Cockroach and dust mite allergens have potent toll-like receptor 4 dependent innate immune-stimulatory activity

Abstract

Rationale Innate immune-stimulatory PAMPs (pathogen associated molecular patterns), such as endotoxin (ET), may contribute to the fundamental differences in allergenicity of common allergens. PAMPs are ligands for pattern recognition receptors, such as toll-like receptor 4 (TLR4). Methods Mouse bone marrow macrophages were stimulated for 24 hours in vitro with dialyzed allergen extracts. IL-10, IL-12 and nitric oxide (NO) levels from supernatants were compared in wild-type (C3H/HEOUJ) and functionally deficient TLR4-mutant (C3H/HEJ) mice. Allergen protein levels were measured by spectrophotometry and endotoxin levels by Limulus Amebocyte Lysate assay. Results Two cockroach and two dust mite extracts were significantly higher in endotoxin [mean 21 EU/mg protein (range 3-61)] than cat, dog, timothy, and alternaria extracts [0.3 EU/mg (0.01-0.91); p=0.03]. The protein content of the “ET-High” vs “ET-Low” allergens did not differ significantly. In wild-type mice, ET-High allergens induced significantly higher levels than ET-Low of IL-10 [171 pg/ml (28-425) vs 0 pg/ml; p=0.02] and IL-12 [1755 pg/ml (72-4736) vs 8 pg/ml (0-23); p=0.03]. Allergen endotoxin content correlated with IL-10 (r=0.76; p=0.03) and IL-12 (r=0.80; p=0.02). When cytokine and NO levels in TLR4-mutant were compared with wild-type, IL-10, IL-12 and NO levels for ET-High allergens were reduced by 81% (p=0.11), 98% (p=0.02), and 100% (p=0.05), respectively. Conclusions High endotoxin levels in cockroach and dust mite allergens are associated with high levels of TLR4-dependent IL-10 and IL-12 production. PAMP-related distinctions in innate immune-stimulatory activity may contribute to important differences in the allergenicity of common allergens.