Critical role of mac-1 sialyl lewis x moieties in regulating neutrophil degranulation and transmigration

Leukocyte cell surface sialyl Lewis x (sLe^x) and related epitopes play an important role in cell rolling and adhesion during diapedesis via interaction with E-selectin. Here, we present evidence that Mac-1 (CD11b/CD18, CR-3) is a major neutrophil glycoprotein decorated with sLe^x and ligation of these carbohydrate moieties by anti-sLe^x antibody significantly impairs neutrophil functions. First, Western blot analysis shows that both CD11b and CD18 subunit of purified Mac-1 are decorated with sLe^x moieties. A significant co-localization of CD11b and sLe^x moieties is observed at neutrophil secondary granules. With stimulation of formyl-Met-Leu-Phe (fMLP), neutrophil surface labeling with anti-sLe^x antibody follows an identical up-regulation pattern of Mac-1. Second, protein-binding assays indicate that sLe^x moieties on Mac-1 are critical for binding interaction of Mac-1 to E-selectin. Removal of sLe^x moieties completely abolishes Mac-1-E-selectin binding. Finally, ligation of Mac-1 sLe^x by anti-sLe^x antibody induces a significant degranulation of neutrophil secondary granules at the absence of chemoattractant stimulation. This “dysregulated” degranulation induced by anti-sLe^x antibody strongly inhibits neutrophil transmigration in response to fMLP. In summary, Mac-1 sLe^x moieties play a critical role in regulating β₂ integrin functions during neutrophil transmigration and degranulation.     

Novel Antibodies Reactive with Sialyl Lewis X in Both Humans and Mice Define Its Critical Role in Leukocyte Trafficking and Contact Hypersensitivity Responses

Sialyl Lewis X (sLe^x) antigen functions as a common carbohydrate determinant recognized by all three members of the selectin family. However, its expression and function in mice remain undefined due to the poor reactivity of conventional anti-sLe^x monoclonal antibodies (mAbs) with mouse tissues. Here, we developed novel anti-sLe^x mAbs, termed F1 and F2, which react well with both human and mouse sLe^x, by immunizing fucosyltransferase (FucT)-IV and FucT-VII doubly deficient mice with 6-sulfo-sLe^x-expressing cells transiently transfected with an expression vector encoding CMP-N-acetylneuraminic acid hydroxylase. F1 and F2 specifically bound both the N-acetyl and the N-glycolyl forms of sLe^x as well as 6-sulfo-sLe^x, a major ligand for L-selectin expressed in high endothelial venules, and efficiently blocked physiological lymphocyte homing to lymph nodes in mice. Importantly, both of the mAbs inhibited contact hypersensitivity responses not only when administered in the L-selectin-dependent sensitization phase but also when administered in the elicitation phase in mice. When administered in the latter phase, F1 and F2 efficiently blocked rolling of mouse leukocytes along blood vessels expressing P- and E-selectin in the auricular skin in vivo. Consistent with these findings, the mAbs blocked P- and E-selectin-dependent leukocyte rolling in a flow chamber assay. Taken together, these results indicate that novel anti-sLe^x mAbs reactive with both human and mouse tissues, with the blocking ability against leukocyte trafficking mediated by all three selectins, have been established. These mAbs should be useful in determining the role of sLe^x antigen under physiological and pathological conditions.     

Amyloid-β Peptide on Sialyl-LewisX-Selectin-Mediated Membrane Tether Mechanics at the Cerebral Endothelial Cell Surface

Increased deposition of amyloid-β peptide (Aβ) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimer''s disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized by sialyl-Lewis^x (sLe^x) were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLe^x and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (F_mtf), and produced a bimodal population of F_mtf, suggesting subcellular mechanical alterations in membrane tethering. The lower F_mtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower F_mtf. In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB.     

Metabolic Inhibition of Sialyl-Lewis X Biosynthesis by 5-Thiofucose Remodels the Cell Surface and Impairs Selectin-Mediated Cell Adhesion

