interleukin-8 and isophorone-diisocyanate

Diisocyanates are commonly used in polyurethanes where use includes industrial, commercial, and residential applications and can exist as respirable contaminants. These respirable contaminants exist in the air we breathe. Yet, there is no rapid assay available to test for potential respiratory sensitizers. To assess these hazards, as well as to decrease animal numbers used in testing, investigations that lead to verifiable in vitro methods are needed. We describe an easy, reliable, verified cell culture model that can be adopted by any lab capable of performing molecular toxicology. The architecturally relevant alveolar model consists of epithelial cells, macrophage cells, and dendritic cells in a simply maintained submerged system ideal for high-throughput testing. Exposures to contaminants that verify biomarker identification include a known pulmonary sensitizer (isophorone diisocyanate) and a positive control for cellular activation (phorbol 12-myristate 13-acetate/ionomycin). The mitochondrial reactive oxygen species generation and cytostructural changes were assessed with confocal laser scanning microscopy; cell morphology was assessed with scanning electron microscopy; biochemical reactions were assessed via protein arrays; genetic alterations were assessed via gene arrays; and cell surface activation markers were assessed via flow cytometry. Results showed that compared to untreated cultures, isophorone diisocyanate increased markers for dendritic cell activation, trafficking, and antigen presentation; number and length of dendritic protrusions; oxidative stress; and genetic and cytokine expression of neutrophil chemoattractants. The chemokines and cytokines CCL7, CXCL5, IL-6, and IL-8 were identified as biomarkers indicative of respiratory sensitization. By including multiple methods to assess endpoints, the in vitro model described can serve as a high-throughput assay to identify substances which may lead to respiratory sensitization.

Topics: Animals; Biomarkers; Cytokines; In Vitro Techniques; Interleukin-8; Isocyanates

This study is the first to analyse the soluble factors secreted by the bronchial epithelium after exposure to isophorone diisocyanate (IPDI) that are responsible for increasing migration and proliferation of primary normal human bronchial smooth muscle cells (BSMCs). We treated immortalised, nontumorigenic human bronchial epithelial cells (cell line BEAS-2B) and primary normal human bronchial epithelial cells (HBEC) with IPDI, and then collected the conditioned culture media (IPDI-BEAS-2B-CM and IPDI-HBEC-CM, respectively), which was added to BSMCs. Exposure of BEAS-2B cells and HBECs to IPDI increased interleukin (IL)-8 production. Culture of BSMCs with IPDI-BEAS-2B-CM and IPDI-HBEC-CM increased BSMC proliferation and migration, which are major features in asthma-related airway remodelling. Induction of BSMC proliferation and migration by IPDI-BEAS-2B-CM and IPDI-HBEC-CM was associated with increased focal adhesion kinase (FAK), Src, extracellular signal-regulated kinase (ERK)1/2 and AKT activation. Blocking FAK with a specific inhibitor significantly decreased BSMC migration and proliferation by inhibiting ERK1/2 activation. FAK and ERK1/2 inhibitor also decreased IPDI-BEAS-2B-CM-, IPDI-HBEC-CM- and recombinant human IL-8-mediated BSMC proliferation and migration, whereas blocking Rnd3 using small interfering RNA failed to affect BSMC proliferation, suggesting that Rnd3 was only involved in the regulation of BSMC migration. Our study suggests that inhibition of IL-8 or IL-8-mediated FAK/ERK/Rnd3 signalling is an attractive therapeutic target for IPDI-mediated asthma.

Topics: Bronchi; Cell Line; Cell Movement; Cell Proliferation; Cells, Cultured; Enzyme Inhibitors; Focal Adhesion Protein-Tyrosine Kinases; Humans; Interleukin-8; Isocyanates; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth; Proto-Oncogene Proteins c-akt; rho GTP-Binding Proteins; RNA, Small Interfering; Signal Transduction; src-Family Kinases