The fungicidal activity of human PMNs against C albicans is partially due to manganese-dependent activity of arginase I. (A) When manganese is limiting (RPMI medium 1640 with 10% serum) arginase does not contribute to the fungicidal activity of human PMNs against C albicans. Human PMNs were cocultured for 1 hour with a wild-type strain of C albicans. The viability of C albicans after phagocytosis was monitored with the XTT assay in 7 independent experiments. PMN arginase was inhibited by preincubation with nor-NOHA (200 μM) and PMN MPO with sodium azide (NaN₃;1 μM) in 7 independent experiments, whereas in 3 separate experiments NADPH oxidase was blocked with DPI (20 μM). To allow for comparison between individual experiments, the percentage of kill inhibition (compared to the viability of C albicans after phagocytosis without the use of inhibitors) is shown. The inhibition of fungicidal activity was statistically significant with the indicated P values, analyzed with the 2-tailed Student t test. n.s. indicates not significant. (B) Arginase activity in human PMNs depends on the manganese concentration present during phagocytosis of C albicans. Experimental set-up was as in panel A. During phagocytosis increasing concentrations of exogenously added MnCl₂ were present. Arginase activity was determined in lysates of PMN–C albicans coincubations after 1 hour. The generation of reactive oxygen intermediates was inhibited by the presence of 1 μM NaN₃ during phagocytosis. No exogenous manganese was added during the enzymatic assay (“native arginase activity”). One representative example of 4 independent experiments is shown; r is correlation coefficient. (C) Fungicidal activity of human PMNs against C albicans correlates with PMN arginase activity. The representative experiment is the same as in panel B. The percentage of C albicans kill (% kill after 1 hour of coincubation compared to the viability of C albicans alone) is shown in correlation to the different PMN arginase activities induced by manganese as demonstrated in panel B. (D) On manganese supplementation (RPMI medium 1640 with 10% serum + 50 μM MnCl₂) arginase does participate in the fungicidal activity of human PMNs against C albicans. Experimental set-up and statistical analysis is as in panel A. Error bars in panels A and D indicate standard deviation. Curves in panels B and C are regression lines.