Detection of ocular mucus in normal human conjunctiva and conjunctiva from patients with cicatricial pemphigoid using lectin probes and histochemical techniques

doi:10.1016/S0014-4835(88)80006-X

Experimental Eye Research

Volume 46, Issue 4, April 1988, Pages 485-497

https://doi.org/10.1016/S0014-4835(88)80006-X Get rights and content

Conjunctival biopsies from patients with cicatricial pemphigoid affecting the conjunctiva andpatients undergoing cataract surgery (normal conjunctiva) were snap-frozen, cryostat sectioned and incubated with fluorescein-conjugated lectins; peanut agglutinin (PNA), Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and succinylated wheat germ agglutinin (S-WGA). Controls consisted of preincubating the lectins with the appropriate blocking sugars before applying the lectins to the sections. PNA and HPA stained the mucus granules contained in the conjunctival goblet cells but did not stain mucus or glycocalyx at the ocular surface distal to the goblet cells. Native WGA and S-WGA had high affinities for conjunctival goblet cells and the apical epithelial cell layers. Native WGA stained mucus and glycocalyx at the ocular surface. This staining of the ocular surface by WGA was confirmed at the transmission electron microscopic level using WGA conjugated to ferritin. Cicatricial pemphigoid patients in this study had reduced numbers of goblet cells; however, those goblet cells which were observed in cicatricial pemphigoid conjunctiva stained positively with HPA, PNA, WGA, and SWGA as did goblet cells in normal conjunctiva.

References (23)

FosterC.S. et al.
Scanning electron microscopy of conjunctival surfaces in patients with ocular cicatricial pemphigoid
Am. J. Ophthalmol.
(1986)
GoldsteinI.J. et al.
The lectins: carbohydrate-binding proteins of plants and animals
Adv. Carbohydrate Chem. Biochem.
(1978)
HollyF.J. et al.
Wettability and wetting of the corneal epithelium
Exp. Eye Res.
(1971)
HollyF.J. et al.
Tear physiology and dry eyes
Surv. Ophthalmol.
(1977)
JohnsonG.D. et al.
A simple method of reducing the fading of immunofluorescence during microscopy
J. Immunol. Meth.
(1981)
KawanoK. et al.
Application of lecting for detection of goblet cell carbohydrates of the human conjunctiva
Exp. Eye Res.
(1984)
AllenM. et al.
The human lacrimal gland
Arch. Ophthalmol.
(1972)
CorfieldA.P. et al.
Occurrence of sialic acids
FosterC.S.
Cicatricial pemphigoid
Trans. Am. Ophthalmol. Soc.
(1987)
GibbonsR.J.
Review and discussion of role of mucus in mucosal defense

GreinerJ.V. et al.

Mucus secretory vesicles in the conjunctival epithelial cells of wearers of contact lenses

Arch. Opthalmol.

(1980)

Cited by (17)

