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kinase C, respectively. Thus, the type of stimuli is also an important factor for active oxygen metabolism in neutrophils. Among these probes used, L-012 exhibited the strongest chemilumi-nescence irrespective of the ligands used for stimulating blood samples and isolated neutrophils.

Because neutrophils in the oral cavity are already primed during and/or after infiltrations to the mucosal surface, they spontaneously generate reactive oxygen species without added stimuli.26 Under unstimulated conditions, chemiluminescence intensity of L-012 was about 3 and 50 times greater than that of MCLA and luminol, respectively (Figure 20.5). When stimulated by PMA, reactive oxygen species generation by neutrophils from the oral cavity increased in a time-dependent manner. PMA enhanced L-012-dependent chemiluminescence by about 12-fold. The enhanced chemiluminescence of L-012 was about 10 and 100 times stronger than that of MCLA and luminol, respectively. Thus, highly sensitive L-012 permits studies of the mechanism of reactive oxygen species

Luminol L012

FIGURE 20.5 Chemiluminescence of PMA-stimulated neutrophils from the oral cavity. Reaction mixtures contained in a final volume of 500 ¡A KRP, 1 x 105 OPMN, and 400 ¡M L-012 (A, 1 and 2), or 4 ¡M MCLA (A, 3 and 4), or 1 mM luminol (B, 5 and 6). At the indicated times (arrows), either 10 nM PMA (2, 4, 6) or saline (1, 3, 5) was added to the mixture at 37°C. Chemiluminescence intensities were recorded as described in Figure 20.3. (From Imada, I. et al., Anal. Biochem., 271, 53, 1999. With permission.)

FIGURE 20.5 Chemiluminescence of PMA-stimulated neutrophils from the oral cavity. Reaction mixtures contained in a final volume of 500 ¡A KRP, 1 x 105 OPMN, and 400 ¡M L-012 (A, 1 and 2), or 4 ¡M MCLA (A, 3 and 4), or 1 mM luminol (B, 5 and 6). At the indicated times (arrows), either 10 nM PMA (2, 4, 6) or saline (1, 3, 5) was added to the mixture at 37°C. Chemiluminescence intensities were recorded as described in Figure 20.3. (From Imada, I. et al., Anal. Biochem., 271, 53, 1999. With permission.)

FIGURE 20.6 Chemiluminescence of rat peritoneal neutrophils. Reaction mixtures contained in a final volume of 500 ¡A KRP, 1 x 105 rat peritoneal neutrophils, and 400 ¡M L-012 (A, 1 and 2), or 4 ¡M MCLA (A, 3 and 4), or 1 mM luminol (B, 5 and 6). At the indicated times (arrows), either 2 Ag/ml opsonized zymosan (OZ) (2, 4, 6) or saline (1, 3, 5) was added to the mixture. During the incubation at 37°C, chemiluminescence was monitored as shown in Figure 20.3. (From Imada, I. et al., Anal. Biochem., 271, 53, 1999. With permission.)

FIGURE 20.6 Chemiluminescence of rat peritoneal neutrophils. Reaction mixtures contained in a final volume of 500 ¡A KRP, 1 x 105 rat peritoneal neutrophils, and 400 ¡M L-012 (A, 1 and 2), or 4 ¡M MCLA (A, 3 and 4), or 1 mM luminol (B, 5 and 6). At the indicated times (arrows), either 2 Ag/ml opsonized zymosan (OZ) (2, 4, 6) or saline (1, 3, 5) was added to the mixture. During the incubation at 37°C, chemiluminescence was monitored as shown in Figure 20.3. (From Imada, I. et al., Anal. Biochem., 271, 53, 1999. With permission.)

generation by leukocytes and of the role of reactive oxygen species in the pathogenesis of various diseases. The present work describes that, under physiological conditions, L-012 reveals strong chemiluminescence in the presence of various types of activated neutrophils.

20.5 GENERATION OF REACTIVE OXYGEN SPECIES BY RAT PERITONEAL NEUTROPHILS

Neutrophils were obtained from the peritoneal cavity of the rat 16 h after intraperitoneal injection

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