Sialyl-Lewis X (sLe^X) is a tetrasaccharide that serves as a ligand for the set of cell adhesion proteins known as selectins. This interaction enables adhesion of leukocytes and cancer cells to endothelial cells within capillaries, resulting in their extravasation into tissues. The last step in sLe^X biosynthesis is the α1,3-fucosyltrasferase (FUT)-catalyzed transfer of an L-fucose residue to carbohydrate acceptors. Impairing FUT activity compromises leukocyte homing to sites of inflammation and renders cancer cells less malignant. Inhibition of FUTs is, consequently, of great interest, but efforts to generate glycosyltransferase inhibitors, including FUT inhibitors, has proven challenging. Here we describe a metabolic engineering strategy to inhibit the biosynthesis of sLe^X in cancer cells using peracetylated 5-thio-L-fucose (5T-Fuc). We show that 5T-Fuc is taken up by cancer cells and then converted into a sugar nucleotide analog, GDP-5T-Fuc, that blocks FUT activity and limits sLe^X presentation on HepG2 cells with an EC₅₀ in the low micromolar range. GDP-5T-Fuc itself does not get transferred by either FUT3 or FUT7 at a measurable rate. We further demonstrate that treatment of cells with 5T-Fuc impaired their adhesive properties to immobilized adhesion molecules and human endothelial cells. 5T-Fuc, therefore, is a useful probe that can be used to modulate sLe^X levels in cells to evaluate the consequences of inhibiting FUT-mediated sLe^X formation. These data also reveal the utility of using sugar analogues that lead to formation of donor substrate analogues within cells as a general approach to blocking glycosyltransferases in cells.     

A Novel Function of CD82/KAI1 in Sialyl Lewis Antigen-Mediated Adhesion of Cancer Cells: Evidence for an Anti-Metastasis Effect by Down-Regulation of Sialyl Lewis Antigens

We have recently elucidated a novel function for CD82 in E-cadherin-mediated homocellular adhesion; due to this function, it can inhibit cancer cell dissociation from the primary cancer nest and limit metastasis. However, the effect of CD82 on selectin ligand-mediated heterocellular adhesion has not yet been elucidated. In this study, we focused on the effects of the metastasis suppressor CD82/KAI1 on heterocellular adhesion of cancer cells to the endothelium of blood vessels in order to further elucidate the function of tetraspanins. The over-expression of CD82 in cancer cells led to the inhibition of experimentally induced lung metastases in mice and significantly inhibited the adhesion of these cells to human umbilical vein epithelial cells (HUVECs) in vitro. Pre-treatment of the cells with function-perturbing antibodies against sLe^a/x significantly inhibited the adhesion of CD82-negative cells to HUVECs. In addition, cells over-expressing CD82 exhibited reduced expression of sLe^a/x compared to CD82-negative wild-type cells. Significant down-regulation of ST3 β-galactoside α-2, 3-sialyltransferase 4 (ST3GAL4) was detected by cDNA microarray, real-time PCR, and western blotting analyses. Knockdown of ST3GAL4 on CD82-negative wild-type cells inhibited expression of sLe^x and reduced cell adhesion to HUVECs. We concluded that CD82 decreases sLe^a/x expression via the down-regulation of ST3GAL4 expression and thereby reduces the adhesion of cancer cells to blood vessels, which results in inhibition of metastasis.     

L-Selectin Ligands That Are O-glycoprotease Resistant and Distinct from MECA-79 Antigen are Sufficient for Tethering and Rolling of Lymphocytes on Human High Endothelial Venules

During the process of lymphocyte recirculation, lymphocytes bind via L-selectin to sulfated sialyl-Lewis^x (sLe^x)–containing carbohydrate ligands expressed on the surface of high endothelial venules (HEV). We have examined the expression of sLe^x on HEV using a panel of mAbs specific for sLe^x and sLe^x-related structures, and have examined the function of different sLe^x-bearing structures using an in vitro assay of lymphocyte rolling on HEV. We report that three sLe^x mAbs, 2F3, 2H5, and CSLEX-1, previously noted to bind with high affinity to glycolipid-linked sLe^x, vary in their ability to stain HEV in different lymphoid tissues and bind differentially to O-linked versus N-linked sLe^x on glycoproteins. Treatment of tissue sections with neuraminidase abolished staining with all three mAbs but slightly increased staining with MECA-79, a mAb to a sulfation-dependent HEV-associated carbohydrate determinant. Treatment of tissue sections with O-sialoglycoprotease under conditions that removed the vast majority of MECA-79 staining, only partially reduced staining with the 2F3 and 2H5 mAbs. Using a novel rolling assay in which cells bind under flow to HEV of frozen tissue sections, we demonstrate that a pool of O-sialoglycoprotease–resistant molecules is present on HEV that is sufficient for attachment and rolling of lymphocytes via L-selectin. This interaction is not inhibited by the mAb MECA-79. Furthermore, MECA-79 mAb blocks binding to untreated sections by only 30%, whereas the sLe^x mAb 2H5 blocks binding by ~60% and a combination of MECA-79 and 2H5 mAb blocks binding by 75%. We conclude that a pool of O-glycoprotease-resistant sLe^x-like L-selectin ligands exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb. We postulate that these ligands may participate in lymphocyte binding to HEV.     

