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

Abstract

Diisocyanate chemicals essential to polyurethane production are a well-recognized cause of occupational asthma. The pathogenesis of diisocyanate-induced asthma, including the pathways by which the chemical is taken up and its distribution in exposed tissue, especially the lung, remains unclear. We developed an antiserum with specificity for methylene diphenyl diisocyanate (MDI) the most abundantly produced and utilized diisocyanate world-wide, and established its ability to detect MDI in situ. Polyclonal MDI-specific IgG was induced by immunizing rabbits with MDI-conjugated to keyhole limpet hemocyanin (KLH) emulsified in complete Freund's adjuvant, followed by two booster injections with incomplete Freund's adjuvant. The antiserum contains IgG that recognize a variety of different MDI conjugated proteins, but not unconjugated or mock exposed proteins by dot blot analysis. The antiserum further demonstrates specificity for proteins conjugated with MDI, but not other commonly used diisocyanates. Immunochemical studies with cytospun airway cells and formalin-fixed paraffin embedded lung tissue sections from mice intranasally exposed to MDI (as reversibly reactive glutathione conjugates, e.g. GSH-MDI) demonstrated the antiserum's ability to detect MDI in tissue samples. The data demonstrate penetration of MDI into the lower airways, localized deposition in the epithelial region surrounding airways, and uptake by alveolar macrophages. The new immunochemical reagent should be useful for further studies delineating the uptake and tissue distribution of MDI, especially as it relates to adverse health effects from exposure.

Keywords: Airways; Antiserum; Methylene-diphenyl-diisocyanate (MDI); Occupational asthma.

Figure 1

Specificity of MDI antisera. Different MDI-conjugated (+ MDI) or unconjugated/mock exposed (− MDI) proteins were spotted onto nitrocellulose membranes, stained for total protein using Ponceau-S (Panel A), and then blotted (Panel B) with a 1:1000 dilution of polyclonal anti-MDI serum, and reagents to detect rabbit IgG. In Panel C, mock exposed control albumin samples, or albumin conjugated with MDI, TDI or HDI (as labeled) were spotted onto nitrocellulose membranes and probed with MDI antisera or biotinylated anti-human albumin (as labeled) and developed with reagents that detect rabbit IgG or biotin respectively. No signal was observed in dot blots with control, pre-immune, normal rabbit serum, or polyclonal rabbit anti-IL-33 (data not shown).

Figure 2

Immunochemical detection of MDI in BAL cells. Lower airway cells from MDI skin sensitized mice exposed to inactivated (hydrolyzed) MDI (Panels A & B) or MDI-GSH (Panels C & D) were stained with protein-A affinity purified control (anti-IL-33) rabbit polyclonal antisera (Panels A & C) or KLH-depleted, protein-A affinity purified MDI antisera (Panels B & D). Note dark staining within cytoplasm of cells with alveolar macrophage and dendritic cell-like morphology.

Figure 3

Immunochemical detection of MDI in situ in the airway. Lung tissue sections from mice exposed via the airways to control inactivated (e.g. hydrolyzed) MDI (A, B) or MDI-GSH conjugates (C–H) were stained with control (anti-IL-33) rabbit serum (A, C, E) or anti-MDI rabbit serum (B, D, F, G, H) as described in the methods section and detected via DAB (brown) peroxidase stain, with blue hematoxylin counter stain. Panels A–F were photographed under 200X magnification. Panels G and H, taken under 1000X magnification, highlight areas shown in the rectangles in Panels D and F respectively. Arrows highlight staining of cells lining the airways, the basement membrane and an alveolar macrophage.