Adhesion-Dependent Release of Elastase From Human Neutrophils in a Novel, Flow-Based Model: Specificity of Different Chemotactic Agents

Abstract

AbstractNeutrophils must adhere to the vessel wall, migrate, and degranulate in an ordered manner to perform their protective function. Disruption of these processes may be pathogenic. Current knowledge of the degranulation process is derived almost exclusively from studies on neutrophils in suspension, in which priming with the nonphysiological agent cytochalasin B is necessary to obtain elastase release in response to activating agents. To avoid this, we have adopted a different approach. Using a novel flow-based adhesion system, we have been able to quantify the release of elastase from the primary granules of activated neutrophils adherent to immobilized platelets or purified receptors without priming. Comparing stimuli, formyl tripeptide (fMLP), interleukin-8 (IL-8), activated complement fragment C5a, and platelet-activating factor (PAF) all induced rapid conversion to CD11b/CD18 (MAC-1) -mediated stationary adhesion when perfused over neutrophils already rolling on platelet monolayers or purified P-selectin. However, fMLP, C5a, and IL-8, but not PAF, induced release of elastase from the adherent cells in minutes. Neutrophils stimulated in suspension showed little degranulation. Treatment of neutrophils with an inhibitor of 5-lipoxygenase–activating protein (MK886) and thus synthesis of leukotrienes (LTs) or with an antagonist of the LTB4 receptor (LY223982) blocked the release of elastase. This indicated that endogenous synthesis of 5-lipoxygenase products such as LTs and autocrine activation of neutrophils was required for fMLP-driven elastase release. We hypothesize that the differential ability of PAF and fMLP to induce elastase release from surface-adherent neutrophils could arise from differential ability to generate leukotrienes, such as LTB4, and would be an appropriate mechanism for the control of elastase release during inflammation in vivo, where it is important that cytotoxic agents are not released until activated neutrophils have migrated into the extravascular tissues.