Leucocytes are white blood cells that help the body fight against bacteria, viruses and tumour cells. However, the activity of leucocytes has been implicated in other clinically important inflammatory conditions such as ischaemic heart disease, stroke, and during cardio-aortic and orthopaedic surgery. The main objectives of this study was to optimise methods for the isolation of leucocyte subpopulations (neutrophils and monocytes), and to assess in vitro the effects of PMA and fMLP on markers of leucocyte adhesion (CD11b, CD62L) and activation (intracellular hydrogen peroxide) (n=10). Leucocyte subpopulations were labelled by incubation with fluorescein isothiocya-nate (FITC) conjugated anti-human CD11b and CD62L antibodies. The cell surface expression of these labelled adhesion molecules were measured by flow cytometry. Intracellular production of hydrogen peroxide by neutrophils and monocytes was measured by flow cytometry, using the fluorochrome dichloroflurorescin diacetate (DCFH-DA). These were visualised by Immunofluorescence microscopy. During this study, methods of isolating leucocyte subpopulations from whole blood were optimised. This ensured that these cells were isolated with consistently high yields, purity and with no changes in cellular function. Following incubation with PMA and fMLP, neutrophils and monocytes displayed an increase in CD11b cell surface expression; a decrease in CD62L cell surface expression; and increased leucocyte activation. Leucocyte activation was represented by the intracellular production of hydrogen peroxide. In conclusion this study confirms that both PMA and fMLP have an intrinsic effect on markers of leucocyte function. These findings are in agreement with previous studies performed

Leucocytes represent a very important host defence against a number of invading pathogens and neoplasia. However, the activity of phagocytic leucocytes has been heavily implicated in the development of ischaemia-reperfusion injury, and as an aetiological factor in the pathology of other clinically important inflammatory conditions. Ischaemia-reperfusion injury occurs in diseases such as stroke and ischaemic heart disease (IHD), and during surgical procedures such as orthopaedic surgery. Investigations presented here employed a model of tourniquet-induced forearm ischaemia-reperfusion injury to investigate the effect on leucocyte adhesion and trapping (n=20). Neutrophil and monocyte leucocyte subpopulations were isolated by density gradient centrifugation techniques. Neutrophil and monocyte cell surface expression of the adhesion molecule CD11b was measured by labelling with fluorescent anti-CD11b monoclonal antibody via flow cytometry. Plasma concentrations of the soluble intercellular adhesion molecule-1 (sICAM-1) and soluble L-selectin (sL-selectin) adhesion molecules were measured using commercially available ELISA kits. Leucocyte trapping was investigated by measuring the concentration of leukocytes in venous blood leaving the arm. During ischaemia-reperfusion there was an increase in CD11b expression on neutrophils (p=0.040) and monocytes (p=0.049), a decrease in sL-selectin (p=0.387) and sICAM-1 (p=0.089) concentrations, and a decrease in peripheral blood leucocyte concentration (p=0.019). Evidence of increased leucocyte adhesion and trapping during ischaemia-reperfusion injury was supported by an increase in CD11b cell surface expression of neutrophils and monocytes. CD11b is expressed on phagocytic leucocytes and binds to ICAM-1 expressed on the surface of vascular endothelium. This increased expression of CD11b on leucocytes may therefore play a central role as the mechanism by which leucocyte trapping in the microcirculation occurs. The measured decrease in plasma concentration of sICAM-1 and sL-selectin suggests that these adhesion molecules retain their functional activity, and may bind to their corresponding cell surface ligands. It is therefore reasonable to believe that ICAM-1 expressed on the endothelium and L-selectin expressed on leucocytes is also binding to their corresponding cell surface ligands. A decrease in the number of leucocytes in the peripheral circulation may be due to increased trapping of leucocytes in the microcirculation. When leucocytes become trapped their concentration in blood leaving the microcirculation decreases, resulting in the measured decrease in leucocyte concentration. In conclusion, this study confirms the important role of leucocytes during ischaemia-reperfusion injury, which could allow for the possibility of future research that may provide therapeutic intervention for inflammatory conditions