Elsevier

Toxicology in Vitro

Volume 15, Issue 6, December 2001, Pages 631-634
Toxicology in Vitro

Mechanisms of isocyanate sensitisation. An in vitro approach

https://doi.org/10.1016/S0887-2333(01)00078-9Get rights and content

Abstract

Although there is an abundance of clinical evidence which suggests that the inhalation of isocyanates can induce occupational asthma, the immunological basis for the disease is not understood. We have investigated immune cell responses to isocyanate using the cell line mono-mac-6, by measuring the production of hydrogen peroxide, and the expression of ICAM-1 following challenge with isocyanates and their corresponding amines. We observed an increase in the levels of intracellular peroxide, in addition to an upregulation of ICAM-1 expression (P<0.05), following cell stimulation with isocyanates, which was not apparent following stimulation with amines. From the results of this study we hypothesise that the production of reactive oxygen species (ROS) by monocytic cells at the site of exposure to an isocyanate may have two potential outcomes. The first is that the ROS may contribute to tissue damage at the site of inflammation, and then secondly, it is possible this production of hydrogen peroxide may also induce the upregulation of adhesion markers on monocytic cells, specifically ICAM-1, which may potentiate the infiltration and adhesion of cells at the site of inflammation.

Introduction

Isocyanates are widely used in industry in the production of paints and polyurethanes and are one of the most common inducers of occupational asthma. There is an abundance of clinical evidence which suggests that inhalation of isocyanates can cause asthma, but these findings often do not correlate with the presence of specific IgE antibodies to isocyanate (Kennedy and Brown, 1992). A recent review (Tee et al., 1998) reported that the specificity of the RAST analysis for specific IgE antibodies to isocyanates is often 100%, although the maximum reported sensitivity was 39%, suggesting that specific IgE to isocyanates is a more specific than sensitive index of occupational asthma. It is presumed that isocyanates combine with body proteins to form isocyanate protein conjugates (Kochman et al., 1990), but Baur et al. (1996) suggested that the hydrolysis products of isocyanates in an aqueous solution (namely monomeric or oligomeric diamines) may contribute to the induction of respiratory health effects. Studies performed on patients with isocyanate induced asthma have also demonstrated increased numbers of eosinophils, T-cells (Redlich et al., 1996) and airway inflammation which may also contribute to the aetiology of symptoms described by exposed individuals.

Isocyanates may also have a pharmacological mode of action, since they have been shown to reduce the ability of beta-adrenergic receptors to produce cyclic adenosine monophosphate interfering with the maintenance of bronchial tone (Mapp et al., 1988). In vitro studies (Wass and Belin, 1989) have also demonstrated that toluene diisocyanate affects prostaglandin receptors which can result in hyper-reactivity of smooth muscle.

Previous research by this laboratory has demonstrated that colophony, a complex mix of low molecular weight chemicals (LMWC), induced an oxidative burst response in HL60 cells that had been differentiated into monocyte- and neutrophil-like cell types (Elms et al., 2000), which suggested that the inhalation of colophony could lead to the production of ROS, contributing to tissue damage in the lung. A further consequence of the induction of oxidative burst, specifically the production of hydrogen peroxide has shown to be the increase in ICAM-1 expression, which has been demonstrated in vitro using endothelial cell lines (Roebuck, Rahman, Lakshminarayanan, Janakidevi, & Malik, 1995, Lakshminarayanan, Beno, Costa, & Roebuck, 1997).

In this study, the isocyanates toluene diisocyanate (TDI), diphenylmethane diisocyanate (HDI) and hexamethylene diisocyanate (HDI) were dissolved in phosphate buffered saline to mimic the physiological conditions of the lung. The potential of the isocyanates and their corresponding amines to induce an increase in peroxide levels and ICAM-1 expression in the monocytic cell line mono-mac-6 was investigated.

Section snippets

Materials and methods

Unless stated otherwise, all reagents and chemicals were purchased from Sigma (Dorset, UK).

Challenge with isocyanates

Treated and untreated cell lines exhibited >85% viability when challenged with isocyanates, amines or glycerol at 10 mm for up to 18 h.

Endpoints—measurement of oxidative burst

An initial time course experiment performed in singlet, demonstrated that following challenge with MDI, intracellular peroxide levels rose between a 5-min and 20-min incubation period (data not included); therefore for subsequent challenge assays 5-min and 20-min time points were used. Peroxide levels were significantly increased (P<0.05) in mono-mac-6 cells

Discussion

Isocyanates are often used in the production of paints and polyurethanes, and are one of the most commonly reported inducers of occupational asthma (Meredith et al., 1991). Although there is an abundance of clinical evidence to support this observation, the mechanistic basis for this is not well defined.

In this study, we have demonstrated a significant increase in the levels of intracellular peroxide in a monocytic cell line challenged with the isocyanates TDI and MDI, above cells challenged

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