Acute lung injury (ALI) affects a significant proportion of patients requiring critical care and is associated with high morbidity and mortality. Treatment is currently only supportive, with no pharmacological treatment yet shown to definitively improve outcome. There is evidence from murine models of ALI that monocytes play a key role in the development of the neutrophilic lung infiltration characteristic of ALI. Depletion of blood monocytes in mice given intra-tracheal lipopolysaccharide (LPS) significantly reduces pulmonary neutrophil influx, systemic neutrophilia and other markers of lung injury. In humans, monocyte-like cells have been documented in the bronchoalveolar lavage (BAL) fluid of patients with a variety of inflammatory lung conditions, including ALI. This thesis describes novel work performed in healthy human subjects to test whether, in an experimental model of human lung inflammation, depletion of circulating blood monocytes can ameliorate systemic and pulmonary inflammation. LPS inhalation is an established method of modelling ALI in healthy human subjects as it safely and consistently induces mild and self-limiting systemic and pulmonary inflammation. A preliminary study in a group of 12 healthy subjects confirmed the safety and efficacy of LPS inhalation compared to saline placebo. LPS inhalation induced a marked blood neutrophilia together with a rise in body temperature and heart rate and elevated BAL neutrophil and pro-inflammatory cytokine concentrations. This study also used flow cytometry to confirm the presence of pulmonary monocyte-like cells (PMLCs) in BAL fluid, which, although distinct from blood monocytes, could be clearly divided into two separate sub-types according to CD14/CD16 expression. LPS inhalation caused a rise in the number of circulating classical monocytes in blood and an expansion in the CD14++CD16- 'inducible' iPMLC subtype (reminiscent of classical blood monocytes), compared to the CD14++CD16+ 'resident' rPMLC subtype. This may represent transmigration of classical monocytes from blood across the pulmonary endothelium. In humans, mononuclear cell (MNC) leukapheresis provides a readily available method of depleting circulating blood monocytes. A second preliminary study, performed in a separate group of 6 healthy subjects, demonstrated that leukapheresis of four total blood volumes could be safely employed to deplete large numbers of circulating blood monocytes. Active recruitment of monocytes into circulating blood during leukapheresis did, however, limit the reduction in total circulating blood monocyte counts. This study also investigated, for the first time, the potential pulmonary effects of leukapheresis. Despite a relative prominence of iPMLCs in BAL fluid after leukapheresis, there was no evidence of significant neutrophil influx or a clinically important pro-inflammatory effect in the alveolar space. A randomised, double blind, placebo-controlled trial was then performed in a third group of 30 healthy human subjects who all inhaled LPS at baseline. There was no evidence that MNC leukapheresis (depletion group, n=15), compared to a sham procedure (sham group, n=15), attenuated the systemic and pulmonary inflammation induced by LPS inhalation, as measured by: blood neutrophil and plasma C-reactive protein (CRP) levels; by the neutrophil, protein and pro-inflammatory cytokine content of BAL fluid; and by [18F]fluorodeoxyglucose positron emission tomography ([18F]FDG PET)-derived measures of global lung inflammation. MNC leukapheresis temporarily prevented the LPS-induced rise in circulating classical monocytes and was also associated with a small reduction in the estimated numbers of MNCs in BAL fluid. It did not, however, appear to affect the LPS-induced expansion in the iPMLC subtype. Further characterisation of the PMLC subtypes by flow cytometry/sorting and cell culture demonstrated that the iPMLC subtype was more pro-inflammatory but less mature and with a lower proliferation potential than the rPMLC subtype. In summary, this work did not support a role for circulating blood monocytes in the evolution of LPS-induced systemic or pulmonary neutrophilia in man. The rise in circulating levels of classical blood monocytes and the dramatic expansion of pro-inflammatory, immature iPMLCs in BAL fluid after LPS inhalation do, however, suggest that monocytes migrate to the lung and are to some extent involved in the pathogenesis of lung inflammation. Compared to murine methods of monocyte depletion, leukapheresis could not achieve such an extensive or sustained reduction in circulating blood monocyte counts, nor was it likely to have influenced other (specifically patrolling or splenic) monocyte pools. Future work in the drive to find treatments for ALI should therefore investigate the potential of pre-emptive leukapheresis or the efficacy and safety of other methods of human monocyte depletion in experimental lung inflammation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:726489 |
| Date | January 2014 |
| Creators | Barr, Laura Caroline |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/23705 |
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