Constitutive expression of GlyCAM-1 core protein in the rat cochlea

Glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) is a mucin-like glycoprotein previously identified on high endothelial venules (HEV) of lymph nodes and also in lactating mammary glands. A specifically glycosilated form of GlyCAM-1 on HEV has been shown to be a ligand for a leukocyte L-selectin, which plays an important role in leukocyte rolling along the inflamed endothelium. Here we report that GlyCAM-1 is also expressed in the cochlea. Immunohistochemistry revealed the lateral wall of the cochlea, tectorial membrane, modiolus, organ of corti, and spiral modiolar vein (SMV) to be strongly stained with polyclonal anti-GlyCAM-1 antibody. Moreover, RT-PCR of the cochlear tissue by the use of specific oligonucleotide primers for rat GlyCAM-1 generated a 378 bp product which was then verified by nucleotide sequencing to represent GlyCAM-1. Electron microscopic investigation revealed the presence of GlyCAM-1 over the entire lumenal surface of the vessels, and the basolateral infoldings in stria vascularis. However, soluble L-selectin or mAb MECA-79 which recognizes a carbohydrate epitope on functional L-selectin ligands bound only to the spiral ligament, tectorial membrane and modiolus. These observations suggest that GlyCAM-1 expressed in the cochlear region is heterogenous in terms of its glycosylation.     

L-selectin and its biological ligands

This review considers the leukocyte adhesive receptor known as L-selectin. This protein, belonging to the selectin family of cell-cell adhesion receptors, mediates adhesion by virtue of a C-type lectin domain at its amino terminus. The protein was discovered as a lymphocyte homing receptor involved in the attachment of lymphocytes to high endothelial venules (HEV) of lymph nodes. Its widespread distribution on all leukocyte populations underlies a more general role in a variety of leukocyte-endothelial interactions. In the HEV interaction, cognate HEV ligands for L-selectin have been identified as two sulfated, sialylated, and fucosylated glycoproteins, known as GlyCAM-1 and Sgp90. These ligands have mucin-like domains which confer important properties for their proposed adhesive function. The carbohydrate features of these ligands, essential for their interaction with L-selectin, are reviewed. The existence of extralymphoid ligands for L-selectin is also discussed.Presented at the XXXV Symposium of the Society for Histochemistry, 29 September 1993, Gargellen, Austria     

Identification and characterization of ligands for L-selectin in the kidney. II. Expression of chondroitin sulfate and heparan sulfate proteoglycans reactive with L-selectin

Ligands for the leukocyte adhesion molecule L-selectin are expressed not only in lymph node high endothelial venules (HEV) but also in the renal distal tubuli. Here we report that L-selectin-reactive molecules in the kidney are chondroitin sulfate and heparan sulfate proteoglycans of 500-1000 kDa, unlike those in HEV bearing sialyl Lewis X-like carbohydrates. Binding of L-selectin to these molecules was mediated by the lectin domain of L-selectin and required divalent cations. Binding was inhibited by chondroitinase and/or heparitinase but not sialidase. Thus, L-selectin can recognize chondroitin sulfate and heparan sulfate glycosaminoglycans structurally distinct from sialyl Lewis X-like carbohydrates.     

Differential requirements for core2 glucosaminyltransferase for endothelial L-selectin ligand function in vivo

L-selectin is a calcium-dependent lectin on leukocytes mediating leukocyte rolling in high endothelial venules and inflamed microvessels. Many selectin ligands require modification of glycoproteins by leukocyte core2 beta1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT-I). To test the role of Core2GlcNAcT-I for L-selectin ligand biosynthesis, we investigated leukocyte rolling in venules of untreated and TNF-alpha-treated cremaster muscles and in Peyer's patch high endothelial venules (HEV) of Core2GlcNAcT-I null (core2(-/-)) mice. In the presence of blocking mAbs against P- and E-selectin, L-selectin-mediated leukocyte rolling was almost completely abolished in cremaster muscle venules of core2(-/-) mice, but not littermate control mice. By contrast, leukocyte rolling in Peyer's patch HEV was not significantly different between core2(-/-) and control mice. To probe L-selectin ligands more directly, we injected L-selectin-coated beads. These beads showed no rolling in cremaster muscle venules of core2(-/-) mice, but significant rolling in controls. In Peyer's patch HEV, beads coated with a low concentration of L-selectin showed reduced rolling in core2(-/-) mice. Beads coated with a 10-fold higher concentration of L-selectin rolled equivalently in core2(-/-) and control mice. Our data show that endothelial L-selectin ligands relevant for rolling in inflamed microvessels of the cremaster muscle are completely Core2GlcNAcT-I dependent. In contrast, L-selectin ligands in Peyer's patch HEV are only marginally affected by the absence of Core2GlcNAcT-I, but are sufficiently functional to support L-selectin-dependent leukocyte rolling in Core2GlcNAcT-I-deficient mice.     

