Sandra Spurr-Michaud

Hemidesmosomes and anchoring fibril collagen appear synchronously during development and wound healing

Bullous pemphigoid antisera and monoclonal antibodies to type VII collagen were used to localize hemidesmosomes and anchoring fibrils, respectively, in tissues of developing eyes and healing corneal wounds of New Zealand white rabbits. In the 17-day fetal rabbit eye, both antibodies colocalize to the epithelial-stromal junction of the lid and conjunctival region, but neither binds to the cornea, and electron microscopy demonstrates hemidesmosomes only where the antibodies bind. By 20 days of fetal development, the antibodies colocalize in cornea, and, by electron microscopy, hemidesmosomes are shown to be present as well. In healing 7-mm corneal wounds, both antibodies colocalize at the wound periphery within 66 h. By electron microscopy, hemidesmosomes along small segments of basal lamina are also shown to be present at the wound periphery at this time. These demonstrations of the synchronous assembly of hemidesmosomes and anchoring fibrils support the hypothesis of linkage of hemidesmosomes through the basement membrane to anchoring fibrils.

MUC16 is lost from the uterodome (pinopode) surface of the receptive human endometrium: in vitro evidence that MUC16 is a barrier to trophoblast adherence.

Abstract In order for the preimplantation embryo to implant into the uterus, the trophoblast cells must initially adhere to the uterine epithelial surface. In preparation, the luminal secretory cells of the epithelium lose their nonadhesive character and their surface microvilli and bulge into the lumen, forming uterodomes (pinopodes; uterodome is used instead of pinopode, since in humans the surface membrane exocytoses rather than endocytoses (Murphy, Hum Reprod 2000; 15:2451–2454). Previous research has led to the hypothesis that loss of the nonadhesive membrane-spanning mucin MUC1 from the uterodome surface allows trophoblast adherence. Immunofluorescence microscopic assay of luminal epithelia on human uterine biopsies taken from LH+0 to LH+13 show that another membrane-spanning mucin, MUC16, was lost from uterodome surfaces in all samples taken during the receptive phase, LH+6 to LH+8 (n = 12), and that MUC1 was present on uterodomes in 4 of 12 samples and on all ciliated cells of the epithelium in the receptive phase. Short interfering RNA (siRNA) knockdown of MUC16 in a uterine epithelial cell line ECC-1 that, like uterine epithelium, expresses MUC16 and MUC1 allowed increased adherence of cells of a trophoblast cell line. In parallel experiments, siRNA knockdown of MUC1 did not affect trophoblast cell adherence. These data indicate that MUC16 is a membrane component of the nonreceptive luminal uterine surface, which prevents cell adhesion, and that its removal during uterodome formation facilitates adhesion of the trophoblast.

Release of Membrane-associated Mucins from Ocular Surface Epithelia

PURPOSE Three membrane-associated mucins (MAMs)--MUC1, MUC4, and MUC16--are expressed at the ocular surface epithelium. Soluble forms of MAMs are detected in human tears, but the mechanisms of their release from the apical cells are unknown. The purpose of this study was to identify physiologic agents that induce ocular surface MAM release. METHODS An immortalized human corneal-limbal epithelial cell line (HCLE) expressing the same MAMs as native tissue was used. An antibody specific to the MUC16 cytoplasmic tail was developed to confirm that only the extracellular domain is released into the tear fluid or culture media. Effects of agents that have been shown to be present in tears or are implicated in the release or shedding of MAMs in other epithelia (neutrophil elastase, tumor necrosis factor [TNF]), TNF-alpha-converting enzyme, and matrix metalloproteinase-7 and -9) were assessed on HCLE cells. HCLE cell surface proteins were biotinylated to measure the efficiency of induced MAM release and surface restoration. Effects of induced release on surface barrier function were measured by rose bengal dye penetrance. RESULTS MUC16 in tears and in HCLE-conditioned medium lacked the cytoplasmic tail. TNF induced the release of MUC1, MUC4, and MUC16 from the HCLE surface. Matrix metalloproteinase-7 and neutrophil elastase induced the release of MUC16 but not of MUC1 or MUC4. Neutrophil elastase removed 68% of MUC16, 78% of which was restored to the HCLE cell surface 24 hours after release. Neutrophil elastase-treated HCLE cells showed significantly reduced rose bengal dye exclusion. CONCLUSIONS Results suggest that the extracellular domains of MUC1, MUC4, and MUC16 can be released from the ocular surface by agents in tears. Neutrophil elastase and TNF, present in higher amounts in the tears of patients with dry eye, may cause MAM release, allowing rose bengal staining.

