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. Author manuscript; available in PMC: 2022 Aug 1.
Published in final edited form as: Exp Mol Pathol. 2021 Jun 1;121:104656. doi: 10.1016/j.yexmp.2021.104656

Characterization of the rabbit conjunctiva: Effects of sulfur mustard

Laurie B Joseph a,*, Marion K Gordon a, Jieun Kang a, Claire R Croutch b, Peihong Zhou a, Diane E Heck c, Debra L Laskin a, Jeffrey D Laskin d
PMCID: PMC9006340  NIHMSID: NIHMS1787565  PMID: 34081961

Abstract

Sulfur mustard (SM; bis (2-chloroethyl) sulfide) is a potent vesicant which causes irritation of the conjunctiva and damage to the cornea. In the present studies, we characterized the ocular effects of SM in New Zealand white rabbits. Within one day of exposure to SM, edema and hazing of the cornea were observed, followed by neovascularization which persisted for at least 28 days. This was associated with upper and lower eyelid edema and conjunctival inflammation. The conjunctiva is composed of a proliferating epithelium largely consisting of stratified columnar epithelial cells overlying a well-defined dermis. Superficial layers of the conjunctival epithelium were found to express keratin 1, a marker of differentiating squamous epithelium, while in cells overlying the basement membrane expressed keratin 17, a marker of stratified squamous epithelium. SM exposure upregulated keratin 17 expression. Mucin 5AC producing goblet cells were interspersed within the conjunctiva. These cells generated both acidic and neutral mucins. Increased numbers of goblet cells producing neutral mucins were evident after SM exposure; upregulation of expression of membrane-associated mucin 1 and mucin 4 in the superficial layers of the conjunctival epithelium were also noted. These data demonstrate that ocular exposure of rabbits to SM causes significant damage not only to the cornea, but to the eyelid and conjunctiva, suggesting multiple targets within the eye that should be assessed when evaluating the efficacy of potential countermeasures.

Keywords: Vesicants, Goblet cells, Mucin 5ac, Mucin 1, Mucin 4, Eyelids

1. Introduction

The conjunctiva is a translucent mucous membrane covering the inner surface of the eyelid where it acts to protect the eye from injury and infection by producing mucins and aqueous proteins which function to lubricate the cornea. The conjunctiva is composed of a stratified non-keratinized columnar and squamous epithelium that is interspersed with goblet cells, and accessory appendages including lymph nodes and lacrimal glands (Knop and Knop, 2007). As apocrine secretory cells, goblet cells release gel-forming and water soluble mucins from their apical surface onto the cornea. Superficial conjunctival epithelial cells also synthesize membrane-associated mucins (Ablamowicz and Nichols, 2016; Fini et al., 2020; Georgiev et al., 2019; Hori et al., 2004). Conjunctival mucins largely consist of O-glycosylated glycoproteins which are important in maintaining tear film diffusivity and stability (Brockhausen et al., 2018; Davidson and Kuonen, 2004; Rodriguez Benavente and Argueso, 2018; Royle et al., 2008). Goblet cell mucins contain carbohydrate chains often substituted with carboxylate- and/or -sulphonate groups that are charged at physiological pH, and/or neutral carbohydrate chains lacking acidic groups. Mucin 5AC (MUC5AC), a secreted mucin which contains both charged and neutral carbohydrates, is the most well characterized goblet cell mucin and has been used as a marker for conjunctival goblet cells (Gipson and Argueso, 2003; Hori, 2018). Epithelial derived mucins such as mucin 1 (MUC1) and mucin 4 (MUC4) are membrane associated (Argueso and Gipson, 2001). Extracellular domains can be shed from epithelial cells and released into tear film where they function to limit adherence of debris and pathogens to the corneal surface (Dartt, 2004; Gipson and Argueso, 2003; Hodges and Dartt, 2013). Decreases in numbers of goblet cells, changes in goblet cell function, and glycosylation of MUC1, are associated with damage to the ocular surface and have been linked to various ocular diseases including Sjogren's Syndrome, dry eye disease, and allergic conjunctivitis (Garcia-Posadas et al., 2016; Garcia et al., 2002; Gipson, 2016; Watanabe, 2002).

Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a bifunctional alkylating agent which has been used in chemical warfare. It is a potent vesicating agent that targets the eyes, causing visual impairment (Ghasemi et al., 2009). Characteristics of acute ocular toxicity following exposure to SM include blepharospasm, lacrimation, irritation, pain and photophobia (Panahi et al., 2017; Solberg et al., 1997; Vidan et al., 2002). Depending on the dose and duration of exposure, SM can damage the cornea causing edema and hazing, ulcerations, and neovascularization (Etezad-Razavi et al., 2006). SM has also been reported to cause conjunctivitis including edema, inflammation, burning, and pain (Fig. 1) (Derby, 1919; Mann and Pullinger, 1942; Maumenee and Scholz, 1948; Panahi et al., 2017; Vidan et al., 2002). SM induced injury to the cornea has also been reported in rabbits; studies in the conjunctiva have not been reported following mustard exposures (Fuchs et al., 2021; Mann and Pullinger, 1942).

Fig. 1.

Fig. 1.

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Drawn-to-life image of a severely burned human eye following battlefield exposure to SM. Drawing is reproduced from “An Atlas of Gas Poisoning”, Medical Research Committee, British Expeditionary Forces, 1918. The atlas was issued to familiarize Officers with key features of chemical warfare gas injuries of the eye. In cooperation with the American Red Cross Society, this report was made available to the American Army Medical Service. The figure is a reproduction of Plate Xia. The original description of the pathology is as follows: “Early in the second day after exposure to mustard gas vapour the eyelids and the external surface of the globe show an intense inflammatory reaction. Tears stream from between the closed oedematous eyelids, which may even be blistered, and there is often severe pain behind the eyes and in the forehead. The conjunctiva is swollen, oedematous, and bright red from injection of the blood vessels. The injury of the cornea, even when severe, is not so obvious, and careful examination is of great importance for its detection. Photophobia and blepharospasm render examination of the eye very difficult”.

In the present studies we characterized the effects of SM on the conjunctiva using a rabbit model. The rabbit conjunctiva was found to largely consists of proliferating epithelial cells expressing both basal and suprabasal keratins overlying a dermis composed of loosely compacted fibrils. Interspersed in the epithelium are MUC5AC expressing goblet cells, which express both acid and neutral glycoproteins. We found that ocular exposure of rabbits to SM resulted in a marked increase in goblet cell production of neutral mucins and upregulation of membrane associated mucins. Identification of structural and biochemical changes in the conjunctiva induced by SM will aid in the identification of targets that can be exploited for the development of therapeutics that mitigate ocular toxicity of mustard vesicants.

2. Materials and methods

2.1. Animals and exposures

All animal experiments and SM exposures were performed at MRIGlobal, Kansas City, MO in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited facility following Institutional Animal Care and Use Committee (IACUC) approval. Animals received humane care in compliance with the institution's guidelines, as outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Briefly, 0.4μL neat SM (5mg = ~30nmol) was applied directly onto the central cornea of the right eye of 16 anesthetized male New Zealand white rabbits weighing between 2.5 and 4.0 kg and no less than 3 months of age (Charles River Laboratories, Kingston, NY). The eyelids were held open for 5min using an ocular speculum and then manually closed 3 times to ensure uniform spreading of SM over the cornea. The eyes were then flushed for 2 min with saline. Unexposed left eyes served as controls. The animals were observed every 10 min over 2 h of offgassing of the SM in the Chemical Surety chemical hood. Corneal neovascularization, opacity or hazing, and thickness (pachymetry) were assessed 1, 3, 7, 14, 21, and 28 days later (Gordon et al., 2010).