Sialyl Lewis-dependent binding of human monocyte-derived dendritic cells to selectins

The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewis^x (sLe^x) in selectin-dependent mo-DC binding. Our data reveal that sLe^x is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLe^x-dependent binding of mo-DC to selectins was further substantiated by using sLe^x free sugar and anti-sLe^x antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLe^x expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.     

The Interaction of N-Glycans in Fcγ Receptor I α-Chain with Escherichia coli K1 Outer Membrane Protein A for Entry into Macrophages: EXPERIMENTAL AND COMPUTATIONAL ANALYSIS*

Neonatal meningitis, caused by Escherichia coli K1, is a serious central nervous system disease. We have established that macrophages serve as permissive niches for E. coli K1 to multiply in the host and for attaining a threshold level of bacterial load, which is a prerequisite for the onset of the disease. Here, we demonstrate experimentally that three N-glycans in FcγRIa interact with OmpA of E. coli K1 for binding to and entering the macrophages. Adoptive transfer of FcγRIa^−/− bone marrow-derived macrophages transfected with FcγRIa into FcγRIa^−/− newborn mice renders them susceptible to E. coli K1-induced meningitis. In contrast, mice that received bone marrow-derived macrophages transfected with FcγRIa in which N-glycosylation sites 1, 4, and 5 are mutated to alanines exhibit resistance to E. coli K1 infection. Our molecular dynamics and simulation studies predict that N-glycan 5 exhibits strong binding at the barrel site of OmpA formed by loops 3 and 4, whereas N-glycans 1 and 4 interact with loops 1, 3, and 4 of OmpA at tip regions. Molecular modeling data also suggest no role for the IgG binding site in the invasion process. In agreement, experimental mutations in IgG binding site had no effect on the E. coli K1 entry into macrophages in vitro or on the onset of meningitis in newborn mice. Together, this integration of experimental and computational studies reveals how the N-glycans in FcγRIa interact with the OmpA of E. coli K1 for inducing the disease pathogenesis.     

Functional Analysis of a Rickettsial OmpA Homology Domain of Shigella flexneri IcsA

Shigella flexneri is a gram-negative bacterium that causes diarrhea and dysentery by invasion and spread through the colonic epithelium. Bacteria spread by assembling actin and other cytoskeletal proteins of the host into “actin tails” at the bacterial pole; actin tail assembly provides the force required to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa S. flexneri outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal domain (the β domain), such that the amino-terminal 706 amino acid residues (the α domain) are exposed on the exterior of the bacillus. The α domain is therefore likely to contain the domains that are important to interactions with host factors. We identify and characterize a domain of IcsA within the α domain that bears significant sequence similarity to two repeated domains of rickettsial OmpA, which has been implicated in rickettsial actin tail formation. Strains of S. flexneri and Escherichia coli that carry derivatives of IcsA containing deletions within this domain display loss of actin recruitment and increased accessibility to IcsA-specific antibody on the surface of intracytoplasmic bacteria. However, site-directed mutagenesis of charged residues within this domain results in actin assembly that is indistinguishable from that of the wild type, and in vitro competition of a polypeptide of this domain fused to glutathione S-transferase did not alter the motility of the wild-type construct. Taken together, our data suggest that the rickettsial homology domain of IcsA is required for the proper conformation of IcsA and that its disruption leads to loss of interactions of other IcsA domains within the amino terminus with host cytoskeletal proteins.     

Limited Addition of the 6-Arm β1,2-linked N-Acetylglucosamine (GlcNAc) Residue Facilitates the Formation of the Largest N-Glycan in Plants

The most abundant N-glycan in plants is the paucimannosidic N-glycan with core β1,2-xylose and α1,3-fucose residues (Man₃XylFuc(GlcNAc)₂). Here, we report a mechanism in Arabidopsis thaliana that efficiently produces the largest N-glycan in plants. Genetic and biochemical evidence indicates that the addition of the 6-arm β1,2-GlcNAc residue by N-acetylglucosaminyltransferase II (GnTII) is less effective than additions of the core β1,2-xylose and α1,3-fucose residues by XylT, FucTA, and FucTB in Arabidopsis. Furthermore, analysis of gnt2 mutant and 35S:GnTII transgenic plants shows that the addition of the 6-arm non-reducing GlcNAc residue to the common N-glycan acceptor GlcNAcMan₃(GlcNAc)₂ inhibits additions of the core β1,2-xylose and α1,3-fucose residues. Our findings indicate that plants limit the rate of the addition of the 6-arm GlcNAc residue to the common N-glycan acceptor as a mechanism to facilitate formation of the prevalent N-glycans with Man₃XylFuc(GlcNAc)₂ and (GlcNAc)₂Man₃XylFuc(GlcNAc)₂ structures.     

