Generation and characterization of a monoclonal antibody to the cytoplasmic tail of MUC16.

MUC16 is a large transmembrane mucin expressed on the apical surfaces of the epithelium covering the ocular surface, respiratory system and female reproductive tract. The transmembrane mucin is overexpressed by ovarian carcinomas, it is one of the most frequently used diagnostic markers for the disease and it is considered a promising target for immunotherapeutic intervention. Immunodetection of the mucin has to date been through antibodies that recognize its exceptionally large ectodomain. Similar to other membrane anchored mucins, MUC16 has a short cytoplasmic tail (CT), but studies of the biological relevance of the C-terminal domain of MUC16 has been limited by lack of availability of monoclonal antibodies that recognize the native CT. Here, we report the development of a novel monoclonal antibody to the CT region of the molecule that recognizes native MUC16 and its enzymatically released CT region. The antibody is useful for immunoprecipitation of the released CT domain as demonstrated with the OVCAR3 ovarian cancer cell line and can be used for detailed cytolocalization in cells as well as in frozen sections of ocular surface and uterine epithelium.

Human conjunctival goblet cells express the membrane associated mucin MUC16: Localization to mucin granules.

MUC16 is an extraordinarily large 22,152 amino acid membrane spanning mucin that has been shown to be present in the glycocalyx of the apical cells of the human cornea and conjunctiva where it interfaces with the tear film. The ectodomain of the molecule has been demonstrated in tears, where it has been presumed to be from surface epithelial cells. Data presented here from multiple assays, including immunohistochemistry, immunoelectron microscopy, in situ hybridization, and RT-PCR of RNA isolated from goblet cells isolated by laser capture microdissection, demonstrate that the membrane tethered mucin is also expressed by conjunctival goblet cells both in humans and in mice. The mucin is present in mucin granules and appears to be localized to the mucin granule membrane. Correlation analyses of the amounts of the goblet cell secreted mucin MUC5AC and the amounts of MUC16 and of MUC1 another membrane tethered mucin ectodomain found in human tear samples demonstrated that MUC5AC amounts correlated to the amounts of MUC16 but not to MUC1. These data suggest that goblet cells are a second source of the mucin in tears. The function of the membrane tethered mucin in the mucin granule remains to be determined.

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 ocular surface phenotype of Muc5ac and Muc5b null mice.

PURPOSE: Recent development of mice null for either Muc5ac or Muc5b mucin allows study of their specific roles at the mouse ocular surface. A recent report indicated that Muc5ac null mice show an ocular surface phenotype similar to that seen in dry eye syndrome. The purpose of our study was to determine the effect of lack of Muc5ac or Muc5b on the ocular surface, and to determine if environmental desiccating stress exacerbated a phenotype. METHODS: Muc5ac null and Muc5b null mice, and their wild-type controls were examined for ocular surface defects by fluorescein staining. The number of goblet cells per area of conjunctival epithelium was counted, and levels of mucin gene expression and genes associated with epithelial stress, keratinization, and differentiation, known to be altered in dry eye syndrome, were assayed. To determine if the null mice would respond more to desiccating stress than their wild-type controls, they were challenged in a controlled environment chamber (CEC) and assessed for changes in fluorescein staining, tear volume, and inflammatory cells within the conjunctival and corneal epithelia. RESULTS: Unlike the previous study, we found no ocular surface phenotype in the Muc5ac null mice, even after exposure to desiccating environmental stress. Similarly, no ocular surface phenotype was present in the Muc5b null mice, either before or after exposure to a dry environment in the CEC. CONCLUSIONS: Our results indicate that deleting either the Muc5ac or Muc5b gene is insufficient to create an observable dry eye phenotype on the ocular surface of these mice.

Gene expression in human accessory lacrimal glands of Wolfring.

