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. Author manuscript; available in PMC: 2019 Sep 1.
Published in final edited form as: Toxicol Lett. 2017 Nov 7;293:77–81. doi: 10.1016/j.toxlet.2017.11.005

Sulfur mustard induced mast cell degranulation in mouse skin is inhibited by a novel anti-inflammatory and anticholinergic bifunctional prodrug

Laurie B Joseph 1, Gabriella M Composto 1, Roberto M Perez 1, Hong-Duck Kim 2, Robert P Casillas 3, Ned D Heindel 4, Sherri C Young 4, Carl J Lacey 4, Jaya Saxena 4, Christophe D Guillon 4, Claire R Croutch 3, Jeffrey D Laskin 1, Diane E Heck 2
PMCID: PMC5938161  NIHMSID: NIHMS921659  PMID: 29127031

Abstract

Sulfur mustard (SM, bis(2-chloroethyl sulfide) is a potent vesicating agent known to cause skin inflammation, necrosis and blistering. Evidence suggests that inflammatory cells and mediators that they generate are important in the pathogenic responses to SM. In the present studies we investigated the role of mast cells in SM-induced skin injury using a murine vapor cup exposure model. Mast cells, identified by toluidine blue staining, were localized in the dermis, adjacent to dermal appendages and at the dermal/epidermal junction. In control mice, 48–61% of mast cells were degranulated. SM exposure (1.4 g/m3 in air for 6 min) resulted in increased numbers of degranulated mast cells 1–14 days post-exposure. Treatment of mice topically with an indomethacin choline bioisostere containing prodrug linked by an aromatic ester-carbonate that targets cyclooxygenases (COX) enzymes and acetylcholinesterase (1% in an ointment) 1–14 days after SM reduced skin inflammation and injury and enhanced tissue repair. This was associated with a decrease in mast cell degranulation from 90% to 49% 1–3 days post SM, and from 84% to 44% 7–14 days post SM. These data suggest that reduced inflammation and injury in response to the bifunctional indomethacin prodrug may be due, at least in part, to abrogating mast cell degranulation. The use of inhibitors of mast cell degranulation may be an effective strategy for mitigating skin injury induced by SM.

Keywords: Acetylcholinesterase, Mast cells, Sulfur mustard, Countermeasures, Epidermis

Introduction

Sulfur mustard (SM, bis(2-chloroethyl sulfide) is a potent vesicating agent that has been used in chemical warfare (Dacre & Goldman, 1996; Graham & Schoneboom, 2013; Shakarjian et al., 2010; Thiermann et al., 2013; Vogt et al., 1984). The most sensitive targets are the skin, eyes and lung (Graham & Schoneboom, 2013; Kehe & Szinicz, 2005). As a bifunctional alkylating agent, SM induces tissue injury by reacting with a variety of cellular macromolecules including nucleic acids, proteins and lipids (Ludlum et al., 1994; Papirmeister et al., 1985). Modifications in DNA result in the formation of mono- and bifunctional adducts which cause both single and double strand breaks resulting in apoptosis and necrosis (Dacre & Goldman, 1996; Joseph et al., 2011; Kehe et al., 2009; Papirmeister et al., 1985). Alkylation of proteins and other components of the skin can cause alterations in tissue structure and function (Laskin et al., 2010). Erythema and pruritus are the first signs of SM-induced dermal injury. Subsequently, degradation of the epidermis can result in the formation of fluid filled blisters and necrotic lesions (Firooz et al., 2011; Graham & Schoneboom, 2013; Hejazi et al., 2016; Kehe et al., 2008; Rice, 2003). Prolonged healing including chronic inflammation with xerosis and tissue remodeling produce scarring which is a hallmark of SM-induced skin toxicity (Ghanei et al., 2010; Graham et al., 2005; Rowell et al., 2009).