Formation of the tectorial membrane of the cochlear canal during the embryogenesis of birds

By means of electron microscopy formation of the tectorial membrane of the cochlear canal and differentiation of the cells participating in the process (supporting cells of the basilar papilla and anterior homogeneous cells--AHC) have been studied in chick embryos. The AHC, to which the tectorial membrane is fixed, produce fine fibrillar material, included into the composition of the tectorial membrane. The cells mentioned form a number of cytoskeletal structures connected with the mechanical function of the tectorial membrane. Besides the network of the tonofilaments, gradually filling cytoplasm of the AHC, some peculiar attachings in the form of collagenous fibrillar bundles are revealed, they reach the AHC from the sublying connective tissue and have a direct contact with the basal membrane of the cells. The beginning of the tectorial membrane formation precedes the formation of the cytoskeletal structures. The latter appear only when the mass of the tectorial membrane, and hence, the mechanical loading on the AHC is great enough.     

L-selectin ligands expressed by human leukocytes are HECA-452 antibody-defined carbohydrate epitopes preferentially displayed by P-selectin glycoprotein ligand-1.

Leukocytes express L-selectin ligands critical for leukocyte-leukocyte interactions at sites of inflammation. The predominant leukocyte L-selectin ligand is P-selectin glycoprotein ligand-1 (PSGL-1), which displays appropriate sialyl Lewis x (sLex)-like carbohydrate determinants for L-selectin recognition. Among the sLex-like determinants expressed by human leukocytes is a unique carbohydrate epitope defined by the HECA-452 mAb. The HECA-452 Ag is a critical component of L-selectin ligands expressed by vascular endothelial cells. However, HECA-452 Ag expression on human leukocyte L-selectin ligands has not been assessed. In this study, the HECA-452 mAb blocked 88-99% of neutrophil rolling on, or attachment to, adherent cells expressing L-selectin in multiple experimental systems. A function-blocking anti-PSGL-1 mAb also inhibited L-selectin binding to neutrophils by 89-98%. In addition, the HECA-452 and anti-PSGL-1 mAbs blocked the majority of P-selectin binding to neutrophils. Western blot analysis revealed that PSGL-1 immunoprecipitated from neutrophils displayed HECA-452 mAb-reactive determinants and that PSGL-1 was the predominant scaffold for HECA-452 Ag display. Leukocyte L-selectin ligands also contained sulfated determinants since culturing ligand-bearing cells with NaClO3 abrogated L-selectin binding. Consistent with this, human neutrophils expressed mRNA encoding five different sulfotransferases associated with the generation of selectin ligands: CHST1, CHST2, CHST3, TPST1, and HEC-GlcNAc6ST. Therefore, the HECA-452-defined carbohydrate determinant displayed on PSGL-1 represented the predominant L-selectin and P-selectin ligand expressed by neutrophils.     

Thyroid hormone-deficient period prior to the onset of hearing is associated with reduced levels of beta-tectorin protein in the tectorial membrane: implication for hearing loss

The genes for alpha- and beta-tectorin encode the major non-collagenous proteins of the tectorial membrane. Recently, a targeted deletion of the mouse alpha-tectorin gene was found to cause loss of cochlear sensitivity (). Here we describe that mRNA levels for beta-tectorin, but not alpha-tectorin, are significantly reduced in the cochlear epithelium under constant hypothyroid conditions and that levels of beta-tectorin protein in the tectorial membrane are lower. A delay in the onset of thyroid hormone supply prior to onset of hearing, recently described to result in permanent hearing defects and loss of active cochlear mechanics (), can also lead to permanently reduced beta-tectorin protein levels in the tectorial membrane. beta-Tectorin protein levels remain low in the tectorial membrane up to one year after the onset of thyroid hormone supply has been delayed until postnatal day 8 or later and are associated with an abnormally structured tectorial membrane and the loss of active cochlear function. These data indicate that a simple delay in thyroid hormone supply during a critical period of development can lead to low beta-tectorin levels in the tectorial membrane and suggest for the first time that beta-tectorin may be required for development of normal hearing.     

