Factors affecting the measurement of chemiluminescence in stimulated human polymorphonuclear leucocytes

Abstract

Optimum conditions were established for the generation and measurement of luminol-dependent chemiluminescence (CL) in human polymorphonuclear leucocytes (PMNL) stimulated with a variety of particulate and soluble agents. Several factors had a particular influence on the kinetics of CL stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Two peaks, both azide-sensitive, were observed at 21 degrees C and 25 degrees C, but these increased in magnitude and merged to give a single, early peak when the temperature was increased at 37 degrees C. Pre-exposure of PMNL to a buffer containing calcium was essential for the expression of both phases of fMLP-stimulated CL, while the second peak decreased dramatically if the cells were stored at 4 degrees C for 4 hours before assay. In contrast, storage of PMNL at 4 degrees C for up to 8 hours in a buffer without divalent cations did not alter the kinetics or magnitude of CL induced by other stimuli, and had the benefit of minimizing the rate of cell aggregation. This study confirms that measurement of luminol-dependent CL in stimulated PMNL is a useful analytical tool, but shows that careful attention to experimental design is required to ensure that the observed CL provides a true measure of the parameter under investigation.

Assaying Granulocyte Phagocytosis by Chemiluminescence: Effect of Storage Time and Temperature of Blood Samples

“…When stored on ice the samples can be kept for 1 and 4 h in pig and horse, respectively, without reduced chemiluminescence, while there was a reduction in the cow samples after 1 h. Similar to the present data, chemiluminescence following human granulocyte phagocytosis started to decline after 3 h of storage of blood samples at RT (RIsTo~ti and REPO, 1989). Purified human granulocytes, on the other hand, showed consistent chemiluminescence for up to 8 h after storage at 4OC (HARBER and TOPLEY, 1986). This may indicate that granulocytes in whole blood do not maintain their functional capacity during storage as well as purified granulocytes.…”

“…Knowledge about the timing of this reduction in vitm is imperative for proper analysis of data on the granulocyte's functional capacity within and between experiments. There are some data available on the timing of the reduction of in vitm functional capacity of human granulocytes (HARBER and TOPLEY, 1986;PAUL et al, 1987;RrSTOLA and REPO, 1989). The present study in healthy farm animals with haematological values within the respective reference Considering 30min to be the minimum time delay to initialize the assay procedure, the results can be summarized in the statement: blood samples can be stored without reduced chemiluminescence following granulocyte phagocytosis at RT for additionally 1 h in horse, 2 h in pig and 4 h in cow.…”

Analysis of Polymorphonuclear Leukocyte Respiratory Burst Activity in Uremic Patients using Whole-Blood Chemiluminescence

“…CL was displayed as realtive light units, 1 unit being equiva lent to 10 photon counts per second (cps). The peak CL was noted in each case and was used as a measure of respiratory burst stimulation [18].…”

Type 1 fimbriate Escherichia coli stimulates a unique pattern of degranulation by human polymorphonuclear leukocytes

“…These soluble stimuli directly or indirectly affect the movement of calcium into or within the cell, whereas zymosan has been shown to induce a calcium-dependent response only when opsonized with immunoglobulin G (25). All these stimuli activate the PMN respiratory burst to various extents (18). In terms of degranulation, however, only high concentrations of A23187 were capable of mobilizing all the granule markers measured, whereas at concentrations below 0.5 ,uM only release of the 20 granule marker was detected.…”

“…The activation of phagocytic cells by particulate stimuli occurs as a result of either specific receptor-ligand binding (26) or a receptor-independent physicochemical interaction of a particle with the cell surface (42). This stimulus-cell interaction has been characterized in terms of respiratory burst activation (18), the release of intracellular proteins (3), and the generation of pro-inflammatory mediators (44). There is considerable evidence that the reactive oxygen metabolites and proteases released by phagocytic cells during this inflammatory response can contribute directly to tissue damage (13,14,23).…”