Matthias P. Wymann

Turning on the respiratory burst

The respiratory burst is a distinguishing property of phagocytes. It is induced by chemotactic stimulation or phagocytosis and reflects the activation of a membrane-bound enzyme system that transfers electrons from cytosolic NADPH to extracellular oxygen, producing superoxide. The products of the burst are essential for the killing of microorganisms, but are also a cause of tissue damage and inflammation. Studies aimed at a better understanding of the regulation of the respiratory burst should help in the search for new ways to treat infections and inflammation.

Chemiluminescence detection of H2O2 produced by human neutrophils during the respiratory burst.

A sensitive luminol-dependent chemiluminescence assay for H2O2 was developed for the indirect determination of the transient changes in NADPH oxidase activity associated with the respiratory burst of human neutrophils. A relatively large, controlled amount of horseradish peroxidase was used in combination with added luminol to rapidly remove and simultaneously detect H2O2 as soon as it is formed, thus preventing its accumulation during burst activity and minimizing the effects of side reactions. Cell-derived myeloperoxidase and possibly catalase were inhibited with 90 microM sodium azide to maintain the total catalytic activity toward H2O2 at a constant level. Chemiluminescence measurements of the respiratory burst activity of human neutrophils stimulated with N-formyl-Met-Leu-Phe (fMLP) were in good agreement with measurements made using an established fluorometric assay based on similar principles (P. A. Hyslop and L. A. Sklar (1984) Anal. Biochem. 141, 280-286). In contrast to fluorometry, the chemiluminescence progress curves reflect the instantaneous rather than the integrated levels of H2O2 at any time and are thus a more direct measure of the activity of the NADPH oxidase. This advantage, as well as higher signal-to-noise ratios and greater inherent sensitivity, distinguishes chemiluminescence as a means of following burst activity. The onset of fMLP-stimulated H2O2 generation was detectable by chemiluminescence within 2 s of stimulation (as opposed to more than double this time by fluorometry), showing that high sensitivity is an important consideration in evaluating respiratory burst kinetics. In contrast to fMLP stimulation, longer and concentration-dependent onset times were observed when phorbol myristate acetate was used as a stimulus.

Shape changes, exocytosis, and cytosolic free calcium changes in stimulated human eosinophils.

Essentially pure preparations of normal density eosinophils obtained from patients with hypereosinophilic syndrome (HES) were stimulated with complement factor 5a (C5a), platelet-activating factor (PAF), FMLP and neutrophil-activating peptide (NAP-1/IL-8). Three responses were studied, the transient rise in cytosolic free calcium concentration ([Ca2+]i) (derived from indo-1 fluorescence), shape changes (measured by laser turbidimetry), and exocytosis of eosinophil peroxidase (EPO) (assessed by H2O2/luminol-dependent chemiluminescence). Responses were obtained with all four agonists, but C5a and PAF were by far more potent than FMLP and NAP-1/IL-8, which induced only minor effects. Pretreatment of the cells with pertussis toxin attenuated [Ca2+]i changes, EPO release and, to a lesser extent, shape changes, indicating that GTP-binding proteins of Gi-type are involved in receptor-dependent signal transduction processes leading to these responses. A clear dissociation was observed in the control of the shape change response and EPO exocytosis. The shape change was not affected by Ca2+ depletion or treatment with the protein kinase inhibitor staurosporine, but exocytosis was prevented by Ca2+ depletion and markedly enhanced by staurosporine. The activation of the contractile system, leading to shape changes and motility, thus appears to be independent of the classical signal transduction pathway involving phospholipase C, a [Ca2+]i rise and protein kinase C activation. Exocytosis is, as expected, Ca2+ dependent and appears to be under a negative control involving protein phosphorylations.

Oscillatory motion in human neutrophils responding to chemotactic stimuli

Human neutrophils treated with the secretion inhibitor 17-hydroxywortmannin were stimulated with fMLP, C5a, PAF or LTB4, and the ensuing shape change was studied. The cells rapidly extended lamellipodia and showed regular oscillatory behaviour. The oscillations were observed in both light transmission and 90 degrees light scattering, had the same frequency in each case, and disappeared within 30-50 seconds. Light scattering theory suggests that they reflect rhythmic changes in the shape and/or size of the chemotactically stimulated cells, possibly related to crawling or swimming movements associated with migration.

The Neutrophil Leukocyte. Properties and Mechanism of Activation

The neutrophil leukocytes constitute the largest population of white blood cells. Their main function is to defend the host organism from microbial invasion. The properties required for this function are chemotactic responsiveness, mobility and the ability to phagocytose and to release microbicidal products. Microorganisms which colonize a tissue are sensed by the neutrophils through chemotactic molecules generated upon infection. In response to such stimuli, neutrophils adhere to the endothelia of the microvessels of the infected area, leave the blood and migrate toward the invaders, eventually phagocytosing and killing them. Killing requires the activation of NADPH-oxidase, a plasma membrane enzyme that generates superoxide (Babior, 1978 ; Baggiolini, 1984), and is aided by the release of enzymes and other storage proteins from the azurophil and specific granules (Baggiolini & Dewald, 1984)). The event initiating this host defense process is the activation of the neutrophils via chemotactic agonists and phagocytosis receptors. The mechanism of activation is generally studied using chemotactic agonists rather than phagocytosable particles. As soluble molecules, the agonists have the advantage of acting instantaneously and uniformly on all target cells.