in granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4 and show that it is not expressed in DC derived from monocytes cultured in GM-CSF and interferon-alpha (IFN-alpha). We also demonstrate that DC-SIGN(-) DC cultured in GM-CSF and IFN-alpha are able to phagocytose BCG and to undergo a maturation program as well as DC-SIGN(+) DC cultured in IL-4 and GM-CSF. We also show that BCG causes the impairment of IL-12 and the induction of IL-10 secretion by DC, irrespective of DC-SIGN expression. Finally, we demonstrate that the capacity to stimulate a mixed leukocyte reaction of na?ve T lymphocytes is not altered by the treatment of both DC populations with BCG. These data suggest that DC-SIGN cannot be considered as the unique DC receptor for BCG internalization, and it is more interesting that the mycobacteria-induced immunosuppression cannot be attributed to the engagement of a single receptor.     

The role of tumor necrosis factor in the regulation of antigen presentation by human monocytes

Human peripheral blood monocytes pretreated with human recombinant tumour necrosis factor alpha (rTNF) showed an enhanced ability to present a soluble antigen, a purified protein derivate of tuberculin, to autologous T lymphocytes as assessed by their increased proliferation in vitro. This enhancing activity was due to TNF and not impurities in TNF preparations as anti-TNF antibodies abolished this phenomenon. The rTNF-treated monocytes showed an increased expression of HLA-DR molecules and enhanced co-stimulatory activity in the murine thymocyte assay. Pretreatment of monocytes before an antigen pulse with anti-TNF mAb inhibited antigen presentation, which indicated that endogenously produced TNF was involved. These studies thus suggest that TNF acts in an autocrine fashion and enhances the ability of monocytes to present protein antigen. It is unclear at present whether this effect is due to the modification in antigen processing, expression of MHC class II molecules, or other factors (IL-1, IL-6, adhesion molecules, etc.) that are important for the induction of T cell response to a nominal antigen. The enhancement of the antigen presenting capacity of monocytes/macrophages may be the additional mechanism of pro-inflammatory activity of TNF.