Occupational asthma markers: Candidate proteins for exposure to toluene diisocyanate

Industrial isocyanate attack

Occupational asthma is one of those furtive industrial hazards, sneaking up on its victims unawares while they go about their jobs. Induced by exposure to chemicals that irritate the airways, OA can be a debilitating condition that lays workers low for extended periods of time.

One common agent of OA is toluene-2,4-diisocyanate (TDI) which is used extensively to produce polyurethane foams, coatings, adhesives and sealants. Unfortunately, if workers are regularly exposed to TDI over a period of months or years, they become sensitised to it and develop immunologically mediated OA. This is triggered by further exposure by skin contact or inhalation, even by very low levels of the chemical.

It is not only industrial workers who are at risk. Consumers using products containing TDI, like sprays and coatings, can be exposed via skin contact or inhalation as well as members of the general public who are in the vicinity of buildings where it is being used. This shortened exposure can lead to irritant-induced OA.

While it might be relatively straightforward to diagnose OA, the causative agent is more difficult to identify. In the case of TDI, scientists in Belgium from KU Leuven and Hasselt University, Diepenbeek, have taken the proteomics route to see if regular contact with TDI over time makes any changes to proteins in the body. A set of biomarkers could be more reliable than the current antibody-based tests which are accurate for less than 50% of cases.

Senior reporter Jeroen Vanoirbeek described how they took a different approach to that normally adopted in proteomics studies of pulmonary diseases. Rather than examining cases where the disease was already established, they searched for changes in the proteome that occurred during the sensitisation phase of OA, before the occurrence of the clinical symptoms.

Mouse model of asthma

The research team used the mouse model of chemical-induced asthma. TDI was applied once or twice to the ears of mice and they were sacrificed three days after the last application. The auricular lymph nodes located beneath the ear were removed and serum was also collected for analysis. A different set of mice was treated with olive oil to act as controls.

The abundant proteins albumin, transferrin and immunoglobulin G were removed from the sera to make it easier to find the less abundant proteins that remained. Then the proteins in the sera and homogenised lymph nodes from the TDI-treated mice and the controls animal were separated by 2D differential gel electrophoresis.

Any proteins which were found to have altered abundances compared with the controls were isolated from the gels and identified by mass spectrometry. Their identities were confirmed by Western blotting of lymph node proteins and ELISA of sera from a new set of TDI-treated and control mice.

Protein panel

The auricular lymph nodes approximately doubled in weight following one or two TDI treatments and they showed large increases in the levels of B and T lymphocytes, indicating a significant local immune response. The serum and lymph node proteomes also began to change after one sensitisation and continued to change after the second, which the researchers took to be the onset of immune response.

There were 38 and 58 differentially expressed proteins in the lymph nodes, and 7 and 16 in the serum, following single or double treatment with TDI. A principal components analysis could distinguish between the lymph nodes from the treated and untreated animals after one and two treatments, but only the serum after two treatments. About 85% of the proteins were identified by mass spectrometry and several of them caught the attention of the research team.

About 8% of the lymph node proteins identified after two treatments were immune-related. Lymphocyte specific protein-1 (LSP-1) had reduced abundance in the TDI-treated mice. Decreased amounts of LSP-1 allow a higher neutrophil influx, an indication of infection.

Coronin 1A was increased in lymph nodes after one TDI treatment and continued to increase after two treatments. This protein controls the release of calcium ions from within cells, which is part of the T lymphocyte activation process in response to antigens.

In the serum samples, only three proteins were found to be differentially expressed after one treatment. One of them was hemopexin, which was increased after one TDI treatment but was decreased after the second. This odd behaviour could not be explained at this time, although the research team agreed that hemopexin must be associated with TDI exposure.

The discovery of several proteins apparently related to dermal exposure to TDI in mice suggests that they will also apply to humans. The next stage will be their evaluation in symptomatic and asymptomatic workers exposed to TDI as well as non-exposed workers in order to evaluate the specificity and sensitivity of one or more of these proteins as biomarkers.

The panel of proteins looks promising, because they were affected by only one or two sensitisations, whereas industrial workers are likely to encounter regular exposures over a longer period of time.