Corneal neovascularization and opacity were evaluated by visual inspection. For neovascularization, corneas were divided into quadrants. Invasive progression of the vessel(s) within each quadrant was measured and attributed to the quadrant of origin. The scores for each cornea quadrant were combined to yield a single neovascularization score. Neovascularization scores were assigned as follows: 0 = no vascularization; 1 = the longest vessel's length was >25% of the radius of the cornea; 2 = longest vessel's length was >50% of the radius of the cornea; 3 = longest vessel's length was >75% of the radius of the cornea; 4 = the longest vessel's length was <75% of the radius of the cornea. Corneal opacity was measured on a scale of 0 to 4, with 0 = no injury/transparent, 1 = minimal loss of transparency, 2 = moderate loss of transparency (iris, vessels or pupil visible), 3 = severe loss of transparency (either iris, vessels or pupil not visible) and 4 = diffuse loss of transparency (e.g., the iris and the pupil were not visible). Corneal thickness was measured in the central regions of the eyes using ultrasonic pachymetry (PACSCAN 300P; Sonomed, Inc., Lake Success, NY).

2.2. Histology and immunohistochemistry

On day 28, animals were euthanized; upper and lower eyelids were removed, trimmed and stored in ice cold phosphate buffered saline (PBS) containing 3% paraformaldehyde/2% sucrose. After fixation, eyelids were embedded in paraffin and 6 μm sections prepared and stained with hematoxylin and eosin (H&E) or Gomori's trichrome containing methyl (aniline) blue (Histopathology Core Facility, Rutgers University, Piscataway, NJ). Mucins synthesized by conjunctival goblet cells were stained using an alcian blue/periodic acid-Schiff kit (Richard-Allan Scientific, Kalamazoo, MI) (Yamabayashi, 1987). For immunohistochemistry, tissue sections were deparaffinized, and blocked with 20% horse serum (Invitrogen, Grand Island, NY) at room temperature for 2 h. After overnight at 4 °C, tissue sections were treated with mouse monoclonal antibodies against proliferating cell nuclear antigen (PCNA, 1:200, MilliporeSigma, Burlington, MA), keratin 1 (1:500, Abcam, Cambridge, MA), keratin 17 (1:500, Abcam), mucin 1 (MUC1, 1:500, LSBio, Seattle, WA), mucin 4 (MUC4, 1:500, Thermo Fisher Scientific, Waltham, MA), mucin 5AC (MUC5AC, 1:100, Abcam), or control mouse IgG (ProSci, Atlanta, GA). Tissue sections were washed and incubated at room temperature for 30 min with horse anti-mouse secondary antibody (Vector Labs, Burlingame, CA). Antibody binding was visualized using a DAB Peroxidase Substrate Kit (Vector Labs). Images of tissue sections were acquired at high resolution using an Olympus VS120 Virtual Microscopy System and analyzed using OlyVIA version 2.9 software (Center Valley, PA).

2.3. Statistical analyses

Data are presented as the mean ± SE and were analyzed using a student t-test. Results are considered significant with p ≤ 0.05.

3. Results

Exposure of rabbits with SM resulted in significant eyelid inflammation and corneal injury. Beginning at 1 day post SM and persisting for at least 28 days, both the upper and lower eyelids were indurated and inflamed with significant edema; conjunctival hyperemia was also noted (Fig. 2). More extensive hyperemia was evident in the upper eyelid. The third eyelid of the rabbit eye was engorged covering about 10–20% of the sclera. With increasing time, corneal opacity decreased, while corneal thickness increased (Fig. 3). By 14 days post-SM, scleral neovascularization was apparent, which continued to increase for at least 28 days (Fig. 3). In contrast, no pathologic changes in the eyelids or cornea were evident in contralateral control eyes.

Fig. 2.

Fig. 2.

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Effects of SM on rabbit eyelids. Rabbit eyes were exposed to air, CTL (left panel) or SM (right panel) as described in the Materials and Methods. Eyelids were everted and photographed 28 days later. Representative eyelids are shown. Asterisk, third eyelid; C, conjunctiva.

Fig. 3.

Fig. 3.

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Effects of SM on clinical profile of corneal injury. Rabbit eyes were exposed to air, CTL (triangle) or SM (circles) as described in the Materials and Methods. Corneal opacity, thickness, and neovascularization were assessed 1, 3, 7, 14, 21 and 28 days later. Data, mean ± SE (n = 16). *Significantly different (p < 0.05) from CTL.