In earlier studies we observed extensive inflammation and damage in mouse skin 1–3 days post SM exposure (Joseph et al., 2016; Joseph et al., 2011). Epithelial cell hypertrophy, hyperplasia, edema, parakeratosis and loss of epidermal structures were noted, along with increases in enzymes generating pro-inflammatory mediators including myeloperoxidase and COX-2. Wounding was associated with a prominent infiltration of inflammatory cells into the tissue, which consisted primarily of neutrophils and macrophages. In the mouse skin, wound healing ensued 7–14 days post exposure with a neoepidermis migrating over the wound edges. Of particular interest were our findings that throughout the pathogenic response to SM, extensive mast cell degranulation occurred (Joseph et al., 2011). Similar degranulation of mast cells in mouse skin has been reported following exposure to nitrogen mustard (Composto et al., 2016; Jain et al., 2014). Mast cells are known to release a variety of mediators that control hemostasis and inflammation during the early stages of tissue injury, and cellular proliferation and remodeling during later stages of wound repair suggesting that they are key to both processes (Joseph et al., 2016; Kennelly et al., 2011; Theoharides et al., 2012; Weller et al., 2006). In studies with nitrogen mustard, we found that a novel bifunctional prodrug consisting of the non-steroidal anti-inflammatory drug (NSAID) indomethacin covalently linked to a choline bioisostere that targets cyclooxygenase enzymes and acetylcholinesterase, effectively inhibited mast cell degranulation (see Figure 1 for structure) (Composto et al., 2016). This NSAID prodrug was also effective in reducing nitrogen mustard-induced skin injury in mouse skin suggesting a link between inhibition of mast cell degranulation and inhibition of vesicant-induced skin toxicity. Other studies in our laboratory have also demonstrated that the NSAID prodrug was effective in mitigating SM-induced skin injury in mouse skin (Chang et al., 2014; Lacey et al., 2016). The purpose of the present studies was to determine if this anti-inflammatory/wound healing drug could also inhibit mast cell degranulation in mouse skin following exposure to SM.

Figure 1.

Figure 1

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Chemical structure of NDH 4338 [NDH4338 4-(((3,3-dimethylbutoxy)carbonyl)oxy) benzyl 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetate] is a carbamate composed of an anti-inflammatory moiety, indomethacin, linked via an aromatic ester–carbonate to a choline bioisostere 3.3-dimethyl-1-butanol.

Materials and Methods

All SM animal studies were performed at MRIGlobal, Kansas City, MO as previously described (Chang et al., 2014; Joseph et al., 2016; Joseph et al., 2011). The indomethacin prodrug, referred to as NDH4338 (Fig. 1), was synthesized as previously reported (Young et al., 2010). Mice (male SKH1-Hr mice, 5–8 weeks of age, Charles River Laboratories, Wilmington, MA) were treated topically with an ointment containing 1% NDH4338 twice per day beginning 1 h after SM exposure (Composto et al., 2016). For analysis of mast cells, full thickness skin punch biopsies were collected 1, 3, 7, and 14 days post exposure. Tissue was trimmed, and stored in ice cold phosphate buffered saline (PBS) containing 2% paraformaldehyde/3% sucrose at 4°C. The skin was then rinsed in ice cold PBS containing 3% sucrose, transferred to ethanol (50%), and paraffin embedded. Six micron histological sections were prepared and stained with toluidine blue O (Sigma Chemical, St. Louis MO) to visualize mast cell metachromatic/basophilic granules. Extrusion of basophilic toluidine blue stained granules was evidence of mast cell degranulation (Composto et al., 2016; Joseph et al., 2016; Joseph et al., 2011; Puebla-Osorio et al., 2017; Tewari-Singh et al., 2009). Mast cell number and the percentage of degranulated mast cells at the dermal/epidermal junction were analyzed in tissue sections as previously described (Composto et al., 2016; Joseph et al., 2011). Data were analyzed using a two way ANOVA and expressed as mean ± S.E. (n = 6). A p value ≤ 0.05 was considered significant.

Results and Discussion

In earlier work we reported that SM causes time-dependent structural changes in the skin 1–14 days post exposure (Joseph et al., 2016; Joseph et al., 2011; Joseph et al., 2014; Wohlman et al., 2016). At early times (˂ 3 days post SM), there is an influx of inflammatory cells and a loss of the stratum corneum; this is associated with the formation of a well-defined eschar. After 3–7 days post-SM, a thickened eschar is evident, along with areas of increased cellularity. Hemorrhage was apparent throughout the dermis. The dermis was compacted such that the reticular and papillary dermis were indistinguishable. After 7–14 days post exposure, areas of inflammation were still evident in the dermis. A neoepidermis covered over 75% of the wound site and was found to contain areas of parakeratosis within the presumptive stratum corneum overlying a disorganized hyperplastic epidermis. Treatment of the mice with NDH4338 reduced the inflammatory cell infiltrate at 3 days post-SM. By 7 days, a well demarcated neoepidermis was observed below a diminished eschar. The dermis was no longer as compact and easily distinguished from the migrating neoepidermis. At 14 days post NDH4338, a normal stratum corneum superior to a well-organized epidermis covering approximately 90% of the wound site was evident.

