Cell surface sialic acid and the regulation of immune cell interactions: the neuraminidase effect reconsidered.
It has been known for over a decade that sialidase (neuraminidase) treatment could substantially enhance the capacity of resting B cells to stimulate the proliferation of allogeneic and antigen specific, syngeneic T cells. Thus, cell-surface sialic acid was implicated as a potential modulator of immune cell interaction. However, little progress has been made in either identifying explicit roles for sialic acid in this system or in hypothesizing mechanisms to explain the "neuraminidase effect." Here we show for the first time that cell surface sialic acid on medium incubated B cells blocks access to costimulatory molecules on the B cell surface, and that this is the most likely explanation for the neuraminidase effect. Further, we show that it is likely to be upregulation of ICAM-1 and its subsequent engagement of LFA-1 rather than loss of cell surface sialic acid that in part regulates access to CD86 and other costimulatory molecules. However, we cannot exclude a role for CD86-bound sialic acid on the B cell in modulating binding to T cell CD28. Because sialidase treatment of resting B cells but not resting T cells enables T cell activation, we suggest that sialidase treatment may still be an analogue for an authentic step in B cell activation, and show that for highly activated B cells (activated with polyclonal anti-IgM plus INF-gamma) there is specific loss 2, 6-linked sialic acid. Potential roles for sialic acid in modulating B cell/T cell collaboration are discussed. (+info)
Characterization of an immunogenic glycocalyx on the surfaces of Cryptosporidium parvum oocysts and sporozoites.
Ruthenium red staining of Cryptosporidium parvum oocysts revealed the presence of a carbohydrate matrix on their outer bilayers that is characteristic of a glycocalyx. Surface labeling of intact oocysts identified material of high molecular weight (>10(6)) that reacted positively with sera from cryptosporidium-infected patients and with immunoglobulin A monoclonal antibodies. (+info)
Conjunctival epithelial cell differentiation on amniotic membrane.
PURPOSE: Amniotic membrane (AM)-reconstructed conjunctival surfaces recover the normal epithelial phenotype with a significantly higher cell density than the control. The present study was undertaken to examine how AM modulates rabbit conjunctival epithelial cell differentiation. METHODS: Rabbit conjunctival epithelial cells (RCEs) were cultured on the basement membrane side of dispase-pretreated AM, with or without seeding rabbit conjunctival fibroblasts (RCFs) on the stromal side. After 7 to 12 days, half of the cultures were raised to the air-liquid interface, and the remainder stayed submerged. A small group of air-lifted cultures containing RCFs was treated with retinoic acid. After 1, 2, and 4 weeks, cultures were terminated and processed for immunostaining with antibodies directed against distinct types of mucins (SMC and AM3), glycocalyx (AMEM2), keratin K3 (AE5), and K12 (AK2). Additionally, western blot analysis was performed for K3 keratin expression. Ultrastructural changes were evaluated by transmission electron microscopy. RESULTS: In general, RCEs grown on AM were uniformly small, negative to AE5 and AK2 antibodies, and positive to AMEM2 and ASPG1 antibodies. Epithelial stratification and cell polarity with prominent microvilli, tight junctions, and hemidesmosomes were more pronounced in air-lifted cultures. RCEs cocultured with RCFs showed scattered AM3-positive goblet cells, which were not increased by retinoic acid. CONCLUSIONS: RCEs cultured on AM primarily exhibit a nongoblet conjunctival epithelial phenotype. Epithelial stratification and cell polarity, features essential for epithelial differentiation, are promoted by air-lifting. This culture model will be useful for studying how growth and differentiation of conjunctival epithelial cells can be modulated further. (+info)
Bismuth-mediated disruption of the glycocalyx-cell wall of Helicobacter pylori: ultrastructural evidence for a mechanism of action for bismuth salts.
The mechanism of bismuth's bactericidal activity against Helicobacter pylori was investigated using transmission electron microscopy (TEM) and analytical electron microscopy (AEM); time-kill kinetic methods evaluated the effect of excess divalent cations. TEM analysis of untreated H. pylori revealed a normal morphology. In contrast, H. pylori exposed to bismuth salts had swollen, distorted cells with membrane-cell wall blebbing and a cytoplasm containing electron-dense, sometimes crystalline aggregates. By AEM, swollen cells contained bismuth at the cell periphery, whereas bacillary forms contained cytoplasmic bismuth localizations. Time-kill studies showed that the bactericidal activity of bismuth could be prevented by pretreatment with divalent cations. The effects of bismuth salts on the glycocalyces-cell walls of H. pylori with reversal of bactericidal activity by divalent cations are identical to those produced by other polycationic agents on various gram-negative bacilli. We conclude that disruption of the glycocalyces-cell walls of H. pylori is one mechanism of action for bismuth salts. (+info)
Permeation of the luminal capillary glycocalyx is determined by hyaluronan.