In further studies, we characterized the structure of the conjunctiva. Contiguous layers of epithelial cells containing stratified columnar epithelial cells with numerous goblet cells were observed in both the upper and lower eyelids of control eyes (Fig. 4). Goblet cells, which were present in the superficial layers of the epithelium, appeared as simple polarized columnar cells with nuclei concentrated at the base. Consistent with previous reports, the apical end of the goblet cells projected into the surface of the conjunctiva (Gipson et al., 2004; Verdugo, 1990). In the upper eyelids of control eyes, the adenoid layer of the substantia propria of the conjunctiva was composed of loosely compacted fibrils overlying a more compact fibrous layer; the dermis of the lower eyelids was more homogeneous and composed of more compacted fibrils (Fig. 4).

Fig. 4.

Fig. 4.

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Structural changes in the rabbit conjunctiva following exposure to SM. Histological sections were stained with H&E (Panel A) or Gomori's trichrome (Panel B) of lower eyelid (left panels) and upper eyelid (right panels), prepared 28 days following exposure of eyes with control (CTL) (upper panels) or SM (lower panels). Gomori's trichrome containing hematoxylin which stains nuclei dark blue/black, eosin which stains keratin and cytoplasm red, and aniline blue which stains collagen I/III royal blue.One representative section from 3 rabbits/exposure group is shown (bar = 50 μm). cap, capillary; Epi, epidermis; G, goblet cells; n, goblet cell nucleus; SP, substantia propria.

Keratins are essential structural elements in epithelial cells; they are also important in regulating proliferation, cell migration, differentiation, and wound repair (Moll et al., 2008). Keratin 1, a marker of differentiating squamous epithelium, was largely expressed in the superficial layers of the conjunctiva and in cells surrounding goblet cells in the upper and lower eyelids (Fig. 5). Keratin 17, a marker of basal epithelium and skin appendages (Zhang, 2018; Zhang et al., 2019), was expressed in basal layers of the conjunctiva in the lower and upper eyelids (Fig. 6). An increase in keratin 17 was noted in the conjunctiva of SM exposed rabbits.

Fig. 5.

Fig. 5.

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Localization of keratin 1 in the rabbit conjunctiva. Histological sections of lower eyelid (left panels) and upper eyelid (right panels), prepared 28 days following exposure of eyes with control (CTL) (upper panels) or SM (lower panels), were stained with antibody to keratin 1. Antibody binding was visualized using a Vectastain Elite ABC kit. One representative section from 3 rabbits/exposure group is shown (bar = 50 μm). G, goblet cells.

Fig. 6.

Fig. 6.

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Localization of keratin 17 in the rabbit conjunctiva. Histological sections of lower eyelid (left panels) and upper eyelid (right panels), prepared 28 days following exposure of eyes with control (CTL) (upper panels) or SM (lower panels), were stained with antibody to keratin 17. Antibody binding was visualized using a Vectastain Elite ABC kit. One representative section from 3 rabbits/exposure group is shown (bar = 50 μm). G, goblet cells.

Goblet cells are known to synthesize and release mucins, these include both neutral and acidic mucins (Davidson and Kuonen, 2004; Gipson, 2016). To assess mucins in goblet cells, alcian blue/periodic acid-Schiff staining was used. At pH 2.5, alcian blue stains goblet cell acidic mucins deep blue and neutral mucins magenta, while goblet cells containing both neutral and acidic mucins stain purple. In conjunctiva from both CTL and SM exposed rabbits, goblet cells expressing neutral, acidic and neutral/acidic mucins were identified (Fig. 7). SM reduced the numbers of goblet cells containing neutral/acidic mucins in both upper and lower eyelid conjunctiva, relative to CTL (Fig. 8). SM exposure also resulted in a significant increase in the number goblet cells producing neutral mucins with no effect on acidic mucins (Figs. 7 and 8).

Fig. 7.

Fig. 7.

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Goblet cells mucins in rabbit conjunctiva. Histological sections of lower eyelid (left panels) and upper eyelid (right panels), prepared 28 days following exposure of eyes with control (CTL) (upper panels) or SM (lower panels), were stained with alcian blue/periodic acid-Schiff (PAS) stain. AM, acidic mucin; NM, neutral mucin; AM/NM, acidic/neutral mucin. One representative section from 3 rabbits/exposure group is shown (bar = 50 μm).