PURPOSE: The accessory lacrimal glands are assumed to contribute to the production of tear fluid, but little is known about their function. The goal of this study was to conduct an analysis of gene expression by glands of Wolfring that would provide a more complete picture of the function of these glands. METHODS: Glands of Wolfring were isolated from frozen sections of human eyelids by laser microdissection. RNA was extracted from the cells and hybridized to gene expression arrays. The expression of several of the major genes was confirmed by immunohistochemistry. RESULTS: Of the 24 most highly expressed genes, 9 were of direct relevance to lacrimal function. These included lysozyme, lactoferrin, tear lipocalin, and lacritin. The glands of Wolfring are enriched in genes related to protein synthesis, targeting, and secretion, and a large number of genes for proteins with antimicrobial activity were detected. Ion channels and transporters, carbonic anhydrase, and aquaporins were abundantly expressed. Genes for control of lacrimal function, including cholinergic, adrenergic, vasoactive intestinal polypeptide, purinergic, androgen, and prolactin receptors were also expressed in gland of Wolfring. CONCLUSIONS: The data suggest that the function of glands of Wolfring is similar to that of main lacrimal glands and are consistent with secretion electrolytes, fluid, and protein under nervous and hormonal control. Since these glands secrete directly onto the ocular surface, their location may allow rapid response to exogenous stimuli and makes them readily accessible to topical drugs.

Biocompatibility and biofilm inhibition of N,N-hexyl,methyl-polyethylenimine bonded to Boston Keratoprosthesis materials.

The biocompatibility and antibacterial properties of N,N-hexyl,methyl-polyethylenimine (HMPEI) covalently attached to the Boston Keratoprosthesis (B-KPro) materials was evaluated. By means of confocal and electron microscopies, we observed that HMPEI-derivatized materials exert an inhibitory effect on biofilm formation by Staphylococcus aureus clinical isolates, as compared to the parent poly(methyl methacrylate) (PMMA) and titanium. There was no additional corneal epithelial cell cytotoxicity of HMPEI-coated PMMA compared to that of control PMMA in tissue cultures in vitro. Likewise, no toxicity or adverse reactivity was detected with HMPEI-derivatized PMMA or titanium compared to those of the control materials after intrastromal or anterior chamber implantation in rabbits in vivo.

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.

Antiadhesive character of mucin O-glycans at the apical surface of corneal epithelial cells.

PURPOSE: Prolonged contact of opposite mucosal surfaces, which occurs on the ocular surface, oral cavity, reproductive tract, and gut, requires a specialized apical cell surface that prevents adhesion. The purpose of this study was to evaluate the contribution of mucin O-glycans to the antiadhesive character of human corneal-limbal epithelial (HCLE) cells. METHODS: Mucin O-glycan biosynthesis in HCLE cells was disrupted by metabolic interference with benzyl-alpha-GalNAc. The cell surface mucin MUC16 and its carbohydrate epitope H185 were detected by immunofluorescence and Western blot. HCLE cell surface features were assessed by field emission scanning electron microscopy. Cell-cell adhesion assays were performed under static conditions and in a parallel plate laminar flow chamber. RESULTS: Benzyl-alpha-GalNAc disrupted the biosynthesis of O-glycans without affecting apomucin biosynthesis or cell surface morphology. Static adhesion assays showed that the apical surface of differentiated HCLE cells expressing MUC16 and H185 was more antiadhesive than undifferentiated HCLE cells, which lacked MUC16. Abrogation of mucin O-glycosylation in differentiated cultures with benzyl-alpha-GalNAc resulted in increased adhesion of applied corneal epithelial cells and corneal fibroblasts. The antiadhesive effect of mucin O-glycans was further demonstrated by fluorescence video microscopy in dynamic flow adhesion assays. Cationized ferritin labeling of the cell surface indicated that anionic repulsion did not contribute to the antiadhesive character of the apical surface. CONCLUSIONS: These results indicate that epithelial O-glycans contribute to the antiadhesive properties of cell surface-associated mucins in corneal epithelial cells and suggest that alterations in mucin O-glycosylation are involved in the pathology of drying mucosal diseases (e.g., dry eye).