Three-step monitoring of glycan and galectin profiles in the anterior segment of the adult chicken eye
2018, Annals of Anatomy
Citation Excerpt :
Initial work on glycosylation in the anterior parts of the eye was performed by staining for mucopolysaccharides in human specimen (Graumann and Rohen, 1958). It was then taken to the level of lectin histochemistry on mammalian specimen, covering either the entire anterior segment or distinct regions such as ciliary body, cornea, iris or trabecular meshwork (for representative studies, please see Refs. Versura et al., 1984; Holmes et al., 1985; Panjwani et al., 1986; Vanden Hoek et al., 1987; Brandon et al., 1988; Wells et al., 1988; Tuori et al., 1994; Chapman et al., 1995; Hietanen et al., 1995; Chan et al., 1999; for review, please see Ref. Prause, 1991). That receptors for glycan epitopes are present could first be demonstrated histochemically with labeled neoglycoproteins in corneal and conjunctival epithelia of pig eyes (Lange et al., 1989) (the second step given above).
A histochemical three-step approach is applied for processing a panel of sections that covers the different regions of fixed anterior segment of the adult chicken eye. This analysis gains insight into the presence of binding partners for functional pairing by galectin/lectin recognition in situ. Glycophenotyping with 11 fungal and plant lectins (step 1) revealed a complex pattern of reactivity with regional as well as glycan- and cell-type-dependent differences. When characterizing expression of the complete set of the seven adhesion/growth-regulatory chicken galectins immunohistochemically (step 2), the same holds true, clearly demonstrating profiles with individual properties, even for the CG-1A/B paralogue pair. Testing this set of labeled tissue lectins as probes (step 3) detected binding sites in a galectin-type-dependent manner. The results of steps 2 and 3 reflect the divergence of sequences and argue against functional redundancy among the galectins. These data shape the concept of an in situ network of galectins. As consequence, experimental in vitro studies will need to be performed from the level of testing a single protein to work with mixtures that mimic the (patho)physiological situation, a key message of this report.
Demonstration of discrete secreted and membrane-bound ocular mucins in the dog
1997, Experimental Eye Research
Our aims were to separate and characterize secreted canine ocular mucins, and to provide definitive evidence of membrane-bound mucins at the canine ocular surface. Mucus was collected by suction from the ocular surface of normal dogs and dispersed in guanidine hydrochloride and a cocktail of protease inhibitors. Caesium chloride density gradient centrifugation separated secreted mucins from membranes, which were collected from the top of the gradients. Membranes were extracted with octyl glucoside and screened using lectins and anti-mucin antibodies. Gradient fractions containing secreted mucins were constituted into pools on the basis of differential lectin and antibody staining. High molecular weight material from each pool was purified by gel filtration. This material, and the membrane extract, were reduced and alkylated. Vacuum blotting of separated materials after agarose gel electrophoresis was used to compare subunit structure. Density gradient profiles indicated three principal secreted glycoprotein peaks: one staining strongly with anti-mucin antibodies. Gel filtration demonstrated that each contained high molecular weight material. Vacuum blots demonstrated the presence of two secreted glycoproteins with differently sized subunits. On the basis of buoyant density, one of these may be lipid complexed. Membrane extracted material stained with anti-mucin antibodies, and vacuum blotting of this material provided evidence for two membrane-bound components. In conclusion, we have shown that normal canine ocular mucus contains two secreted mucins, each exhibiting different subunit structure; one of these mucins may undergo lipid complexation. Normal canine ocular mucus also contains two membrane-bound mucins: one of which is unique among membrane mucins in showing subunit structure.
Correlation of goblet cell density and mucosal epithelial membrane mucin expression with rose bengal staining in patients with ocular irritation
1997, Ophthalmology
Purpose: This study was designed to compare goblet cell densities and mucosal epithelial membrane mucin (MEM) expression in impression cytology specimens obtained from control subjects and patients with one of the following clinically defined diseases: aqueous tear deficiency (ATD) associated with Sjögren syndrome, ATD not associated with Sjögren syndrome, inflammatory Meibomian gland disease associated with rosacea, and Meibomian gland atrophy. These data were correlated with ocular surface rose Bengal staining scores, Schirmer scores, and HLA-DR antigen staining of conjunctival epithelial cells.
Methods: Goblet cell density and MEM expression were studied by impression imprints with immunohistochemical staining using an anti-mucosal epithelial membrane mucin antibody in the temporal and inferior bulbar and inferior tarsal conjunctiva of study subjects.
Results: Goblet cell density adjacent to the temporal limbus was significantly reduced at 3 mm posterior to the temporal limbus in both aqueous tear deficiency groups compared with the other groups and in patients with Sjögren syndrome compared with all other groups. In the inferior tarsus, goblet cell density was significantly reduced in patients with non-Sjögren syndrome ATD as compared with all other groups, except those with inflammatory Meibomian gland disease. Mucosal epithelial membrane mucin expression in the bulbar and tarsal conjunctiva was absent in a greater percentage of patients with Sjögren syndrome compared with all other groups. Total ocular surface rose Bengal staining scores were significantly higher in patients with Sjögren syndrome as compared with all other groups and in patients with non-Sjögren syndrome ATD as compared with control groups. Rose Bengal staining scores and Schirmer I test results (without anesthesia) were inversely correlated with bulbar, but not tarsal, conjunctival goblet cell densities, and with the absence of bulbar conjunctival MEM expression.
Conclusions: These results suggest that reduced goblet cell density and mucosal epithelial cell mucin expression could explain increased rose Bengal staining in patients with aqueous tear deficiency. In addition, MEM may be regarded as a marker for normal differentiation of ocular surface epithelia, with its absence signifying the development of squamous metaplasia.
Corneal and Ocular Surface Histochemistry
1993, Progress in Histochemistry and Cytochemistry
The normal human tear glycoprotein profile detected with lectin probes
1991, Experimental Eye Research
Tear samples were collected from 46 healthy volunteers evenly distributed according to sex and age (mean age 43·5 years). Samples were denatured in a Tris-HCl sample buffer containing 2-mercaptoethanol and SDS, and applied to a gradient SDS-polyacrylamide gel for electrophoresis. The proteinaceous material was transferred to nitrocellulose by a semi-dry blotting technique, and the glycoprotein content subsequently visualized by incubation with four lectins (WGA, PHA, PSA and SBA) and staining with avidin horseradish-peroxidase.
Glycoprotein bands were generally found to be significantly less frequent in persons under the age of 30 years. Apart from this the technique gave a uniform picture of the glycoprotein profile, with only modest differences according to age and/or sex. The technique may therefore be suitable for the detection of differences in the glycoprotein composition indicative of disease.
Lectin cytochemistry of the rabbit conjunctiva and lacrimal sac
1999, Japanese Journal of Ophthalmology
Four different types of lectin were applied to the rabbit conjunctiva and the lacrimal sac to examine the distribution of glycoconjugates. The conjunctival epithelium is comprised of goblet cells and nongoblet cells. The mucus granules of the goblet cells were stained with soybean agglutinin (SBA), and the cell body of the nongoblet cells was labeled with concanavalin A (Con A). The glycocalyx of the apical surfaces of the goblet cells and of the nongoblet cells was labeled with Maculura pomifera agglutinin. The lacrimal sac mucosa is comprised of superficial light and dark cells, and basal cells. The cell bodies of the light cells were stained with SBA. The glycocalyx of the apical surfaces of the light and dark cells was characteristically labeled with Con A. These results suggest that the composition of glycoconjugates is markedly different between the conjunctiva and the lacrimal sac, especially in the cell surface glycocalyx.

View all citing articles on Scopus

View full text

Detection of ocular mucus in normal human conjunctiva and conjunctiva from patients with cicatricial pemphigoid using lectin probes and histochemical techniques

Am. J. Ophthalmol.

Adv. Carbohydrate Chem. Biochem.

Exp. Eye Res.

Surv. Ophthalmol.

J. Immunol. Meth.

Exp. Eye Res.

The human lacrimal gland

Arch. Ophthalmol.

Occurrence of sialic acids

Cicatricial pemphigoid

Trans. Am. Ophthalmol. Soc.

Review and discussion of role of mucus in mucosal defense

Mucus secretory vesicles in the conjunctival epithelial cells of wearers of contact lenses

Arch. Opthalmol.