Fig. 8.

Fig. 8.

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Quantification of neutral, acidic and neutral/acidic mucins in goblet cells. Histological sections of lower and upper eyelids, prepared 28 days following exposure of eyes with control or SM, were stained with alcian blue/periodic acid-Schiff (PAS) stain. In each conjunctiva, the percentage of PAS positive goblet cells containing neutral, acidic and acidic/neutral (combination) mucins were determined. Bars, mean ± S.E. (n = 6). * Significantly different from control (p < 0.05).

We next immunostained rabbit eyelids for the conjunctival gel-forming water soluble mucin, MUC5AC, and two membrane-associated mucins, MUC1 and MUC4 ((Mantelli and Argueso, 2008). Similar expression of MUC5AC was noted in goblet cells in both upper and lower eyelids of control and SM exposed animals (Fig. 9). In contrast, conjunctival epithelial cells expressed MUC1 and MUC4 (Figs. 10 and 11). While MUC1 was constitutively expressed in suprabasal epithelial cells including areas surrounding goblet cells in control eyelids (upper panel Fig. 10), it was upregulated in eyelids post SM exposure (lower panel Fig. 10). An increase in MUC1 was noted on the apical surface of the conjunctiva epithelium in the lower eyelid; diffuse MUC1 expression was observed throughout the epithelium and sporadically in the upper portion of the substantia propria in the upper eyelid. Low levels of MUC4, which decorated apical epithelial membranes, were expressed in control rabbit conjunctiva (upper panel Fig. 11). Following SM exposure, increased MUC4 was evident on the apical surface of the conjunctival epithelium of the upper and lower eyelids. The cytoplasm of individual conjunctival epithelial cells also expressed increased MUC4 following SM exposure.

Fig. 9.

Fig. 9.

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Expression of MUC5AC in the rabbit conjunctiva. Histological sections of lower eyelid (left panels) and upper eyelid (right panels), prepared 28 days following exposure of eyes with control (CTL) (upper panels) or SM (lower panels), were stained with antibody to Muc5AC. Antibody binding was visualized using a Vectastain Elite ABC kit. One representative section from 3 rabbits/exposure group is shown (bar = 50 μm). G, goblet cells.

Fig. 10.

Fig. 10.

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Effects of SM on MUC1 in the rabbit conjunctiva. Histological sections of the upper eyelid, prepared 28 days following exposure of eyes with control (CTL) (left panels) or SM (right panels), were stained with antibody to MUC1. Antibody binding was visualized using a Vectastain Elite ABC kit. One representative section from 3 rabbits/exposure group is shown (bar = 50 μm). G, goblet cells.

Fig. 11.

Fig. 11.

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Effects of SM on MUC4 in the rabbit conjunctiva. Histological sections of the upper eyelid, prepared 28 days following exposure of eyes with control (CTL) (left panels) or SM (right panels), were stained with antibody to MUC4. Antibody binding was visualized using a Vectastain Elite ABC kit. One representative section from 3 rabbits/exposure group is shown (bar = 50 μm). G, goblet cells.

Conjunctival epithelial cells were also found to express PCNA, a marker of cellular proliferation. In both the upper and lower eyelids of control and SM exposed animals, PCNA was largely identified in the nuclei of epithelial cells surrounding goblet cells and in conjunctival basal cells (Fig. 12). The nuclei of goblet cells also expressed PCNA. An increase in the number of PCNA expressing conjunctival cells along the basal layer was noted in the upper eyelids following SM exposure.

Fig. 12.

Fig. 12.

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Effects of SM on expression of PCNA in the rabbit conjunctiva. Histological sections of lower eyelid (left panels) and upper eyelid (right panels), prepared 28 days following exposure of eyes with control (CTL) (upper panels) or SM (lower panels), were stained with antibody to PCNA. Antibody binding was visualized using a Vectastain Elite ABC kit. One representative section from 3 rabbits/exposure group is shown (bar = 50 μm). G, goblet cells; n, goblet cell nucleus.