Effect of pro-inflammatory mediators on membrane-associated mucins expressed by human ocular surface epithelial cells

Membrane-associated mucins are altered on the ocular surface in non-Sjögrens dry eye. This study sought to determine if inflammatory mediators, present in tears of dry eye patients, regulate membrane-associated mucins MUC1 and -16 at the level of gene expression, protein biosynthesis and/or ectodomain release. A human corneal limbal epithelial cell line (HCLE), which produces membrane-associated mucins, was used. Cells were treated with interleukin (IL)-6, -8, or -17, tumor necrosis factor-alpha (TNF-alpha), and Interferon-gamma (IFN-gamma), or a combination of TNF-alpha and IFN-gamma, or IFN-gamma and IL-17, for 1, 6, 24, or 48 h. Presence of receptors for these mediators was verified by RT-PCR. Effects of the cytokines on expression levels of MUC1 and -16 were determined by real-time PCR, and on mucin protein biosynthesis and ectodomain release in cell lysates and culture media, respectively, by immunoblot analysis. TNF-alpha and IFN-gamma each significantly induced MUC1 expression, cellular protein content and ectodomain release over time. Combined treatment with the two cytokines was not additive. By comparison, one of the inflammatory mediators, IFN-gamma, affected all three parameters-gene expression, cellular protein, and ectodomain release-for MUC16. Combined treatment with TNF-alpha and IFN-gamma showed effects similar to IFN-gamma alone, except that ectodomain release followed that of TNF-alpha, which induced MUC16 ectodomain release. In conclusion, inflammatory mediators present in tears of dry eye patients can affect MUC1 and -16 on corneal epithelial cells and may be responsible for alterations of surface mucins in dry eye.

The role of calcium in mucin packaging within goblet cells

Recent reports hypothesize that calcium plays an important role in providing cationic shielding to keep negatively charged mucins condensed and tightly packed within mucus granules of goblet cells. Vitamin D controls mineral ion homeostasis and intestinal calcium absorption, which is mediated by the nuclear vitamin D receptor (VDR). Hypocalcemia is observed in mice in which the VDR has been ablated. The purpose of this study was to test the hypothesis that normal levels of calcium are required for the physiological packaging of mucins, by comparing the morphology and mucin extractability of conjunctival goblet cells of VDR-ablated to wild-type control mice. Whole eyes from C57/129/sv hybrid wild-type, VDR-ablated, and VDR-ablated mice fed a diet high in calcium to normalize serum ionized calcium levels were fixed in situ and processed for light and transmission electron microscopy (TEM). Mucin extractability from sections of mouse eyes was assessed by lectin-blot, using helix pomatia agglutinin (HPA), and mucin content within goblet cells was assessed by immunohistochemistry, using an antibody specific to the goblet cell mucin Muc5AC. Altered mucin packaging in the goblet cells of VDR-ablated mice as compared to control mice was observed by both light and electron microscopy. In the VDR-ablated mice, the mucin packets varied in size and staining. In contrast, in the controls, the secretory granules appeared regular and uniform. By TEM, mucin packets in the VDR-ablated mice showed dispersed fibrillar and less electron-dense material compared to the homogeneous and more electron-dense packets in wild type. The appearance of mucin packets in the VDR-ablated mice with restored calcium levels was comparable to those of the wild-type control mice. HPA binding to mucin extracted from sections of VDR mouse eyes was reduced when compared to that from wild type. By immunohistochemistry, there was markedly less binding of the antibody to the mucin Muc5AC to goblet cells of VDR-ablated mice compared to controls.VDR-ablated mice presented altered conjunctival mucin packaging. There were lower levels of extractable and immunohistochemically localizable mucin in VDR-ablated mouse conjunctivas than in the wild-type controls. Restoration of ionized calcium levels in the VDR-ablated mice prevented altered mucin packaging, supporting the hypothesis that calcium is required for the physiological packaging of mucins in goblet cells.