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

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.

Functions of MUC16 in corneal epithelial cells.

PURPOSE: The membrane-associated mucin MUC16, a heavily O-glycosylated transmembrane protein, is expressed by the ocular surface epithelia and localized on the tips of the surface microplicae. Although its functions in the ocular surface glycocalyx are unknown, it is thought that MUC16 provides a disadhesive barrier to the epithelial membrane. Two other membrane-associated mucins expressed by ocular surface epithelia, MUC1 and MUC4, are multifunctional and have signaling capabilities through their cytoplasmic tails and EGF-like domains, respectively. The MUC16 cytoplasmic tail has not been characterized, but, because it contains a polybasic amino acid sequence, it potentially interacts with the actin cytoskeleton through ezrin/radixin/moesin (ERM) actin-binding proteins. METHODS: The interaction of MUC16 with the actin cytoskeleton through ERMs was investigated using cytoplasmic tail peptides and ERM pull-down experiments. MUC16 functions were determined using RNA interference in immortalized human corneal-limbal epithelial (HCLE) cells. The effect of MUC16 knockdown on microplicae structure in HCLE cells was determined using scanning and immunoelectron microscopy. HCLE cells were incubated with rose bengal dye to measure the role of MUC16 in ocular surface barrier function. Binding of fluorescently labeled Staphylococcus aureus to HCLE cells was measured to determine the role of MUC16 in the protection of pathogen adherence on the ocular surface epithelium. RESULTS: MUC16 cytoplasmic tail peptides bound the N-terminus of ERMs, with no detectable binding of MUC1 and MUC4 peptides. No effect on surface membrane projections could be detected in HCLE cells after MUC16 suppression; however, HCLE cells incubated with rose bengal showed that exclusion of the dye was significantly reduced in cells with MUC16 suppression. In addition, S. aureus binding to HCLE cells was significantly increased with MUC16 suppression. CONCLUSIONS: These results suggest that MUC16 is a multifunctional molecule linked to the actin cytoskeleton. The expression of MUC16 in the ocular surface glycocalyx helps provide a disadhesive protective barrier for the epithelial surface.

Mucin gene expression in human male urogenital tract epithelia.

BACKGROUND: Mucins are large, hydrophilic glycoproteins that protect wet-surfaced epithelia from pathogen invasion as well as provide lubrication. At least 17 mucin genes have been cloned to date. This study sought to determine the mucin gene expression profile of the human male urogenital tract epithelia, to determine if mucins are present in seminal fluid and to assess the effect of androgens on mucin expression. METHODS AND RESULTS: Testis, epididymis, vas deferens, seminal vesicle, prostate, bladder, urethra and foreskin were assessed for mucin expression by RT-PCR (for 14 mucin genes) and immunohistochemistry (nine antibodies for five mucins). Epithelia of the vas deferens, prostate and urethra expressed the greatest number of mucins, each with mRNA for between 5 and 8 mucins. Except for MUC20 in epididymis, mRNA for MUC1 and MUC20, both membrane-associated mucins, was detected in all tissues analysed. By comparison, MUC6 was more restricted in expression, being primarily detected in seminal vesicle. MUC1, MUC5B and MUC6 were detected in seminal fluid samples by immunoblot analysis. Androgens had no effect on mucin expression in cultured human prostatic epithelial cells. CONCLUSIONS: Each region of urogenital tract epithelium expressed a unique mucin gene repertoire. Secretory mucins are present in seminal fluid, and androgens do not appear to regulate mucin gene expression in prostatic epithelial cells in culture.

Mucin characteristics of human corneal-limbal epithelial cells that exclude the rose bengal anionic dye.