4. Discussion

Early observations during World War I emphasized the fact that conjunctival irritation was a prominent feature of battlefield exposure to SM. Usually initiated 6–12 h post exposure, the conjunctiva was described as “congested, the lids very much swollen, with moderate lacrimation” (Derby, 1919; Warthin and Weller, 1919). In a number of cases, irritation was severe and persisted for prolonged periods of time (Warthin and Weller, 1919). Early rabbit studies performed to better understand the mechanism of SM injury demonstrated that exposure of the central cornea with liquid SM resulted in conjunctival edema, increased lacrimation, congestion of palpebral conjunctival vessels and erythema within 1 h and persisted for several weeks (Livingston and Walker, 1940; Mann and Pullinger, 1942; Maumenee and Scholz, 1948; Warthin and Weller, 1919). We found analogous changes in the rabbit conjunctiva following SM exposure; this was coordinate with corneal toxicity including edema and hazing, and neovascularization (Gordon et al., 2010; Kadar et al., 2009). It should be noted that damage to the conjunctiva is likely due to direct effects of SM on the tissue. However, it is also possible that mediators released from the injured cornea and in the surrounding tear fluid including matrix metalloproteinases, cyclooxygenase- and lipoxygenase-derived lipid mediators and growth factors and cytokines such as tumor necrosis factor α contribute to conjunctival damage (DeSantis-Rodrigues et al., 2016; Goswami et al., 2019; Horwitz et al., 2018; Kehe et al., 2008).

The rabbit conjunctiva is composed of a thin layer of epithelial cells overlaying a dermal matrix (Yamagiwa et al., 2020). In both the upper and lower eyelids, the epithelium of the conjunctiva is composed of stratified squamous and columnar epithelial cells, 3–5 layers thick, interspersed with goblet cells. Keratin 1 was found primarily in the superficial cells of the conjunctiva while keratin 17 was expressed in cells overlying the basement membrane. Keratin 1 is a maturation specific keratin; by combining with other keratins (e.g., keratin 9 or keratin 10) it forms intermediate filaments in tissues such as the skin where it functions to provide strength and resiliency during differentiation (Leigh et al., 1993). Expression of keratin 1 is likely important in maintaining the structural integrity of the epithelial cells as they ascend from the basal layer and mature to form the superficial epithelium of the conjunctiva. In contrast, keratin 17 was expressed in epithelial cells overlying the basement membrane. As a multifunctional protein, keratin 17 regulates many cellular processes including proliferation, differentiation and wound healing (Zhang et al., 2019). Earlier studies have shown that keratin 17 is upregulated in proliferative skin disorders including psoriasis and cancer (Yang et al., 2019). Increased expression of keratin 17 has been observed in skin following exposure with SM where it is thought to function in wound repair (Laskin et al., 2020). These data are consistent with our findings in conjunctiva where SM increased expression of keratin 17, possibly indicating that SM damages basal conjunctival epithelial cells.

Goblet cells were localized within the superficial epithelial cell layers of the conjunctiva; these cells terminated at the apical surface of the conjunctiva in both upper and lower eyelids. An important secreted mucin identified in conjunctival goblet cells is MUC5AC, a major contributor to the rheological properties of mucus that plays an important role in protecting the cornea against pathogens and airborne toxins (Garcia et al., 2002; Gipson and Argueso, 2003; Spurr-Michaud et al., 2007). Although we found homogeneous expression of MUC5AC in rabbit conjunctival goblet cells, there was considerable heterogeneity in the overall characteristics of goblet cell mucins as shown by alcian blue/periodic acid-Schiff staining (Adams and Dilly, 1989; Yamabayashi and Tsukahara, 1987). Thus, goblet cells were found to express acidic and/or neutral mucins; both acidic and neutral mucins coat the surface of the conjunctiva and cornea where they bind toxicants and protect against injury (Argueso and Gipson, 2012; Ricciuto et al., 2008; Yamabayashi and Tsukahara, 1987). Following SM exposure, an increase in goblet cells producing neutral mucins was observed. This may be important in protecting the cornea during the resolution of inflammation and wound repair (Dachir et al., 2017).