A Metalloproteinase Secreted by Streptococcus pneumoniae Removes Membrane Mucin MUC16 from the Epithelial Glycocalyx Barrier

The majority of bacterial infections occur across wet-surfaced mucosal epithelia, including those that cover the eye, respiratory tract, gastrointestinal tract and genitourinary tract. The apical surface of all these mucosal epithelia is covered by a heavily glycosylated glycocalyx, a major component of which are membrane-associated mucins (MAMs). MAMs form a barrier that serves as one of the first lines of defense against invading bacteria. While opportunistic bacteria rely on pre-existing defects or wounds to gain entry to epithelia, non opportunistic bacteria, especially the epidemic disease-causing ones, gain access to epithelial cells without evidence of predisposing injury. The molecular mechanisms employed by these non opportunistic pathogens to breach the MAM barrier remain unknown. To test the hypothesis that disease-causing non opportunistic bacteria gain access to the epithelium by removal of MAMs, corneal, conjunctival, and tracheobronchial epithelial cells, cultured to differentiate to express the MAMs, MUCs 1, 4, and 16, were exposed to a non encapsulated, non typeable strain of Streptococcus pneumoniae (SP168), which causes epidemic conjunctivitis. The ability of strain SP168 to induce MAM ectodomain release from epithelia was compared to that of other strains of S. pneumoniae, as well as the opportunistic pathogen Staphylococcus aureus. The experiments reported herein demonstrate that the epidemic disease-causing S. pneumoniae species secretes a metalloproteinase, ZmpC, which selectively induces ectodomain shedding of the MAM MUC16. Furthermore, ZmpC-induced removal of MUC16 from the epithelium leads to loss of the glycocalyx barrier function and enhanced internalization of the bacterium. These data suggest that removal of MAMs by bacterial enzymes may be an important virulence mechanism employed by disease-causing non opportunistic bacteria to gain access to epithelial cells to cause infection.

Mucins and contact lens wear.

Purpose: This study was designed to determine whether long-term tolerant contact lens (CL) wear causes changes in the expression of mucin mRNA by the conjunctival epithelium and mucin protein content in tears and to determine whether specific mucins adhere to contact lenses. Methods: Twenty long-term (≥5 years ) and tolerant CL wearers (2 with hard and 18 with soft contact lenses) were compared with 23 non-CL wearers. One hour after CL removal, tear fluid was collected after instillation of 60 μL of sterile water onto the ocular surface, and protein concentration was determined. Impression cytology was performed on the bulbar temporal region of conjunctiva to collect cells for RNA isolation. Real-time polymerase chain reaction was performed using TaqMan primer and probes for MUC1, 4, 5AC, and 16. ELISA was performed on the collected tears to detect MUC5AC and the mucin carbohydrate epitope H185. For the analysis of adherent mucins on CL, discarded daily-wear contact lenses were collected, rinsed, and incubated overnight at 4°C in mucin isolation buffer. Immunoblot analysis of adherent mucins was performed to detect MUC1, 4, 5AC, 16, and H185. Results: No significant changes in the levels of mucin mRNA from impression cytology samples were detected when comparing CL and non-CL wearers. The amount of total protein in tears collected from CL wearers (39.9 ± 27.2 μg) was significantly less than that from non-CL wearers (95.1 ± 73.8 μg, P = 0.001). The level of MUC5AC mucin and the H185 epitope in tears per unit protein in CL wearers was not significantly different from non-CL wearers. Low levels of membrane-associated mucins, the secreted mucin MUC5AC, and the carbohydrate epitope, H185, were detected in protein extracts from discarded CLs. Compared with MUC1, 4, and 5AC, there was less MUC16 adherent to the CLs. Conclusion: Neither mucin mRNA expression by conjunctival epithelia nor mucin content per unit protein in tears was altered by long-term tolerant CL wear; however, the amount of protein in the tears was significantly less. Shed membrane-associated mucins and the goblet cell mucins adhere to CLs.