An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum

Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn²⁺ bound (PDB_ID:4PCA), and SAM- Mn²⁺ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.     

Crystal structure of peptidyl-tRNA hydrolase from mycobacterium smegmatis reveals novel features related to enzyme dynamics

Peptidyl-tRNA hydrolase from Mycobacterium smegmatis is a single domain 21 kDa protein involved in the hydrolysis of prematurely produced peptidyl-tRNAs to ensure the viability of cells in bacteria, thus making it a potentially important drug target. In order to aid the development of potent drugs for controlling bacterial infections, the three-dimensional structure of peptidyl-tRNA hydrolase from Mycobacterium smegmatis has been determined. The protein adopts a compact α/β globular fold with a twisted β-sheet surrounded by α-helices. The functionally important C-terminal stretch has been unambiguously modeled for the first time in the unliganded structure of peptidyl-tRNA hydrolase. The segment, Gly138 - Val150 is mobile because it lacks significant interactions with the rest of the protein molecule. This conformational flexibility is reflected through different values of distances between a reference atom Ala147 C^α of the segment Gly138 - Val150 to Gly114 C^α from another segment from opposite side of the substrate binding channel in Mycobacterium smegmatis (7.8 Ǻ), Mycobacterium tuberculosis (9.5 Ǻ) and Escherichia coli (11.8 Ǻ). Similarly, the conformation of loop Gly109 - Gly117 with respect to another loop Asp95 - Asp100 also shows variability of the substrate binding cleft as the distance between Asp98 O^δ2 to Gly113 C^α in Mycobacterium smegmatis is 4.5 Ǻ while the corresponding distances in Mycobacterium tuberculosis and Escherichia coli are 3.1 Ǻ and 6.7 Ǻ respectively. The hydrogen bonded interactions between Asn116, His22 and Asp95 indicate a stereochemically favorable arrangement of these residues for catalytic action.     

Outer Membrane Protein A (OmpA) of Shigella flexneri 2a Induces TLR2-Mediated Activation of B Cells: Involvement of Protein Tyrosine Kinase,ERK and NF-κB

B cells are critically important in combating bacterial infections and their differentiation into plasma cells and memory cells aids bacterial clearance and long-lasting immunity conferred by essentially all vaccines. Outer membrane protein A (OmpA) of Shigella flexneri 2a has been demonstrated to induce the production of IgG and IgA in vivo following immunization of mice through intranasal route, but the direct involvement of B cells in OmpA-mediated immune regulation was not determined. Consequently, we investigated whether OmpA can modulate B cell functions and identified the molecular events involved in OmpA-induced B cell immune response in vitro. We show that OmpA of S. flexneri 2a activates B cells to produce protective cytokines, IL-6 and IL-10 as well as facilitates their differentiation into antibody secreting cells (ASCs). The immunostimulatory properties of OmpA are attributed to the increased surface expression of MHCII and CD86 on B cells. We also report here that B cell activation by OmpA is mediated strictly through recognition by TLR2, resulting in initiation of cascades of signal transduction events, involving increased phosphorylation of protein tyrosine kinases (PTKs), ERK and IκBα, leading to nuclear translocation of NF-κB. Importantly, a TLR2 antibody diminishes OmpA-induced upregulation of MHCII and CD86 on B cell surface as well as significantly inhibits B cell differentiation and cytokine secretion. Furthermore, we illustrate that B cell differentiation into ASCs and induction of cytokine secretion by OmpA are dependent on PTKs activity. Moreover, we identify that OmpA-induced B cell differentiation is entirely dependent on ERK pathway, whereas both NF-κB and ERK are essential for cytokine secretion by B cells. Overall, our data demonstrate that OmpA of S. flexneri 2a amplifies TLR signaling in B cells and triggers B cell immune response, which is critical for the development of an effective adaptive immunity to an optimal vaccine antigen.     