The endothelial cell glycocalyx influences blood flow and presents a selective barrier to movement of macromolecules from plasma to the endothelial surface. In the hamster cremaster microcirculation, FITC-labeled Dextran 70 and larger molecules are excluded from a region extending almost 0.5 micrometer from the endothelial surface into the lumen. Red blood cells under normal flow conditions are excluded from a region extending even farther into the lumen. Examination of cultured endothelial cells has shown that the glycocalyx contains hyaluronan, a glycosaminoglycan which is known to create matrices with molecular sieving properties. To test the hypothesis that hyaluronan might be involved in establishing the permeation properties of the apical surface glycocalyx in vivo, hamster microvessels in the cremaster muscle were visualized using video microscopy. After infusion of one of several FITC-dextrans (70, 145, 580, and 2,000 kDa) via a femoral cannula, microvessels were observed with bright-field and fluorescence microscopy to obtain estimates of the anatomic diameters and the widths of fluorescent dextran columns and of red blood cell columns (means +/- SE). The widths of the red blood cell and dextran exclusion zones were calculated as one-half the difference between the bright-field anatomic diameter and the width of the red blood cell column or dextran column. After 1 h of treatment with active Streptomyces hyaluronidase, there was a significant increase in access of 70- and 145-kDa FITC-dextrans to the space bounded by the apical glycocalyx, but no increase in access of the red blood cells or in the anatomic diameter in capillaries, arterioles, and venules. Hyaluronidase had no effect on access of FITC-Dextrans 580 and 2,000. Infusion of a mixture of hyaluronan and chondroitin sulfate after enzyme treatment reconstituted the glycocalyx, although treatment with either molecule separately had no effect. These results suggest that cell surface hyaluronan plays a role in regulating or establishing permeation of the apical glycocalyx to macromolecules. This finding and our prior observations suggest that hyaluronan and other glycoconjugates are required for assembly of the matrix on the endothelial surface. We hypothesize that hyaluronidase creates a more open matrix, enabling smaller dextran molecules to penetrate deeper into the glycocalyx. (+info)
Mammalian reproductive tract mucins.
Mucin glycoproteins are major constituents of the glycocalyx that covers mucosal epithelium. Two broad classes of mucins exist: membrane-associated and secretory. Of the secreted mucins, those with cysteine-rich regions are thought to polymerize through disulphide bonds. Among these gel-forming mucins are MUC2, MUC5AC, MUC5B and possibly MUC6. MUC7 lacks cysteine-rich domains and is thought to be secreted as a soluble monomer. Incomplete sequence information prevents classification of other mucins. Tandem repeats of amino acids rich in serine, threonine and proline are a common element in mucin core proteins, giving rise to relatively rigid, linear molecules with great potential for glycosylation. Ten distinct mucin genes have been identified in humans so far. Patterns of expression vary greatly. While MUC9, or oviductin, appears to be restricted to oviduct, the transmembrane mucin MUC1 is widely expressed. Proven functions for the different mucins are largely unknown, although potential functions are addressed in this review. Genetic and protein sequence information and expression profiles are also summarized, followed by a description of mucin assembly. Special attention is given to mucin expression in male and female reproductive tracts. (+info)
The glycocalyx of the sperm surface.
The surface of mammalian spermatozoa is covered by a dense coating of carbohydrate-rich molecules forming a 20-60 nm thick glycocalyx. The majority of sugar residues are attached to proteins which are either integrated within the sperm membrane, or are more or less loosely associated with it. It is estimated that there may be several hundred different glycoproteins comprising the glycocalyx, some of which are synthesized within the testis. Others, however, are produced by the epithelia of the efferent ducts, epididymis and possibly other accessory glands, and become associated with the spermatozoa post-testicularly during transit through, and storage in, the male tract. The acquisition of the mature glycocalyx is associated with the attainment of full sperm fertilizing ability. Until its complete molecular structure is elucidated, the complex function of the glycocalyx remains obscure, though it may be related to membrane maturation and immunoprotection in the female tract, as well as to sperm-zona binding and fertilization. (+info)
Adhesion-induced receptor segregation and adhesion plaque formation: A model membrane study.
A model system to study the control of cell adhesion by receptor-mediated specific forces, universal interactions, and membrane elasticity is established. The plasma membrane is mimicked by reconstitution of homophilic receptor proteins into solid supported membranes and, together with lipopolymers, into giant vesicles with the polymers forming an artificial glycocalix. The homophilic cell adhesion molecule contact site A, a lipid-anchored glycoprotein from cells of the slime mold Dictyostelium discoideum, is used as receptor. The success of the reconstitution, the structure and the dynamics of the model membranes are studied by various techniques including film balance techniques, micro fluorescence, fluorescence recovery after photobleaching, electron microscopy, and phase contrast microscopy. The interaction of the functionalized giant vesicles with the supported bilayer is studied by reflection interference contrast microscopy, and the adhesion strength is evaluated quantitatively by a recently developed technique. At low receptor concentrations adhesion-induced receptor segregation in the membranes leads to decomposition of the contact zone between membranes into domains of strong (receptor-mediated) adhesion and regions of weak adhesion while continuous zones of strong adhesion form at high receptor densities. The adhesion strengths (measured in terms of the spreading pressure S) of the various states of adhesion are obtained locally by analysis of the vesicle contour near the contact line in terms of elastic boundary conditions of adhesion: the balance of tensions and moments. The spreading pressure of the weak adhesion zones is S approximately 10(-9) J/m(2) and is determined by the interplay of gravitation and undulation forces whereas the spreading pressure of the tight adhesion domains is of the order S approximately 10(-6) J/m(2). (+info)