Consistent with previous reports, conjunctival epithelial cells, but not goblet cells, were found to express the membrane-associated mucins, MUC1 and MUC4 (Argueso and Gipson, 2001; Mantelli and Argueso, 2008; Pflugfelder et al., 2000). Increases in MUC1 and MUC4 were evident in the epithelial cells post-SM exposure. MUC1 and MUC4 are anchored to conjunctival epithelial cells where they are important in lubricating the cornea and creating a barrier to pathogens (van Putten and Strijbis, 2017). Structurally, they consist of a highly glycosylated extracellular domain which forms part of the glycocalyx, a short transmembrane domain, and a cytoplasmic tail; alterations in the extracellular domain have been linked to inflammatory diseases of the eye (Bafna et al., 2010; Hanson and Hollingsworth, 2016; Uchino, 2018). Abnormal glycosylation of the MUC1 extracellular domain is associated with Sjogren's dry eye syndrome, while in non-Sjogren's dry eye disease, MUC1, MUC4, as well as MUC5AC, are decreased (Argueso et al., 2003; Uchino, 2018). Aberrant production of MUC1 and MUC4 following exposure to SM may compromise tear film and ocular surface lubrication and contribute to corneal injury (Ablamowicz and Nichols, 2016; Argueso, 2020). In this regard, long term ocular effects of SM have been reported to result in tissue damage including delayed onset mustard keratitis with dry eye syndrome and decreased tear meniscus, each of which has been associated with altered mucin production (Baradaran-Rafii et al., 2011; Ghasemi et al., 2009; Panahi et al., 2018).

PCNA, a DNA polymerase cofactor important in DNA replication and repair, maintenance of chromatin structure and assembly, and cell cycle progression, is constitutively expressed in the nuclei of proliferating cells (Composto et al., 2016; Joseph et al., 2011). In both the upper and lower eyelids, PCNA was expressed in epithelial cells and in goblet cells. SM exposure resulted in increased expression of PCNA in cells in the basal layers of the upper eyelid. Enhanced proliferation of epithelial cells in the conjunctiva maybe due to SM-induced inflammation. In this regard, inflammation is known to be associated with the production of growth factors important in controlling epithelial cell proliferation (Composto et al., 2016; Laskin et al., 2020).

Under homeostatic conditions, goblet cells are known to proliferate and replace sloughed cells (Dartt and Masli, 2014; Doughty, 2017; Garcia-Posadas et al., 2016); excessive sloughing and/or suppression of goblet cell proliferation may lead to dry eye disease. Of note is the fact that battlefield exposure to SM has been reported to cause dry eye syndrome, possibly due to the loss of goblet cells (Baradaran-Rafii et al., 2011; Ghasemi et al., 2008). In line with this, using techniques in impression cytology in a rabbit model, SM has been reported to cause a loss of conjunctival goblet cells (Amir et al., 2000; Dachir et al., 2017; Naderi et al., 2009).

In summary, the conjunctiva is a specialized non-keratinizing epithelium that is a continuation of the stratified squamous epithelium of the eyelid. In rabbits, basal layers in the tissue are distinct from superficial layers with respect to patterns of keratin expression and the nature of mucins produced by goblet cells and the stratified epithelium. Presumably, superficial epithelial cells and goblet cells are actively proliferating, shed from the conjunctiva, and are continuously replaced by proliferating and differentiating basal cells. At the present time, factors that promote goblet cell formation are not known but their formation may be triggered by mechanisms that sense the need for mucins required to maintain homeostasis of the ocular surface. Our data show that SM causes changes in the conjunctival dermal matrix, secreted and membrane associated mucin profiles which correlate with corneal injury including edema, hazing and neovascularization. Further studies are needed to determine mechanisms of injury as this may lead to the development of therapeutics to mitigate the ocular effects of SM.

Acknowledgements

This work was supported by the U.S. Department of Health and Human Services, National Institutes of Health under grants AR055073, ES005022 and R25ES020721.

Footnotes

Declaration of Competing Interest

The authors have no conflict of interest for the subject matter of this paper.

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