Respiratory sensitisation in humans: Characterisation of pulmonary epithelial:dendritic cell interactions

We are exposed to a range of chemicals in day-to-day life, at home and at work, which may have unwanted health effects. In this study we are interested in the sensitivity of the lung to chemicals that are inhaled, usually while at work. Not everybody is affected, but when those who are affected are exposed to the same chemical again they may experience an asthma attack, and they may also react to other agents that they inhale from the air (this accounts for approximately 10% of adult-onset asthma). This is called respiratory sensitisation. It is important to determine whether existing or newly-developed chemicals will cause respiratory sensitisation, and if so, the magnitude of the response. It is also important to understand the cellular mechanisms that are involved. The immunological responses of the lung are complex, and no validated tests exist to determine the potential of chemicals to initiate these responses. Animal experiments are often used to help make these assessments, but they are not always very accurate, although they do help us understand some of the mechanisms involved. Furthermore, the 7th Amendment to the European Union Cosmetics Directive (European Commission, 2003) recommends that we develop new ways of testing compounds so that we reduce animal experimentation. The strategy is called the 3Rs, Reduction, Refinement and Replacement of the use of animals in scientific tests. One aim of this study is to establish testing methods that avoid using animals by using human cells. Use of human cells is important as it will ensure development of a more representative model for assessing the potency of these chemicals in man. The cells in the lung that respond to inhaled particles are the lung's 'skin', epithelial cells, as well as cells that act like a vacuum cleaner, internalising and neutralising inhaled material that settles on the 'skin'; which are called macrophages. Epithelial cells and macrophages send messages to dendritic cells that sit underneath the lung epithelial cells. Dendritic cells also extend long 'probes' onto the epithelial surface where they can directly interact with any inhaled chemicals. Depending on the messages received, dendritic cells then activate the immune response to fight infection and other foreign particles that reach the lung. However, sometimes the messages go wrong, and this is when the lung becomes very sensitive to some types of inhaled particles and chemicals. We aim to isolate cells from adult human lung tissue that is removed during an operation to remove a tumour. Normal regions of lung that are attached to, but not affected by, the tumour can be used. We will then culture epithelial, macrophage and dendritic cells alone, and together in a 3-dimensional model resembling the lung. We will expose the cells to chemicals that we know are respiratory sensitisers, as well as chemicals that do not cause respiratory sensitisation, to examine the cellular messaging pathways involved in respiratory sensitisation. We also want to determine whether there are unique responses that identify chemical sensitisers so that these culture models can be developed and used for routine testing of existing and new compounds that might affect the lung. In addition, with this knowledge, it should be possible to develop drugs that prevent these abnormal responses. These models could be used to avoid the use of animals to investigate respiratory sensitisers to understand mechanisms of respiratory sensitisation.

Respiratory chemical sensitisation usually occurs as a result of occupational exposure and forms 10% of adult-onset asthma. Animal models may be used to examine putative sensitisers but have not been validated and do not accurately measure and rank respiratory sensitisers. We aim to use primary human lung alveolar epithelial type II cells (HATII) and macrophages, as well as human monocte-derived dendritic cells (DC) to establish in vitro models to a) investigate molecular mechanisms b) identify unique markers of chemical respiratory sensitisation which may be used for in vitro testing to avoid use of animals. Techniques already established by us will be used to isolate epithelial cells and macrophages from normal regions of human lung tissue following resection for carcinoma; established techniques will be used to prepare monocyte-derived DC. Ultimately, if primary human lung DC can be isolated, their responses will be compared with the monocyte-derived DC to validate the models. The cells will be exposed to known respiratory sensitisers as well as control chemicals that do not elicit a pulmonary response (eg contact sensitisers). Each cell type will be exposed alone, to generate conditioned media, to test the effects of released mediators on DC maturation, migration and function (eg measuring chemokine, cytokine and cell receptor switching). Co-cultures will be established of all three cell types with macrophages situated apically on HATII, on transwell membranes, and DC below the membranes. Responses to apically administered chemicals will be examined. Analysis will include use of leading-edge technologies including targeted gene microarray analysis of gene expression, multiplex analysis of chemokine, cytokine, surface receptor and growth factor protein release/expression, confocal microscopy and FACS analysis to provide a comprehensive understanding of the molecular signalling pathways involved.