Stratified squamous epithelia produce mucin-like glycoproteins

The stratified squamous epithelia of the ocular surface, larynx, and vagina are mucus-coated epithelia, apices of which are subject to abrasive pressure from epithelia-epithelia interactions from eyelid, vocal cords, or vaginal folds, respectively. Mucus coats on these epithelia have generally been considered to be derived from the specialized mucin-producing cells embedded either in the epithelia or in adjacent tissues. Here we report the isolation, partial characterization, and cellular localization of a mucin-like glycoprotein produced by these stratified epithelia. In all three epithelia, the mucin-like molecule is present on cytoplasmic vesicles in subapical cells. As cells differentiate to their apical-most position adjacent to their mucus coat, the mucin-like molecule moves to the cell membrane where it is particularly prominent on microplicae folds. Lectin affinity chromatography was used to isolate the molecule from rat vaginal and corneal epithelium. Isolated material was approximately 60% carbohydrate and 40% protein. The major monosaccharide was N-acetylgalactosamine with lesser amounts of N-acetylglucosamine, galactose, mannose, xylose and fucose. Amino acid analysis demonstrated the predominant amino acids to be glycine, serine, threonine and proline. These data plus PAS and Alcian blue binding to the isolate indicate a mucin-like glycoprotein.

Comparison of the Transmembrane Mucins MUC1 and MUC16 in Epithelial Barrier Function

Membrane-anchored mucins are present in the apical surface glycocalyx of mucosal epithelial cells, each mucosal epithelium having at least two of the mucins. The mucins have been ascribed barrier functions, but direct comparisons of their functions within the same epithelium have not been done. In an epithelial cell line that expresses the membrane-anchored mucins, MUC1 and MUC16, the mucins were independently and stably knocked down using shRNA. Barrier functions tested included dye penetrance, bacterial adherence and invasion, transepithelial resistance, tight junction formation, and apical surface size. Knockdown of MUC16 decreased all barrier functions tested, causing increased dye penetrance and bacterial invasion, decreased transepithelial resistance, surprisingly, disruption of tight junctions, and greater apical surface cell area. Knockdown of MUC1 did not decrease barrier function, in fact, barrier to dye penetrance and bacterial invasion increased significantly. These data suggest that barrier functions of membrane-anchored mucins vary in the context of other membrane mucins, and MUC16 provides a major barrier when present.

The Boston Keratoprosthesis: comparing corneal epithelial cell compatibility with titanium and PMMA.

Purpose: To determine in vitro whether titanium is superior in corneal cell compatibility to standard polymethyl-methacrylate (PMMA) for the Boston Keratoprosthesis (KPro). Methods: Human corneal-limbal epithelial (HCLE) cells were cultured 24, 48, 72, 96, 120, 144, or 168 hours in culture plates alone (controls) or with PMMA or titanium discs. Experiments were performed in triplicate and repeated (final n = 6). To determine if a soluble, toxic factor is emitted from materials, concurrent experiments at 48 and 144 hours were performed with discs placed in Transwell Supports, with HCLE cells plated beneath. As an additional test for soluble factors, cells were incubated 24 hours with disc-conditioned media, and number of viable cells per well was quantified at each timepoint by proliferation assay. To determine if delayed cell proliferation was attributable to cell death, HCLE cell death was measured under all conditions and quantified at each timepoint by cytotoxicity assay. The effects of material on HCLE cell proliferation over time was determined by repeated measures ANOVA. P < 0.05 was statistically significant. Results: HCLE cell proliferation was greater in wells with titanium discs compared to PMMA. Differences between the test discs and control non-disc cocultures were statistically significant over time for both cell proliferation (P = 0.001) and death (P = 0.0025). No significant difference was found using Transwells (P = 0.9836) or disc-conditioned media (P = 0.36). Conclusion: This in vitro HCLE cell model demonstrates significantly increased cell proliferation and decreased cell death with cell/titanium contact compared to cell/PMMA contact. Moreover, differences are unlikely attributable to a soluble factor. Prospective in vivo analysis of the two KPro biomaterials is indicated.