Kinetics of Isomerization and Inversion of Aspartate 58 of αA-Crystallin Peptide Mimics under Physiological Conditions

Although proteins consist exclusively of L-amino acids, we have reported that aspartyl (Asp) 58 and Asp 151 residues of αA-crystallin of eye lenses from elderly cataract donors are highly inverted and isomerized to D-β, D-α and L-β-Asp residues through succinimide intermediates. Of these Asp isomers, large amounts of D-β- and L-β-isomers are present but the amount of D-α-isomer is not significant. The difference in abundance of the Asp isomers in the protein may be due to the rate constants for the formation of the isomers. However, the kinetics have not been well defined. Therefore, in this study, we synthesized a peptide corresponding to human αA-crystallin residues 55 to 65 (T⁵⁵VLD⁵⁸SGISEVR⁶⁵) and its isomers in which L-α-Asp at position 58 was replaced with L-β-, D-β- and D-α-Asp and determined the rate of isomerization and inversion of Asp residues under physiological conditions (37°C, pH7.4). The rate constant for dehydration from L-α-Asp peptide to L-succinimidyl peptide was 3 times higher than the rate constant for dehydration from L-β-Asp peptide to L-succinimidyl peptide. The rate constant for hydrolysis from L-succinimidyl peptide to L-β-Asp peptide was about 5 times higher than the rate constant for hydrolysis from L-succinimidyl peptide to L-α-Asp peptide. The rate constant for dehydration from L-α-Asp peptide to L-succinimidyl peptide was 2 times higher than the rate constant for dehydration from D-α-Asp peptide to D-succinimidyl peptide. The rate constants for hydrolysis from L-succinimidyl peptide to L-β-Asp peptide and for hydrolysis from D-succinimidyl peptide to D-β-Asp peptide were almost equal. Using these rate constants, we calculated the change in the abundance ratios of the 4 Asp isomers during a human lifespan. This result is consistent with the fact that isomerized Asp residues accumulate in proteins during the ageing process.     

Deoxygenated Disaccharide Analogs as Specific Inhibitors of ��1�C4-Galactosyltransferase 1 and Selectin-mediated Tumor Metastasis