PURPOSE: Rose bengal is an organic anionic dye used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. The current study was undertaken to evaluate rose bengal staining in a human corneal-limbal epithelial (HCLE) cell line known to produce and glycosylate membrane-associated mucins. METHODS: HCLE cells were grown to confluence in serum-free medium and switched to DMEM/F12 with 10% serum to promote differentiation. Immunolocalization of the membrane-associated mucins MUC1 and MUC16 and the T-antigen carbohydrate epitope was performed with the monoclonal antibodies HMFG-2 and OC125 and jacalin lectin, respectively. To assess dye uptake, cultures were incubated for 5 minutes with 0.1% rose bengal and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of hyperosmotic stress on rose bengal staining in vitro was evaluated by increasing the ion concentration (Ca+2 and Mg+2) in the rose bengal uptake assay. RESULTS: The cytoplasm and nucleus of confluent HCLE cells cultured in media without serum, lacking the expression of MUC16 but not MUC1, as well as human corneal fibroblasts, which do not express mucins, stained with rose bengal. Culture of HCLE cells in medium containing serum resulted in the formation of islands of stratified cells that excluded rose bengal. Apical cells of the stratified islands produced MUC16 and the T-antigen carbohydrate epitope on their apical surfaces. Colocalization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charges. Increasing the amounts of divalent cations in the media reduced the cellular area protected against rose bengal uptake. CONCLUSIONS: These results indicate that stratification and differentiation of corneal epithelial cells, as measured by the capacity to produce the membrane-associated mucin MUC16 and the mucin-associated T-antigen carbohydrate on their apical surfaces provide protection against rose bengal penetrance in vitro and suggest a role for membrane-associated mucins and their oligosaccharides in the protection of ocular surface epithelia.

Mucin gene expression is not regulated by estrogen and/or progesterone in the ocular surface epithelia of mice.

PURPOSE: Dry eye syndrome is prevalent in post-menopausal women, and post-menopausal women secrete less mucus in their reproductive tracts. Using a mouse model, the purpose of this study was to determine if estrogen and/or progesterone regulates Muc4 and Muc5AC gene expression in the ocular surface epithelia, as the hormones do in reproductive tract epithelia. METHODS: Adult C57BL/6 mice were ovariectomized, and 19 days later, pellets containing estrogen, progesterone, or a combination were inserted subcutaneously. Ocular surface and reproductive tract tissues were harvested following seven days of hormone treatment. A control group consisted of ovariectomized mice that received no hormone treatment. Real-time reverse transcription-polymerase chain reaction was used to determine the tissue expression levels of mucin mRNA of each treatment group relative to the control. Muc4 mRNA expression levels were determined for the reproductive tract, and both Muc4 and Muc5AC expression levels were determined for the ocular surface epithelia. Muc4 and Muc5AC gene expression in ocular surface and Muc4 in reproductive tract epithelia was demonstrated by In Situ hybridization, and Muc4 and Muc5AC protein was demonstrated in the epithelia of animals in the experimental groups. RESULTS: The mRNA expression levels of Muc4 and Muc5AC and the immunofluorescence localization pattern in the ocular surface epithelia were not significantly different in any hormone treatment group when compared to the control ovariectomized group. By comparison, mice that were administered estrogen had a significant increase of Muc4 mRNA in the reproductive tract epithelia, progesterone given in combination with estrogen antagonized the upregulatory effects of estrogen in the reproductive tract, and the amount of Muc4 mRNA in the reproductive tract of progesterone-treated animals was not different from ovariectomized controls. Immunofluorescence localization of Muc4 in the reproductive tract epithelia of the experimental groups correlated to message levels, with lack of Muc4 protein detected in the control and progesterone groups. CONCLUSION: In comparison to reproductive tract epithelia, Muc4 and Muc5AC are not hormonally regulated by estrogen or progesterone in the ocular surface epithelia of mice. These data demonstrate that regulation of epithelial mucin genes is tissue specific.

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.

MUC16 mucin is expressed by the human ocular surface epithelia and carries the H185 carbohydrate epitope.