Mast cells were identified in the dermis, adjacent to dermal appendages and at the dermal/epidermal junction (Fig. 2) (Puebla-Osorio et al., 2017). Approximately 50–60% of the mast cells in control skin were degranulated; the percentage of degranulated mast cells increased to 85–95% in mouse skin 1–14 days post SM (Fig. 2, left and center panels, and Fig. 3). In general, the number of mast cells in the skin remained constant after SM exposure, although a small decrease was noted after 3 days, possibly due to extensive tissue injury at this time. In skin treated with NDH4338 after SM exposure, a significant reduction in mast cell degranulation was noted and the percentage of degranulated cells was similar to control skin (Fig. 2, right panel, and Fig. 3). These data indicate that SM readily increased mast cell degranulation. Moreover, the indomethacin prodrug was effective in suppressing mast cell degranulation.

Figure 2.

Figure 2

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Toluidine blue staining of mast cells in hairless mouse skin following SM exposure. Histological sections prepared 1, 3, 7, and 14 days post exposure to control or SM without and with NDH4338 treatment were stained with toluidine blue (original magnification, ×400). Left Panel: Control mouse skin. Center Panel: Mouse skin treated with SM. Right Panel: Mouse skin treated with SM and NDH4338. Black arrows, mast cells. Yellow arrows, degranulated mast cells. Insets show intact and degranulated mast cells. One representative section from 4 mice/treatment group is shown.

Figure 3.

Figure 3

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Effect of SM on mast cell degranulation. Histological sections prepared 1, 3, 7, and 14 days post exposure to control or SM without and with NDH4338 treatment were stained with toluidine blue. Left Panel: Total number of mast cells. Right Panel: Percentage of degranulated mast cells. Each bar is the mean ± S.E. (n = 4) of 15 oil immersion fields, *Significantly different from control (p<0.05). +Significantly different from SM exposure (p<0.05).

Earlier work from our laboratories and others has shown that treatment of mouse skin with SM or SM analogs such as nitrogen mustard or the half mustard 2-chloroethyl ethyl sulfide causes degranulation of mast cells (Composto et al., 2016; Jain et al., 2011; Joseph et al., 2011; Mouret et al., 2015). A question arises as to the role of mast cells in mustard-induced injury and repair. Mast cell activation is known to contribute to a number of physiological processes in the skin including inflammation, the formation of granulation tissue, the resolution of inflammation, cell proliferation and wound healing (Kalesnikoff & Galli, 2011; Kennelly et al., 2011). Mediators released by mast cells such as histamine, serotonin and heparin are important in regulating many inflammatory reactions including dilation of capillaries and venules, changes in blood vessel permeability, and endothelial cell activation (Conti et al., 2017; Rao & Brown, 2008; Theoharides et al., 2012). This can account for the classic signs of inflammation at sites of SM exposure including heat, redness, swelling and pain. Among the many functions of heparin, suppressing coagulation in the local microenvironment is likely important in regulating leukocyte trafficking to sites of injury (Saban et al., 1997; Stockmann et al., 2014). In this regard, treatment of skin with SM and related analogs is known to cause extensive infiltration of neutrophils and macrophages (Dannenberg et al., 1985; Jain et al., 2014; Joseph et al., 2011; Mouret et al., 2015; Tewari-Singh et al., 2009). This is, at least in part, mediated by the release of chemokines such as IL-8, MCP-1, RANTES, CXCL10, CXCL1 and CXCL8 by mast cells that control cell migration to sites of injury (Cardamone et al., 2016; Ramos et al., 2003). Additional mediators released by mast cells including bioactive lipids such as prostaglandin D2, leukotriene B4, leukotriene C4 and thromboxane A2, as well as chemokines and growth factors such as interferon-γ, IL-1β, IL-6, M-CSF, TNFα, βFGF and VEGF control leukocyte activation (Taketomi & Murakami, 2017). These mediators are also important in wound repair, regulating keratinocyte migration, proliferation and differentiation (Weller et al., 2006).

Mast cells also release a number of proteases important in regulating inflammation and tissue remodeling including tryptase and chymase, cathepsin G, granzyme B, kallikrein-related protease-8 and various carboxypeptidases (Prieto-Garcia et al., 2012). Proteinase-proteoglycan complexes such as the heparin-containing tryptase-serglycin proteoglycans (SGPG) display anticoagulant activity in the wound and are important in fibrin deposition and fibroblast and keratinocyte proliferation and differentiation (Stevens & Adachi, 2007). These enzymes are also important in controlling collagen cross-linking and extracellular matrix turnover during wound healing. In this regard, these enzymes activate metalloproteinases such as matrix metalloproteinase-9 (MMP-9), which is known to be expressed in skin following SM exposure (Gerecke et al., 2009; Shakarjian et al., 2006; Tewari-Singh et al., 2012). The SGPG enzymes are also important in generating signaling molecules by interacting with protease-activated receptors releasing peptides and growth factors, as well as proangiogenic cytokines involved in tissue remodeling during the wound healing process (Gorbacheva et al., 2017).

