Potential biomarkers for predicting the phenotype of isocyanate-induced occupational asthma

Abstract

Background and objective: Considering the poor prognosis of occupational asthma (OA), it is critical to identify useful serologic markers to recognize susceptible subjects among isocyanate-exposed workers. Previously, we reported upregulated expression of vitamin D binding protein (VDBP) and downregulated expression of annexin V in the bronchoalveolar lavage fluid (BALF) of patients with isocyanate-induced OA (isocyanate-OA) compared to that of asymptomatic exposed controls (AECs) through proteomic analysis. However, the use of these proteins for identifying patients with isocyanate-OA was not validated. Toluene diisocyanate (TDI) is the most common cause of isocyanate-OA, and is a one of the leading cause of OA worldwide. TDI exposure induces oxidative stress and epithelial cell-derived inflammation, which are thought to play a role in the pathogenesis of TDI-induced OA (TDI-OA). Recent studies suggested a role of clusterin (CLU) and progranulin (PGRN) in oxidative stress-mediated airway inflammation. In this study, we evaluated the clinical relevance of serum VDBP and annexin V for identifying suspected subjects from isocyanate-exposed individuals. In addition, we aimed to investigate the CLU and PGRN involvement in airway inflammation in TDI-OA with their clinical significance. Materials and Methods: Sixty-one patients with isocyanate-OA, 180 AECs, and 58 unexposed healthy normal controls (NCs) were enrolled. To validate the clinical relevance of VDBP and annexin V, their levels were measured in the sera of each group by enzyme-linked immunosorbent assay (ELISA). The sensitivities and specificities of the VDBP for detecting isocyanate-OA were determined using receiver operating characteristic (ROC) curves. In TDI-exposed subjects, the serum levels of CLU and PGRN were measured using ELISA and the diagnostic values were evaluated using ROC curves. To investigate their roles, we evaluated their production by human airway epithelial cells (HAECs) in response to TDI exposure as well as the effects of co-culturing with neutrophils. Results: Serum VDBP levels were significantly higher in the isocyanate-OA group compared to that in the AEC and NC groups (P<0.001). However, there was no significant difference between study groups with respect to annexin V levels. To identify isocyanate-OA among isocyanate-exposed workers, the optimal serum cut-off level of VDBP was selected as≥311 µg/mL using ROC curve. The sensitivity and specificity for predicting isocyanate-OA were 69% and 81%, respectively. When isocyanate-exposed subjects were classified into two groups: low and high serum level groups, based on the cut-off value of VDBP (≥311 µg/mL), the high-level VDBP group showed significantly lower PC20 methacholine levels than that of the low group (P=0.001). Lower serum CLU and PGRN levels were noted in the TDI-OA group than in the AEC and NC groups (P<0.05). The sensitivity and specificity for predicting TDI-OA phenotype with combined use of CLU (≤125 μg/mL) and PGRN (≤68.4 ng/mL) levels were 72.4% and 89.8%, respectively. TDI-human serum albumin (HAS) stimulation induced the release of CLU/PGRN from HAECs significantly in a dose-dependent manner, which positively correlated with the levels of IL-8 and folliculin levels. However, co-culture with neutrophils significantly decreased CLU/PGRN production from HAECs. Intracellular ROS production in epithelial cells co-cultured with neutrophils tended to increase initially, but decreased gradually at a higher ratio of neutrophils. Conclusion: The present study suggests that VDBP level could be used as a serologic marker for detection of isocyanate-OA among isocyanate exposed workers, as well as for predicting the severity of airway hyper-responsiveness. In addition, this is the first study to demonstrating the involvement of CLU and PGRN in the pathogenesis of TDI-OA probably through protective roles against TDI-induced oxidative stress-mediated inflammation. We also suggest that the combined CLU/PGRN serum level may be used as a potential serological marker for identifying patients with TDI-OA among TDI-exposed workers.