PURPOSE: H185 antibody has been shown to recognize a carbohydrate epitope on a membrane-associated mucin in the apical surfaces of the corneal and conjunctival epithelia. The distribution of this antibody is altered on the surfaces of conjunctival epithelial cells of dry eye patients. The purpose of this work was to determine whether the H185 antibody recognizes the recently cloned membrane-associated mucin MUC16 (formerly CA125 antigen). METHODS: To determine whether ocular surface epithelia express MUC16, the relative expression of the MUC16 mucin gene was determined by real-time PCR on reverse transcription products from RNA isolated from human corneal and conjunctival tissues, as well as from immortalized human corneal-limbal epithelial cell (HCLE) cultures. To determine the distribution of MUC16 mRNA and protein in the ocular surface epithelia, in situ hybridization and immunohistochemistry were performed on sections of corneal and conjunctival epithelia using, respectively, a MUC16 antisense oligoprobe and the antibodies OC125, VK-8, and R16 raised against the MUC16 mucin. Determination of whether MUC1 and MUC16 mucins carry the H185 carbohydrate epitope was achieved with the respective mucins isolated from HCLE protein extracts, using one- or two-step immunoprecipitation assays and immunodepletion experiments followed by Western blot analysis. RESULTS: MUC16 mucin transcripts were detected in the human ocular surface epithelia and in corneal cell cultures. MUC16 mRNA and protein localized to the apical cell layers of the cornea and to the suprabasal region of the conjunctival epithelium. In HCLE cultures, MUC16 protein was detected in apical cells of islands of stratified cells. Immunofluorescence microscopy demonstrated exact colocalization of the MUC16 mucin and the H185 carbohydrate epitope in sections of human corneal tissue. Immunoprecipitated MUC16 mucin was recognized by the H185 antibody and vice versa, indicating that MUC16 mucin carries the H185 epitope. Immunodepletion with H185 antibody resulted in no OC125 antibody reactivity. No cross-reactivity between immunoprecipitated MUC1 and the H185 antibody was observed. CONCLUSIONS: This study demonstrates that the membrane-associated mucin MUC16 is expressed by the human ocular surface epithelia and that MUC16 carries the H185 carbohydrate epitope. Future studies on the expression of MUC16 and the characterization of the molecular structure of the H185 carbohydrate epitope will determine their biological significance on the healthy ocular surface and in dry eye syndrome.

Mucin gene expression in immortalized human corneal-limbal and conjunctival epithelial cell lines.

