A new EPR-based method has been developed to obtain selective information on pO2 in a specified intracellular compartment (phagosome). The method utilises the selective incorporation of the oxygen-sensitive probe 4-(Trimethylammonium) 2,2,6,6,-tetramethylpiperidine-D17-1-Oxyl iodide (D-CAT1) into phagosomes of macrophages stimulated with zymosan. Since the signal arising from the neutral nitroxide 4-oxo-2,2,6,6,-(15N)-tetramethylpiperidine-D16-1-Oxyl (15N-PDT) does not overlap with that from the D-CAT1, these signals can be monitored simultaneously. Lipopolysaccharide (LPS) is the endotoxin from the outer membrane of Gram-negative bacteria that is associated with the high morbidity and mortality in patients with septic shock. Our previous studies have shown that LPS can influence mitochondrial oxygen consumption in a variety of cell types and alter the oxygen utilisation of organs in experimental septic shock. It is also suggested that LPS can augment the respiratory burst associated with phagocytosis in macrophages. D-CAT1 was added to cells of the murine macrophage cell lines RAW 264.7 followed by zymosan stimulation to induce phagocytosis. After washing, the probe remained in the phagosome; without stimulation of phagocytosis the probe was not incorporated and could be removed by washing. 15N-PDT was added to the same samples to give the effective extracellular oxygen concentration (as less than 5% of the PDT signal arises from inside the cells). Cells with intraphagosomal D-CAT1 and 15N-PDT were then treated with LPS and the oxygen concentrations measured for intraphagosomal and extracellular sites after callibration of the two probes in pure air and nitrogen. The results show that LPS reduces intraphagosomal oxygen concentrations by almost one half. Furthermore, EPR spin-trapping experiments (using DMPO to trap - OH and -OOH radicals) showed that LPS stimulates a sustained respiratory burst in these macrophages above that induced by a zymosan alone. These results suggest that LPS can influence macrophage phagocytosis, and in certain cases the low oxygen concentration within phagosomes can potentially limit macrophage microbicidal funtion during infections.
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