The disaccharide peracetylated GlcNAcβ1–3Galβ-O-naphthalenemethanol (disaccharide 1) diminishes the formation of the glycan sialyl Lewis X (Neu5Acα2–3Galβ1–4(Fucα1–3) GlcNAc; sLe^X) in tumor cells. Previous studies showed that the mechanism of action of disaccharide 1 involves three steps: (i) deacetylation by carboxyesterases, (ii) action as a biosynthetic intermediate for downstream enzymes involved in sLe^X assembly, and (iii) generation of several glycans related to sLe^X. In this report, we show that GlcNAcβ1–3Galβ-O-naphthalenemethanol binds to the acceptor site of human β1–4-galactosyltransferase much like the acceptor trisaccharide, GlcNAcβ1–2Manβ1–6Man, which is present on N-linked glycans. The 4′-deoxy analog, in which the acceptor hydroxyl group was replaced by -H, did not act as a substrate but instead acted as a competitive inhibitor of the enzyme. The acetylated form of this compound inhibited sLe^X formation in U937 monocytic leukemia cells, suggesting that it had inhibitory activity in vivo as well. A series of synthetic acetylated analogs of 1 containing -H, -F, -N₃, -NH₂, or -OCH₃ instead of the hydroxyl groups at C-3′- and C-4′-positions of the terminal N-acetylglucosamine residue also blocked sLe^X formation in cells. The reduction of sLe^X by the 4′-deoxy analog also diminished experimental tumor metastasis by Lewis lung carcinoma in vivo. These data suggest that nonsubstrate disaccharides have therapeutic potential through their ability to bind to glycosyltransferases in vivo and to alter glycan-dependent pathologic processes.The sialylated, fucosylated tetrasaccharide, sLe^X,³ is a common carbohydrate determinant present in many O-GalNAc-linked mucins and N-linked glycans that act as selectin ligands (see Ref. 1 and references therein). Expression of sLe^X endows tumor cells with the capacity to bind to platelets and endothelial cells in the vasculature via P- and E-selectins, thus facilitating hematogenous metastasis possibly through protection against innate immune cells and by adhesion to the blood vessel wall. Strategies for blocking selectin-carbohydrate interactions include (i) competition by soluble recombinant forms of selectins, glycoprotein ligands, and glycolipids, (ii) peptides based on the primary sequence of the carbohydrate binding site, (iii) anti-selectin antibodies, (iv) oligosaccharides related to Lewis^X, (v) inositol polyanions and sulfated sugars, (vi) heparin, and (vii) molecular mimics of sLe^X, including oligonucleotides (reviewed in Refs. 2 and 3). Analogs of acceptor substrates of the various glycosyltransferases involved in glycan biosynthesis provide another class of potential inhibitors (reviewed in Refs. 4 and 5). Although many of these analogs are effective in vitro, they generally do not exhibit inhibitory activity in cells due to poor membrane permeability. The large number of polar hydroxyl groups and the lack of membrane transporters for oligosaccharides in most cells presumably prevent their uptake (6).In contrast to many of the inhibitors described above, peracetylated disaccharides (e.g. acetylated Galβ1–4GlcNAcβ-O-naphthalenemethanol (NM), acetylated Galβ1–3GalNAcα-O-NM, and acetylated GlcNAcβ1–3Galβ-O-NM) inhibit sLe^X biosynthesis in cells (6–9). These compounds are taken up by cells by passive diffusion and acted on by cytoplasmic or membrane-associated carboxyesterases, which remove the acetyl groups. The compounds gain access to the biosynthetic enzymes located in the Golgi complex, where they serve as substrates, priming oligosaccharide synthesis and generating products related to O-GalNAc-linked mucin oligosaccharides. Priming in this manner diverts the assembly of the O-linked chains from endogenous glycoproteins, resulting in inhibition of expression of terminal Lewis antigens that are recognized by selectins. Inhibition occurs at a much lower dose than for monosaccharide-based agents, such as GalNAcβ-O-benzyl (∼25 μm versus 1–2 mm, respectively) (10, 11). Furthermore, the disaccharides appear to selectively affect sLe^X formation, since sLe^a expression was unaffected (12). By blocking selectin ligand expression, these compounds block both experimental and spontaneous metastasis (12, 13).In this study, we have examined acetylated disaccharide analogs that have been modified so that after deacetylation their activity as substrates would be altered. Characterization of the 4′-deoxy derivative using β1–4-galactosyltransferase 1 as a model showed that it acts by competitively inhibiting the enzyme. Interestingly, the peracetylated form of this analog maintains the capacity to inhibit sLe^X expression in U937 lymphoma cells and Lewis lung carcinoma (LLC) cells and block tumor formation in vivo. Thus, the deoxy analog presumably inhibits one or more galactosyltransferases in vivo, thereby blocking sLe^X formation and experimental tumor cell metastasis without generation of oligosaccharide products.     

A Fluorescence Reporter Model Defines “Tip-DCs” as the Cellular Source of Interferon β in Murine Listeriosis

Production of type I interferons, consisting mainly of multiple IFNα subtypes and IFNβ, represents an essential part of the innate immune defense against invading pathogens. While in most situations, namely viral infections, this class of cytokines is indispensable for host survival they mediate a detrimental effect during infection with L. monocytogenes by rendering macrophages insensitive towards IFNγ signalling which leads to a lethal bacterial pathology in mice. Due to a lack of suitable analytic tools the precise identity of the cell population responsible for type I IFN production remains ill-defined and so far these cells have been described to be macrophages. As in general IFNβ is the first type I interferon to be produced, we took advantage of an IFNβ fluorescence reporter-knockin mouse model in which YFP is expressed from a bicistronic mRNA linked by an IRES to the endogenous ifnb mRNA to assess the IFNβ production on a single cell level in situ. Our results showed highest frequencies and absolute numbers of IFNβ⁺ cells in the spleen 24 h after infection with L. monocytogenes where they were located predominately in the white pulp within the foci of infection. Detailed FACS surface marker analyses, intracellular cytokine stainings and T cell proliferation assays revealed that the IFNβ⁺ cells were a phenotypically and functionally further specialized subpopulation of TNF and iNOS producing DCs (Tip-DCs) which are known to be essential for the early containment of L. monocytogenes infection. We proved that the IFNβ⁺ cells exhibited the hallmark characteristics of Tip-DCs as they produced iNOS and TNF and possessed T cell priming abilities. These results point to a yet unappreciated ambiguous role for a multi-effector, IFNβ producing subpopulation of Tip-DCs in controlling the balance between containment of L. monocytogenes infection and effects detrimental to the host driven by IFNβ.     

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen’s naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205⁺ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4⁺ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ⁺ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205⁺ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.