It should be noted that mast cell degranulation persists following exposure of mouse skin to SM, not only in the initial stages of inflammation and tissue injury, but also through the resolution of inflammation and wound repair. As this is a time dependent process, it is likely that the release of mediators by mast cells over time is tightly controlled. Distinct mast cell populations may also contribute to the production of mediators during the different phases of SM-induced skin injury and wound healing. This may depend on resident mast cells, as well as mast cells infiltrating into the tissue during the inflammatory process (Sibilano et al., 2014).

NDH4338 was found to suppress mast cell degranulation at all time points up to 14 days post SM treatment. This was associated with inhibition of inflammation as well as reduced injury and enhanced tissue repair. These findings suggest that NDH4338 displays multiple mechanisms of action and that mast cells are not the only target for the drug. NDH4338 contains a non-steroidal anti-inflammatory agent that can inhibit tissue cyclooxygenases reducing the production of proinflammatory prostaglandins (Saxena et al., 2015). Indomethacin can also suppress the activity of neutrophils and macrophages, possibly by modulating chemokine/cytokine signaling (Minakami et al., 1993). NDH4338 also contains a reversible acetylcholinestase inhibitor. Earlier studies have shown that skin function can be regulated by non-neuronal cholinergic activity (Kurzen et al., 2007). Acetylcholinesterase has been identified in mast cells. One could speculate that this enzyme is important in regulating mast cell function following injury and during wound repair (Nechushtan et al., 1996). Previous studies have shown that inhibitors of acetylcholinesterase can suppress proinflammatory cytokine release and this may also contribute to the ability of NDH4338 to counter SM-induced skin injury (Pavlov et al., 2009; Saeed et al., 2005). In this regard, earlier studies have shown that a non-steroidal anti-inflammatory agent linked via a hydrocarbon chain to an acetylcholinesterase inhibitor was also an effective inhibitor of inflammation and injury induced by SM in a mouse ear skin model (Amitai et al., 2006). The advantages of this drug, as well as NDH4338, is that they can readily target multiple pathways mediating SM-induced cutaneous injury by regulating both cyclooxygenases and acetylcholine, the substrate for acetylcholinesterase. Acetylcholine is also known to be produced by infiltrating leukocytes and keratinocytes during inflammation and it may regulate proliferation and differentiation of epidermal cells during wound healing (Grando, 1997).

In summary, our data indicate that mast cell degranulation is an important component of SM-induced injury and repair. Thus, a bifunctional indomethacin prodrug linked to a choline bioisostere that displays activity as a cyclooxygenase inhibitor and an acetylcholinesterase inhibitor, was shown to suppress both nitrogen mustard- and SM-induced skin inflammation and tissue injury and mast cell degranulation (Composto et al., 2016; Joseph et al., 2016; Joseph et al., 2011). At the present time, the precise mechanism by which NDH4338 inhibits mast cell degranulation is not known. A number of drugs that display anti-inflammatory activity function as mast cell stabilizers thereby preventing the degranulation of mast cells (Finn & Walsh, 2013). It is possible that NDH4338 acts as a mast cell stabilizer. Further studies are needed to determine the precise sites of action of NDH4338 and whether it acts directly to inhibit mast cell function.

Highlights.

  • Sulfur mustard induces skin injury

  • Sulfur mustard exposure degranulates mast cells in the dermis

  • Bifunctional indomethacin prodrug abrogates mast cell degranulation

Acknowledgments

This work was supported by the National Institutes of Health CounterACT program through the National Institutes of Arthritis and Musculoskeletal and Skin Diseases U54AR055073, by ES005022 and by T32ES007148.

List of Abbreviations

AChE

acetylcholinesterase

COX

cyclooxygenase

D/E

dermal epidermal junction

MMP9

matrix metalloproteinase 9

NDH4338

choline bioisostere composed of indomethacin and an AChE inhibitor

NSAID

non-steroidal anti-inflammatory drug

PBS

phosphate buffered saline

SGPG

serglycin proteoglycans

SM

sulfur mustard

Footnotes

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