Glutathione Reaction Products with a Chemical Allergen, Methylene-diphenyl Diisocyanate, Stimulate Alternative Macrophage Activation and Eosinophilic Airway Inflammation

Abstract

Isocyanates have been a leading chemical cause of occupational asthma since their utility for generating polyurethane was first recognized over 60 years ago, yet the mechanisms of isocyanate asthma pathogenesis remain unclear. The present study provides in vivo evidence that a GSH mediated pathway underlies asthma-like eosinophilic inflammatory responses to respiratory tract isocyanate exposure. In naïve mice, a mixture of GSH reaction products with the chemical allergen, methylene-diphenyl diisocyanate (MDI), induced innate immune responses, characterized by significantly increased airway levels of Chitinase YM-1 and IL-12/IL-23β (but not α) subunit. However, in mice immunologically sensitized to MDI via prior skin exposure, identical GSH–MDI doses induced substantially greater inflammatory responses, including significantly increased airway eosinophil numbers and mucus production, along with IL-12/IL-23β, chitinases, and other indicators of alternative macrophage activation. The “self”-protein albumin in mouse airway fluid was uniquely modified by GSH–MDI at position 414K, a preferred site of MDI reactivity on human albumin. The 414K–MDI conjugation appears to covalently cross-link GSH to albumin via GSH's NH2-terminus, a unique conformation possibly resulting from cyclized mono(GSH)–MDI or asymmetric (S,N′-linked) bis(GSH)–MDI conjugates. Together, the data support a possible thiol mediated transcarbamoylating mechanism linking MDI exposure to pathogenic eosinophilic inflammatory responses.

Immunochemical detection of the occupational allergen, methylene diphenyl diisocyanate (MDI), in situ

“…Additional staining was also noted in cells that resemble alveolar macrophages, consistent with data from studies of BAL cells shown in Figure 2. The data validate the recently described effectiveness of delivering reactive MDI to the lower airways in rodents, via intranasal application of reversibly reactive MDI-GSH conjugates (Wisnewski, Liu et al 2015). …”

“…The present immunocytochemical studies with anti-MDI polyclonal antiserum expand upon recent studies demonstrating the capacity of reversibly reactive MDI-GSH conjugates to induce eosinophilic airway inflammation in sensitized animals (Wisnewski, Liu et al 2015). The data confirm penetration of MDI to the lower airways following intranasal application of MDI-GSH conjugates under light anesthesia; unlike prior studies, which have found limited lower airway penetration of pure isocyanate vapors (or solutions in organic solvent), presumably due to the scrubbing effects of rodent upper airways (Kennedy, Singh et al 1993, Kennedy, Wilson et al 1994, Pauluhn 2014).…”

“…Immunochemical staining of cytospun BAL cells from mice sensitized and exposed to MDI, as reversibly reactive MDI-GSH conjugates (Wisnewski, Liu et al 2015), identified MDI primarily within the cytoplasm of cells with alveolar macrophage or dendritic cell morphology, but not polymorphonuclear or lymphocyte morphology (Figure 2). In contrast, no staining was observed in BAL cells recovered from MDI sensitized mice that had been exposed to inactivated (e.g.…”

“…Studies were performed on replicate samples of cytospun airway cells obtained by bronchoalveolar lavage (BAL) and lung tissue sections of MDI exposed or control mice acquired during prior studies as previously reported (Wisnewski, Liu et al 2015). Cytospun BAL cells fixed with −20°C methanol and formalin-fixed paraffin-embedded tissue sections were blocked with 20% goat serum and stained with 2 μg/ml of KLH-depleted, protein-A affinity purified anti-MDI rabbit IgG, or 2 μg/ml of isotype control protein-A purified polyclonal rabbit IgG raised against IL-33 or 2 μg/ml protein-A purified normal rabbit IgG (not shown).…”

Toluene diisocyanate: Induction of the autotaxin-lysophosphatidic acid axis and its association with airways symptoms

“…Additional indications include CD14 gene polymorphism associations to diisocyanate asthma (Bernstein et al, 2011), and the finding that MDI-GSH reaction products induce e.g. the mouse chitinase YM-1 (Wisnewski et al, 2015). It has also been suggested that B1-lymphocytes, transferred from dermally TDI-sensitized mice, contribute to an innate-like rapid defense upon a TDI challenge in recipient mice (De Vooght et al, 2013).…”