PURPOSE: The corneal and conjunctival epithelia, which cover the ocular surface, play an important role in preventing pathogen penetrance into the eye and maintaining a wet-surface phenotype by producing highly hydrophilic mucin molecules for their apical surfaces. Ocular surface infections, wounding, and pathologies resulting in dry eye threaten sight and can cause blindness. Understanding the ocular surface defense mechanisms that mucins provide has been hampered by the lack of immortalized human corneal and conjunctival epithelial cell lines that retain mucin gene expression patterns of the native tissue. The purpose of this work was to characterize newly developed immortalized corneal and conjunctival cell lines using mucin gene expression as markers of differentiation. METHODS: The cell lines were derived as described by a previously published process. Primary cultures of corneal-limbal and conjunctival epithelia were sequentially transduced to express a dominant negative p53 protein and a p16(INK4A/Rb)-resistant, mutant cdk4 protein, which enabled the cells to bypass a senescence mechanism recently identified for primary cultures of keratinocytes. These cells were then transduced to express the catalytic subunit of telomerase to permit them to retain their telomeres and divide indefinitely. Cellular morphology and expression of mucin genes in the two cell lines, designated HCLE for the human corneal-limbal line and HCjE for the human conjunctival cell line, were determined after culture on plastic, type I collagen, or Matrigel, in coculture with fibroblasts, and in severe combined immunodeficient (SCID) mice. Expression of the epithelial cell mucins was assayed by reverse transcription, real-time polymerase chain reaction, immunoblot analysis, or immunohistochemistry and compared with expression in native cornea and conjunctiva. RESULTS: When grown in high-calcium medium on plastic and type I collagen, cells of both lines stratified, exhibiting multiple cell layers. In Matrigel, both cell lines formed cell aggregates that contained lumens. In the SCID mice, the conjunctival cell line formed stratified layers under the kidney capsule. The corneal cell line expressed keratins K3 and K12, the keratins that are corneal-epithelial-specific, and both cell lines expressed K19. As in native tissue, the HCLE and HCjE cell lines expressed the membrane-associated mucins, MUC1, -4, and -16, although their levels were generally lower. Levels of MUC4 and -16 mRNA were the most comparable to native tissue, particularly when cultured on plastic. Apical cells of the stratified cultures were the cells that expressed the membrane-associated mucins MUC1 and -16. Goblet-cell-specific MUC5AC mRNA and protein was detected in a small population of HCjE cells only when using type I collagen as a substrate or when cells were cocultured with fibroblasts. Both cell lines produced glycosylated mucins as indicated by binding of H185 antibody, an antibody that recognizes a carbohydrate epitope on mucins. CONCLUSIONS: The immortalized corneal (HCLE) and conjunctival (HCjE) cell lines exhibit the mucin gene expression repertoire of their native epithelia. These cell lines will be useful in determining regulation of ocular surface mucin gene expression and, potentially, goblet cell differentiation.

The cell-layer- and cell-type-specific distribution of GalNAc-transferases in the ocular surface epithelia is altered during keratinization.

PURPOSE: It has been hypothesized that the biosynthesis of O-linked glycans on proteins, particularly on the highly O-glycosylated mucins, by the corneal and conjunctival epithelium is necessary for the protection and maintenance of a healthy ocular surface. The initial step in O-glycosylation is the enzymatic addition of N-acetyl galactosamine (GalNAc) to serine and threonine residues by a large family of polypeptide GalNAc-transferases (GalNAc-Ts). The purpose of this study was to determine the cellular distribution of GalNAc-Ts in the normal ocular surface epithelia and to compare their distribution with that in pathologically keratinized conjunctival epithelia. METHODS: Five conjunctival biopsy specimens and 5 corneas from normal individuals, and 14 conjunctival specimens from patients with ocular cicatricial pemphigoid (OCP) were used. Based on the histologic characteristics of their epithelia, OCP specimens were divided into two groups: less advanced, nonkeratinized (n = 6), and late-stage, keratinized (n = 8). Five monoclonal antibodies raised against the GalNAc-T1, -T2, -T3, -T4, and -T6 isoenzymes, were used for immunofluorescence microscopic localization according to standard protocols. RESULTS: Immunohistochemical studies revealed the presence of GalNAc-T2, -T3, and -T4 isoforms within the stratified epithelium of the cornea and the conjunctiva. The GalNAc-T4 isoenzyme was found in the apical cell layers, whereas GalNAc-T2 was found in the supranuclear region of the basal cell layers of both cornea and conjunctiva. GalNAc-T3 was distributed throughout the entire ocular surface epithelium, whereas GalNAc-T1 was found in scattered cells in conjunctiva only. Binding of antibody to GalNAc-T6 was restricted exclusively to conjunctival goblet cells. There were distinct alterations in expression patterns of GalNAc-T2, -T6, and -T1 in nonkeratinized OCP epithelia compared with normal epithelia. Both GalNAc-T2 and -T6 were expressed in the apical stratified epithelia, and T1 was detected in all cell layers in five of six biopsy specimens. By comparison with nonkeratinized OCP epithelia, a marked reduction in the binding of GalNAc-T antibody was observed in the late-stage keratinized conjunctival epithelia of patients with OCP. In all samples, apical GalNAc-T2 was absent, and GalNAc-T6 was entirely absent. Only one of eight samples was positive for GalNAc-T1. CONCLUSIONS: The presence of GalNAc-T isoenzymes in the human corneal and conjunctival epithelia is cell-layer and cell-type specific. The increased distribution of GalNAc-Ts observed in early stages of the keratinization process in patients with OCP suggests a compensatory attempt of the ocular surface epithelium to synthesize mucin-type O-glycans to maintain a wet-surface phenotype. This early increase in isoenzymes in nonkeratinized OCP epithelia is reduced as keratinization proceeds in the disease.