N-acetylglucosamine 6-O-sulfotransferase-1 regulates expression of L-selectin ligands and lymphocyte homing

Lymphocyte homing is initiated by the binding of L-selectin on lymphocytes to its ligands on high endothelial venules (HEV). Sialyl 6-sulfo Lewis X is a major capping group of L-selectin ligands. N-Acetylglucosamine (GlcNAc) 6-sulfation is essential for the ligand activity, and is catalyzed by GlcNAc 6-O-sulfotransferases (GlcNAc6STs) of which GlcNAc6ST-1 and GlcNAc6ST-2 are expressed in HEV. Here, we report that mice deficient in GlcNAc6ST-1 were impaired in the elaboration of sialyl 6-sulfo Lewis X in HEV and that an epitope of L-selectin ligands recognized by the MECA-79 anti-body was greatly reduced or abolished in the abluminal aspect of HEV. Lymphocyte homing to peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches was significantly reduced in GlcNAc6ST-1 null mice. These results demonstrate that GlcNAc6ST-1 is involved in lymphocyte homing in vivo, and indicate that GlcNAc6ST-1 and -2 play complementary roles. The importance of GlcNAc6ST-1 is particularly high-lighted by its involvement in lymphocyte homing to Peyer's patches where GlcNAc6ST-2 expression is undetectable.     

The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via alpha-actinin: receptor positioning in microvilli does not require interaction with alpha-actinin

The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L- selectin interacts directly with the cytoplasmic actin-binding protein alpha-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified alpha-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of alpha-actinin to the L- selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha- actinin binds directly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo to link actin filaments to the L- selectin cytoplasmic domain. In contrast, a deletion mutant of L- selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L-selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of alpha-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L- selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L- selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton.     

Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions

Interaction of leukocytes in flow with adherent leukocytes may contribute to their accumulation at sites of inflammation. Using L- selectin immobilized in a flow chamber, a model system that mimics presentation of L-selectin by adherent leukocytes, we characterize ligands for L-selectin on leukocytes and show that they mediate tethering and rolling in shear flow. We demonstrate the presence of L- selectin ligands on granulocytes, monocytes, and myeloid and lymphoid cell lines, and not on peripheral blood T lymphocytes. These ligands are calcium dependent, sensitive to protease and neuraminidase, and structurally distinct from previously described ligands for L-selectin on high endothelial venules (HEV). Differential sensitivity to O-sialo- glycoprotease provides evidence for ligand activity on both mucin-like and nonmucin-like structures. Transfection with fucosyltransferase induces expression of functional L-selectin ligands on both a lymphoid cell line and a nonhematopoietic cell line. L-selectin presented on adherent cells is also capable of supporting tethering and rolling interactions in physiologic shear flow. L-selectin ligands on leukocytes may be important in promoting leukocyte-leukocyte and subsequent leukocyte endothelial interactions in vivo, thereby enhancing leukocyte localization at sites of inflammation.     

Electron-microscopic localization of type II,IX, and V collagen in the organ of Corti of the gerbil

Summary The presence of types II, IX and V collagen was probed in the organ of Corti of the adult gerbil cochlea by use of immunocytochemistry at the light- and electron-microscopic levels. Type II collagen is found in the connective tissues of the osseous spiral lamina and spiral limbus. In the region of the sensory hair cells it is present in the tectorial membrane and antibodies bind to the thick unbranched radial fibers. Type IX collagen co-localizes with type II collagen in the tectorial membrane, where antibodies bind to the thick unbranched radial fibers. Type V collagen is present in the connective tissue of the spiral limbus, the osseous spiral lamina, the eighth nerve, and the tectorial membrane. In the tectorial membrane, the staining with antibodies to type V collagen is more diffuse than that seen for types II and IX collagen and antibodies to type V bind to the thin, highly branched fibers in which the thick fibers are embedded. The results indicate that collagens characteristic of cartilage are localized in the organ of Corti. Within the tectorial membrane, types II and IX collagen form heterotypic thick fibers embedded in a reticular network of type V collagen fibers. These collagens form a highly structured matrix which contributes to the rigidity of the tectorial membrane and allow it to withstand the physical stresses associated with transmission of the stimuli necessary for sensory transduction.     

