Comparison of cytoplasmic calcium transients in human neutrophils responding to opsonized myobacterium avium and myobacterium tuberculosis nonvirulent mutant
Stutzman, Michael J
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Objective: The phagocytic process initiates the efficient and effective defense mechanism of the innate human immune system. However, certain pathogenic organisms such as Cryptococcus neoformans (Cj) and Mycobacterium tuberculosis (Mtb) evade this defense and survive or proliferate in the phagosome in human phagocytic cells (8, 18, 19, 30). Research from this lab has demonstrated that Cf survives because it hyperacidities the phagolysosome (18, 19). When treated with chloroquine, an antimalarial drug which alkalinizes the phagosome, the phagocytes' antifungal properties can work and Cf is killed (18, 19). Two important conclusions were strongly supported by this research: the survival of some pathogenic organisms is dependent on the phagosomal environment, and it is possible to manipulate this environment to restore the killing function of phagocytes. However, the specific mechanisms by which each organism that evades killing by phagocytes alters their normal function remain to be determined. The hypothesis of this research is that these mechanisms are unique to the organism causing them. It is well known that a cytosolic calcium transient (Δ[Ca2+]i) is an early rapid receptor-mediated response in polymorphonuclear neutrophils (PMN) (1, 2, 5-7, 12, 15, 21, 23-28). Using Mycobacterium avium, and H37Ra, a nonvirulant mutant of Mtb, as models, this study evaluated potential similarities and differences between receptor elicited Δ[Ca2+]i in human PMN. Using homologous serum-opsonized Mycobacterium avium, and H37Ra, a nonvirulant mutant of Mtb, as models, this study evaluated their receptor elicited Δ[Ca2+]i in human PMN. Methods: Upon informed consent, human PMN were isolated from volunteer blood following the Dextran/Ficoll Paque™ protocol established by Boyem (3). The ester Indo-Am was used to measure the change in the concentration of intracellular calcium in the PMN suspension via fluorometry. At a concentration of 2 million PMN/mL phosphate-buffered saline (PBS), the cells were loaded with 5 microliters of 1 mM IndoAM stock in dimethyl sulfoxide/ mL of cell suspension. The Mycobacterium avium and H37Ra used in these experiments were grown separately in liquid media in the presence of Tween 80. After being washed and effectively resuspended in PBS, the dose was quantitated via optical density (OD) at 436 nmm. For opsonization, the desired volume of mycobacterium was diluted with a 1:1 ratio of autologous human serum. Prior to stimulation, PMN were incubated at 37°C in Krebs Ringer Phosphate Buffer (KRP) at a concentration of 2 million cells/mL KRP to allow for the replenishment of intracellular calcium stores. In certain cases, a 10 microliter dose of 500 mM ethylene glycol tetraacetic acid (EGTA), a calcium chelator, was added to the suspension 15 seconds prior to the second stimulation. Fluorescence was measured with a Hitachi F4500 fluorometer, equipped with a thermostated cuvette holder and stirrer. It was set for an excitation wavelength of 357 nm, with a slit of 5.0 nm. The cycle time was 1.8 seconds. The photomultiplier, adjacent to the excitation beam, was set at 700 V. The emission slit was also set at 5.0 nm to detect emissions at 405 and 485 nm. The software used to control these conditions and compile the data was FL Solutions™. Results: Both M. avi and H37Ra elicited Δ[Ca2+]i in human PMN. This response increased with dose to a maximal Δ[Ca2+]i transient. Stimulating doses larger than that which evoked this maximal response appeared to activate lower affinity receptors, as indicated by a higher retention of [Ca2+]i after the peak response, relative to controls. EGTA slightly affected both the higher and lower affmity receptor-mediated responses. To further investigate the possibility of low affmity receptors, runs in which an equal second dose of stimulus was administered five minutes after the first were conducted. Both species of mycobacteria induced a much smaller second response, but their dependence on extracellular calcium and relation to the dose differed slightly. Conclusions: These results support the hypothesis that early intracellular events in activated PMN differ between their responses to various stimuli, such as M. avi and H37Ra. Furthermore, the presence of a lower affinity receptor involved in a mycobacterium induced activation of PMN is implied by these experiments. The classification of this receptor(s) is unknown, as is its significance regarding the fate of the phagocytized organism, but these matters will be of interest in the near future. It has also been demonstrated that calcium influx likely occurs, but does not appear to be a major contributor, to the signal transduction induced by high affmity receptors in this type of response. It is unclear whether low affinity receptors elicit some calcium influx, though this may be a possibility. Moving forward, similar experiments will be conducted by flow cytometry kinetics, using multiple probes to determine several parameters (e.g. receptor occupancy, Δ[Ca2+]i, ROS, pH, and elastase release) for each PMN.
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