Human neutrophil-mediated nonoxidative antifungal activity against Cryptococcus neoformans

It has long been appreciated that polymorphonuclear leukocytes (PMN) kill C ryptococcus neoformans, at least in part via generation of fungicidal oxida nts. The aim of this study was to examine the contribution of nonoxidative mechanisms to the inhibition and killing of C. neoformans. Treatment of hum an PMN with inhibitors and scavengers of respiratory burst oxidants only pa rtially reversed anticryptococcal activity, suggesting that both oxidative and nonoxidative mechanisms were operative. To define the mediators of nono xidative anticryptococcal activity, PMN were fractionated into cytoplasmic, primary (azurophil) granule, and secondary (specific) granule fractions. I ncubation of C. neoformans with these fractions for 18 h resulted in percen ts inhibition of growth of 67.4 +/- 3.4, 84.6 +/- 4.4, and 29.2 +/- 10.5 (m ean a standard error, n = 3), respectively. Anticryptococcal activity of th e cytoplasmic fraction was abrogated by zinc and depletion of calprotectin. Antifungal activity of the primary granules was significantly reduced by p ronase treatment, boiling, high ionic strength, and magnesium but not calci um. Fractionation of the primary granules by reverse phase high-pressure li quid chromatography on a C, column over an acetonitrile gradient revealed m ultiple peaks with anticryptococcal activity. Of these, peaks 1 and 6 had s ubstantial fungistatic and fungicidal activity. Peak 1 was identified by ac id-urea polyacrylamide gel electrophoresis (PAGE) and mass spectroscopy as human neutrophil proteins (defensins) 1 to 3. Analysis of peak 6 by sodium dodecyl sulfate-PAGE revealed multiple bands. Thus, human PMN have nonoxida tive anticryptococcal activity residing principally in their cytoplasmic an d primary granule fractions. Calprotectin mediates the cytoplasmic activity , whereas multiple proteins, including defensins, are responsible for activ ity of the primary granules.