Collagen XVIII,a basement membrane heparan sulfate proteoglycan,interacts with L-selectin and monocyte chemoattractant protein-1

Leukocyte infiltration during inflammation is mediated by the sequential actions of adhesion molecules and chemokines. By using a rat ureteral obstruction model, we showed previously that L-selectin plays an important role in leukocyte infiltration into the kidney. Here we report the purification, identification, and characterization of an L-selectin-binding heparan sulfate proteoglycan (HSPG) expressed in the rat kidney. Partial amino acid sequencing and Western blotting analyses showed that the L-selectin-binding HSPG is collagen XVIII, a basement membrane HSPG. The binding of L-selectin to isolated collagen XVIII was specifically inhibited by an anti-L-selectin monoclonal antibody, EDTA, treatment of the collagen XVIII with heparitinase or heparin but not by chemically desulfated heparin. A cell binding assay showed that the L-selectin-collagen XVIII interaction mediates cell adhesion. Interestingly, collagen XVIII also interacted with a chemokine, monocyte chemoattractant protein-1, and presented it to a monocytic cell line, THP-1, which enhanced the alpha(4)beta(1) integrin-mediated binding of the THP-1 cells to vascular cell adhesion molecule-1. Thus, collagen XVIII may provide a link between selectin-mediated cell adhesion and chemokine-induced cellular activation and accelerate the progression of leukocyte infiltration in renal inflammation.     

Nasal-associated lymphoid tissue: phenotypic and functional evidence for the primary role of peripheral node addressin in naive lymphocyte adhesion to high endothelial venules in a mucosal site.

Nasal-associated lymphoid tissue (NALT), a mucosal inductive site for the upper respiratory tract, is important for the development of mucosal immunity locally and distally to intranasally introduced Ag. To more fully understand the induction of nasal mucosal immunity, we investigated the addressins that allow for lymphocyte trafficking to this tissue. To investigate the addressins responsible for naive lymphocyte binding, immunofluorescent and immunoperoxidase staining of frozen NALT sections were performed using anti-mucosal addressin cell adhesion molecule-1 (MAdCAM-1), anti-peripheral node addressin (PNAd), and anti-VCAM-1 mAbs. All NALT high endothelial venules (HEV) expressed PNAd, either associated with MAdCAM-1 or alone, whereas NALT follicular dendritic cells expressed both MAdCAM-1 and VCAM-1. These expression profiles were distinct from those of the gut mucosal inductive site, Peyer's patches (PP). The functionality of NALT HEV was determined using a Stamper-Woodruff ex vivo assay. The anti-L-selectin MEL-14 mAb blocked >90% of naive lymphocyte binding to NALT HEV, whereas the anti-MAdCAM-1 mAb, which blocks almost all naive lymphocyte binding to PP, minimally blocked binding to NALT HEV. NALT lymphocytes exhibited a unique L-selectin expression profile, differing from both PP and peripheral lymph nodes. Finally, NALT HEV were found in increased amounts in the B cell zones, unlike PP HEV. These results suggest that NALT is distinct from the intestinal PP, that initial naive lymphocyte binding to NALT HEV involves predominantly L-selectin and PNAd rather than alpha4beta7-MAdCAM-1 interactions, and that MAdCAM-1 and VCAM-1 expressed by NALT follicular dendritic cells may play an important role in lymphocyte recruitment and retention.     

Quantitative X-ray tomography of the mouse cochlea

Imaging with hard X-rays allows visualizing cochlear structures while maintaining intrinsic qualities of the tissue, including structure and size. With coherent X-rays, soft tissues, including membranes, can be imaged as well as cells making use of the so-called in-line phase contrast. In the present experiments, partially coherent synchrotron radiation has been used for micro-tomography. Three-dimensional reconstructions of the mouse cochlea have been created using the EM3D software and the volume has been segmented in the Amira Software Suite. The structures that have been reconstructed include scala tympani, scala media, scala vestibuli, Reissner's membrane, basilar membrane, tectorial membrane, organ of Corti, spiral limbus, spiral ganglion and cochlear nerve. Cross-sectional areas of the scalae were measured. The results provide a realistic and quantitative reconstruction of the cochlea.     

Identification and characterization of ligands for L-selectin in the kidney. III. Characterization of L-selectin reactive heparan sulfate proteoglycans

L-Selectin, a leukocyte adhesion molecule, mediates leukocyte rolling on the endothelium and plays a critical role in leukocyte recruitment at inflammatory sites as well as in lymphocyte homing. We have previously shown that L-selectin reactive chondroitin sulfate and heparan sulfate proteoglycans (HSPGs) are both expressed in the distal tubules of the kidney and that versican is one of the chondroitin sulfate-type ligands. In the present study, we characterized the heparan sulfate-type ligand(s) in more detail. The molecular sizes of HSPGs were approximately 600 kDa with core protein sizes of 160 and 180 kDa. Western blotting analysis showed that L-selectin reactive HSPGs were neither agrin nor perlecan, major basement membrane HSPGs in the kidney. The binding to L-selectin was mediated by the lectin domain of L-selectin in a Ca2+-dependent manner and required heparan sulfate side chains, but not sialic acid. To our knowledge, this is the first biochemical characterization of the L-selectin reactive heparan sulfate proteoglycan(s) in the distal tubules of the kidney.     

