Abstract
BACKGROUND: Isocyanates, a leading cause of occupational asthma, are known to induce adaptive immune responses; however, innate immune responses, which generally precede and regulate adaptive immunity, remain largely uncharacterized. OBJECTIVE: The aim of the study was to identify and characterize the cellular, molecular and systemic innate immune responses induced by hexamethylene diisocyanate (HDI). METHODS: Human peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with HDI-albumin conjugates or control antigen, and changes in phenotype, gene and protein expression were characterized by flow cytometry, microarray, Western blot and ELISA. Cell uptake of isocyanate was visualized microscopically using HDI-albumin conjugates prepared with fluorescently labelled albumin. In vivo, human HDI exposure was performed via a specific inhalation challenge, and subsequent changes in PBMCs and serum proteins were measured by flow cytometry and ELISA. Genotypes were determined by PCR. RESULTS: Human monocytes take up HDI-albumin conjugates and undergo marked changes in morphology and gene/protein expression in vitro. The most significant (P-values 0.007-0.05) changes in microarray gene expression were noted in lysosomal genes, especially peptidases and proton pumps involved in antigen processing. Chemokines that regulate monocyte/macrophage trafficking (MIF, MCP-1) and pattern-recognition receptors that bind chitin (chitinases) and oxidized low-density lipoprotein (CD68) were also increased following isocyanate-albumin exposure. In vivo, HDI-exposed subjects exhibited a drastic increase in the percentage of PBMCs with the same HDI-albumin responsive phenotype characterized in vitro (HLA-DR(+)/CD11c(+) with altered light scatter properties). An exposure-dependent decrease (46+/-11%; P<0.015) in serum concentrations of chitinase 3-like-1 was also observed in individuals who lack the major (type 1) human chitinase (due to genetic polymorphism), but not in individuals possessing at least one functional chitinase-1 allele. CONCLUSIONS: Previously unrecognized innate immune responses to HDI and HDI-albumin conjugates could influence the clinical spectrum of exposure reactions.