Variation in the amount of T antigen and N-acetyllactosamine oligosaccharides in human cervical mucus secretions with the menstrual cycle.

It has been hypothesized that the carbohydrate portion of mucins present in the endocervical canal plays an important role in conferring specific physicochemical properties (e.g. viscosity and hydration) to the mucus gel through the menstrual cycle. Our recent finding showing an increase in the amount of MUC5B mucin protein at midcycle has raised the question of whether the mucin O-glycan content also varies to confer specific hydrodynamic properties to secreted mucins during ovulation. Using lectins as carbohydrate probes, we have identified two common mucin oligosaccharide structures, T antigen and N-acetyllactosamine, within secretory granules in human endocervical glands during the proliferative phase of the menstrual cycle. Analysis of endocervical secretions by enzyme-linked lectin assay revealed that the amounts of T antigen and N-acetyllactosamine are maximal at midcycle. Lectin blot assay of immunoprecipitated MUC5B demonstrated that the mucin is a carrier of the T antigen and N-acetyllactosamine oligosaccharides in cervical mucus secretions. The amounts of T antigen and N-acetyllactosamine oligosaccharides on MUC5B increased during the first half of the cycle, peaked at midcycle, and dramatically dropped at the end of the cycle. The peak in MUC5B mucin protein and carbohydrate content coincides with the change in mucus character that occurs at midcycle. The role of O-glycans on mucins may be to hold water within the endocervical canal during ovulation to facilitate sperm migration.

Goblet cell numbers and epithelial proliferation in the conjunctiva of patients with dry eye syndrome treated with cyclosporine.

OBJECTIVES: To compare conjunctival goblet cell numbers as well as epithelial turnover in patients with non-Sjögren syndrome--associated keratoconjunctivitis sicca (NSS-KCS) and those with SS-KCS before and after 6 months of treatment with topical cyclosporine A (CsA) ophthalmic emulsion. METHODS: Conjunctival biopsy specimens from 16 patients with NSS-KCS and 12 with SS-KCS were obtained at baseline and after 6 months' therapy with CsA or vehicle alone. Conjunctival biopsy specimens were also obtained from 11 normal subjects. Periodic acid--Schiff staining determined the number of goblet cells present. Immunofluorescence microscopy for Ki-67 localization was used to evaluate the number of actively cycling cells. RESULTS: Periodic acid--Schiff staining showed fewer goblet cells at baseline in both dry eye populations when compared with normal subjects (P<.001). After 6 months of CsA treatment, conjunctival biopsy specimens of both NSS-KCS and SS-KCS groups revealed an increase in goblet cells compared with baseline (P<.05). More Ki-67--positive cells were observed in NSS-KCS conjunctiva at baseline than in normal conjunctiva (P<.05) whereas numbers of these cells in SS-KCS conjunctiva were similar to normal at baseline. After 6 months of CsA treatment, conjunctival biopsy specimens of NSS-KCS revealed a decrease in Ki-67--labeled cells compared with baseline (P<.001). In contrast, no substantial change was observed for CsA treatment in patients with SS-KCS. CONCLUSIONS: Treatment of dry eye syndrome for 6 months with topical CsA resulted in an increase in goblet cell numbers in patients with NSS-KCS and SS-KCS and a decrease in epithelial turnover in those with NSS-KCS. Reducing ocular surface inflammation might have an effect on the proliferative activity of the epithelium.