differential L-selectin binding activities of human hematopoietic cell L-selectin ligands, HCELL and PSGL-1

Expression of L-selectin on human hematopoietic cells (HC) is associated with a higher proliferative activity and a more rapid engraftment after hematopoietic stem cell transplantation. Two L-selectin ligands are expressed on human HCs, P-selectin glycoprotein ligand-1 (PSGL-1) and a specialized glycoform of CD44 (hematopoietic cell E- and L-selectin ligand, HCELL). Although the structural biochemistry of HCELL and PSGL-1 is well characterized, the relative capacity of these molecules to mediate L-selectin-dependent adhesion has not been explored. In this study, we examined under shear stress conditions L-selectin-dependent leukocyte adhesive interactions mediated by HCELL and PSGL-1, both as naturally expressed on human HC membranes and as purified molecules. By utilizing both Stamper-Woodruff and parallel-plate flow chamber assays, we found that HCELL displayed a 5-fold greater capacity to support L-selectin-dependent leukocyte adherence across a broad range of shear stresses compared with that of PSGL-1. Moreover, L-selectin-mediated leukocyte binding to immunopurified HCELL was consistently >5-fold higher than leukocyte binding to equivalent amounts of PSGL-1. Taken together, these data indicate that HCELL is a more avid L-selectin ligand than PSGL-1 and may be the preferential mediator of L-selectin-dependent adhesive interactions among human HCs in the bone marrow.     

The petrosal and inner ear of the Late Jurassic cladotherian mammal Dryolestes leiriensis and implications for ear evolution in therian mammals

Dryolestes leiriensis is a Late Jurassic fossil mammal of the dryolestoid superfamily in the cladotherian clade that includes the extant marsupials and placentals. We used high resolution micro‐computed tomography (µCT) scanning and digital reconstruction of the virtual endocast of the inner ear to show that its cochlear canal is coiled through 270°, and has a cribriform plate with the spiral cochlear nerve foramina between the internal acoustic meatus and the cochlear bony labyrinth. The cochlear canal has the primary bony lamina for the basilar membrane with a partially formed (or partially preserved) canal for the cochlear spiral ganglion. These structures, in their fully developed condition, form the modiolus (the bony spiral structure) of the fully coiled cochlea in extant marsupial and placental mammals. The CT data show that the secondary bony lamina is present, although less developed than in another dryolestoid Henkelotherium and in the prototribosphenidan Vincelestes. The presence of the primary bony lamina with spiral ganglion canal suggests a dense and finely distributed cochlear nerve innervation of the hair cells for improved resolution of sound frequencies. The primary, and very probably also the secondary, bony laminae are correlated with a more rigid support for the basilar membrane and a narrower width of this membrane, both of which are key soft‐tissue characteristics for more sensitive hearing for higher frequency sound. All these cochlear features originated prior to the full coiling of the therian mammal cochlea beyond one full turn, suggesting that the adaptation to hearing a wider range of sound frequencies, especially higher frequencies with refined resolution, has an ancient evolutionary origin no later than the Late Jurassic in therian evolution. The petrosal of Dryolestes has added several features that are not preserved in the petrosal of Henkelotherium. The petrosal characters of dryolestoid mammals are essentially the same as those of Vincelestes, helping to corroborate the synapomorphies of the cladotherian clade in neural, vascular, and other petrosal characteristics. The petrosal characteristics of Dryolestes and Henkelotherium together represent the ancestral morphotype of the cladotherian clade (Dryolestoidea + Vincelestes + extant Theria) from which the extant therian mammals evolved their ear region characteristics. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166 , 433–463.     

An endothelial ligand for L-selectin is a novel mucin-like molecule.

The adhesive interaction between circulating lymphocytes and the high endothelial venules (HEV) of lymph nodes (LN) is mediated by lymphocyte L-selectin, a member of the selectin family of cell adhesion proteins. Previous work has identified a sulfated 50 kd glycoprotein (Sgp50) as an HEV ligand for L-selectin. We now report the purification of this glycoprotein and the utilization of the derived N-terminal amino acid sequence to clone a cDNA. The predicted sequence reveals a novel, mucin-like molecule containing two serine/threonine-rich domains. The mRNA encoding this glycoprotein is preferentially expressed in LN. Antibodies against predicted peptides immunoprecipitate Sgp50 and stain the apical surface of LN HEV. These results thus define a tissue-specific mucin-like endothelial glycoprotein that appears to function as a scaffold that presents carbohydrates to the L-selectin lectin domain.