The role of granulocyte antibodies in monocyte and granulocyte activation

Abstract: Granulocytes and monocytes are important cells during the first phase of inflammation. They are activated and recruited from the blood vessels to inflammatory sites via the regulation of receptors involved in adhesion to endothelium. Through phagocytosis and the production of reactive oxygen metabolites and cytokines, they are crucial for the initiation of an immune response. Antibodies against surface and intracellularly expressed granulocyte antigens are important in a number of clinical conditions. Detection of anti neutrophil cytoplasmic antibodies (ANCA) is a diagnostic marker for systemic vasculitis and there is also increasing evidence for a pathophysiological role of the antibody. The objective of this thesis was to analyse the functional responses of granulocytes and monocytes to activation, with special attention paid to the relevance of granulocyte antibodies. In the first study the expression of receptors associated to adhesion to endothelium (CD62L and CD11b), binding of complement and immunoglobulin (CD35 and CD16) and the production of reactive oxygen metabolites were analysed on granulocytes after storage for different periods of time. Next we evaluated a flow cytometric method for the detection of granulocyte antibodies against surface and intracellular antigens. In the third study we determined the in vitro expression of CD62L and CD 11b and the production of reactive oxygen metabolites in monocytes in response to incubation with ANCA. Finally, in a prospective clinical study the monocyte activation and the concentration of soluble inflammation markers and soluble adhesion molecules were analysed in patients with acute anti-proteinase 3 (PR3) positive systemic vasculitis. In our first study we demonstrated a marked decrease in granulocyte CD62L expression concomitant with CD11b up-regulation at 24 hours storage. The expression of receptors associated with phagocytosis and the capacity to produce oxygen metabolites remained more stable. In the next study we showed that antibodies against granulocyte antigens can be detected by flow cytometry and by using both un-permeabilized and permebilized target cells, antibodies against surface and intracellularly expressed antigens can be distinguished. We found a decreased CD62L expression and enhanced production of oxygen radicals in monocytes upon in vitro ANCA stimulation. Compared to ANCA positive IgG-fractions, ANCA positive sera induced a more pronounced CD62L down-regulation, which supports the contribution of other soluble serum factors beside IgG. In contrast with our in vitro finding, monocytes from patients with acute ANCA-positive vasculitis showed reduced capacity to produce oxygen radicals. This may be a consequence of a prolonged period of immune activation and a more dynamic situation in vivo. High concentration of anti-PR3 correlated to decreased CD62L and increased CD11b expression on monocytes. Soluble inflammation markers (sCD14, IL-6, sTNFR1, IL-8, IL- 10) as well as soluble adhesion molecules (sVCAM-1, sICAM-11) were enhanced compared to in healthy controls and similar compared to in a control group with acute ANCA-negative infection. The regulation of adhesion receptors is crucial in the initial attachment to the endothelium in an inflammatory response and a dysregulated CD62L/CD11b expression on monocytes may have pathophysiological implications for the endothelial damage seen in vasculitis.