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 (414)K, a preferred site of MDI reactivity on human albumin. The (414)K-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.

Figure 1

Airway inflammation evoked by GSH–MDI reaction products. Cytospun, stained airway lavage cells from representative naïve (a, b) or MDI sensitized mice (c, d) exposed via the respiratory tract to control stimuli [GSH-m = GSH reacted without MDI (a); MDI-m = MDI reacted without GSH (c)] or GSH–MDI reaction products (b, d). Asterisks highlight eosinophils, and arrows highlight neutrophils or lymphocytes. (e) Mean number of cells (×10⁻³) ± standard error (Y-axis) derived from airway lavage samples of naïve or MDI sensitized mice exposed to control stimuli, GSH-m, MDI-m, or GSH–MDI. Data are derived from three separate experiments with a total of N = 18 mice/group.

Figure 2

Lung tissue inflammation and mucus production evoked by GSH–MDI reaction products. Lung tissue sections from naïve mice exposed to control stimuli, GSH-m (a), or GSH–MDI (b) or from MDI sensitized mice exposed to control stimuli, MDI-m (c), or GSH– MDI (d) were subject to periodic acid–Schiff (PAS) staining to highlight mucus production (airways with asterisk). (e) PAS stained lung tissue section from a representative MDI sensitized, GSH–MDI exposed host under higher magnification to highlight the mucus containing goblet cells lining the airways and submucosal eosinophils.

Figure 3

GSH–MDI increases airway levels of IL-12/IL-23β. (a) Monoclonal antibody-based array (key to the left) was used to screen for changes in cytokine levels of pooled airway fluid samples (N = 6 each) from MDI sensitized hosts exposed to control stimuli MDI-m (MDI reacted without GSH) vs GSH–MDI (middle and far right respectively). (b) Graph representing the mean concentration in pg/ mL ± standard error of different cytokines in airway fluid (Y-axis) from N = 18 each naïve or MDI sensitized mice from three separate experiments, exposed to GSH–MDI or control stimuli, as labeled. (c) Anti-IL-12/IL-23β western blot on airway fluid from naïve or MDI sensitized mice exposed to GSH–MDI or control stimuli (GSH-m = GSH reacted without MDI; MDI-m = MDI reacted without GSH) was performed under nonreducing conditions. Arrows highlight banding due to monomeric p40 (lower) and homodimeric p80 (upper) IL-12/ IL-23β.

Figure 4

GSH–MDI increases airway chitinase YM-1 and calcium-activated chloride channel CLCA1. Pooled airway fluid from N = 6 naïve or MDI sensitized mice exposed to GSH–MDI (+) or control stimuli GSH-m (−) for naïve or MDI-m for sensitized mice were western blotted under reducing (YM-1, IgG Fc) or nonreducing conditions (CLCA1). Arrows highlight the 39 kDa band corresponding to YM-1, the ∼130 kDa mature glycosylated CLCA1, and the ∼50 kDa heavy chain of IgG, as a loading control.

Figure 5

Modification of airway fluid albumin by GSH–MDI in vivo. (a) Pooled airway fluid samples from N = 6 each naïve or MDI sensitized mice exposed to GSH–MDI (+) or control stimuli (−), GSH-m and MDI-m, respectively, were subject to reducing SDS-PAGE and western blotted with biotin labeled MDI-specific mAb DA5. Arrow highlights dominant band ∼68 kDa. (b) Chemical structure depicting the unique GSH–MDI modification of airway fluid albumin detected through LC-MS/MS (see Supporting Information Tables S1 and S2).