Expression of the cell adhesion molecule, L-selectin, on polymorphonuclear leukocytes during and after their release from the bone marrow

The emigration of polymorphonuclear leukocytes (PMN) to sites of inflammation requires a series of leukocyte-endothelial interactions which can be divided in four sequential steps. These include, tethering of flowing leukocytes; triggering or activation of leukocytes; firm adhesion to the endothelium and; emigration of leukocytes out of the vessels into inflamed tissue. The selectin family of adhesion molecules are involved in the tethering of leukocytes and one of these, L-selectin, is expressed on nearly all leukocytes including PMN. The recruitment of the PMN to sites of inflammation is highly dependent on the expression of this molecule on their cell surfaces. However, the role of L-selectin in the trafficking of the PMN from the bone marrow into the blood is less clear. The objective of this thesis was to determine the expression of L-selectin on PMN during their release from the bone marrow into the circulation and their eventual removal into the tissues. Immunocytochemical and immunohistochemical techniques were used to determine the expression of L-selectin on the PMN in cytological and histological specimens respectively. Indirect immunofluorescent flow cytometry was used to determine the expression of L-selectin on circulating PMN. Changes in L-selectin expression on PMN during their release from the bone marrow was studied during cardiopulmonary bypass in humans. These studies demonstrated that the expression of L-selectin on the mature segmented PMN in bone marrow is higher than on circulating PMN and that some of this L-selectin shed when PMN cross from the bone marrow hematopoietic compartment into the bone marrow venous sinusoids. However, L selectin expression on PMN in the venous sinusoids remains high and this results in an increased expression of L-selectin on circulating PMN during active bone marrow release. This means that circulating PMN expressing the highest levels of L-selectin, have been recently released from the bone marrow. A new method for labelling PMN in vivo using the thymidine analogue, 5’bromo-2-deoxyuridine was used to demonstrate that circulating PMN continuously lose L-selectin while they remain in the circulation. These studies explain the variable expression of L-selectin on the circulating PMN with the newly released PMN expressing the highest and the older PMN the lowest levels of L-selectin. This implies that the expression of L-selectin on circulating PMN is not just a marker of cell activation state, but also of cell age. This creates the opportunity to study the functional capabilities and the trafficking of different populations of the circulating PMN.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Abundance of Non-Polarized Lung Macrophages with Poor Phagocytic Function in Chronic Obstructive Pulmonary Disease (COPD)

Lung macrophages are the key immune effector cells in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). Several studies have shown an increase in their numbers in bronchoalveolar lavage fluid (BAL) of subjects with COPD compared to controls, suggesting a pathogenic role in disease initiation and progression. Although reduced lung macrophage phagocytic ability has been previously shown in COPD, the relationship between lung macrophages’ phenotypic characteristics and functional properties in COPD is still unclear. (1) Methods: Macrophages harvested from bronchoalveolar lavage (BAL) fluid of subjects with and without COPD (GOLD grades, I–III) were immuno-phenotyped, and their function and gene expression profiles were assessed using targeted assays. (2) Results: BAL macrophages from 18 COPD and 10 (non-COPD) control subjects were evaluated. The majority of macrophages from COPD subjects were non-polarized (negative for both M1 and M2 markers; 77.9%) in contrast to controls (23.9%; p < 0.001). The percentages of these non-polarized macrophages strongly correlated with the severity of COPD (p = 0.006) and current smoking status (p = 0.008). Non-polarized macrophages demonstrated poor phagocytic function in both the control (p = 0.02) and COPD (p < 0.001) subjects. Non-polarized macrophages demonstrated impaired ability to phagocytose Staphylococcus aureus (p < 0.001). They also demonstrated reduced gene expression for CD163, CD40, CCL13 and C1QA&B, which are involved in pathogen recognition and processing and showed an increased gene expression for CXCR4, RAF1, amphiregulin and MAP3K5, which are all involved in promoting the inflammatory response. (3) Conclusions: COPD is associated with an abundance of non-polarized airway macrophages that is related to the severity of COPD. These non-polarized macrophages are predominantly responsible for the poor phagocytic capacity of lung macrophages in COPD, having reduced capacity for pathogen recognition and processing. This could be a key risk factor for COPD exacerbation and could contribute to disease progression.Medicine, Faculty ofNon UBCMedicine, Department ofRespiratory Medicine, Division ofReviewedFacult