Damage to Pseudohyphal Forms of Candida albicans by Neutrophils in the Absence of Serum In Vitro

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Abstract

Large forms of Candida are characteristically present in invasive lesions and are often cleared by host defenses. Therefore, an in vitro system was developed to study interactions between leukocytes and pseudohyphae. By light, phase contrast, and electron microscopic observations, in the absence of serum, neutrophils attached to and spread over the surfaces of partially ingested pseudohyphae, which then appeared damaged. Using a new assay which measured neutrophil-induced inhibition of uptake of [14C]cytosine by Candida, damage to Candida in the absence of serum was 53.04±2.96% by neutrophils from 27 normal subjects. With serum, damage to Candida increased because of opsonization by low levels of anti-Candida immunoglobulin G in normal sera. Damage to Candida was inhibited by colchicine, cytochalasin B, and 2-deoxyglucose, which interfered with spreading of neutrophils over the surfaces of Candida. Dibutyryl cyclic AMP, theophylline, and isoproterenol also inhibited damage to Candida. Hydrocortisone was inhibitory in levels (10 μM) achievable with pharmacologic doses in man. Light, fluorescence, and electron microscopy indicated that neutrophils degranulated after contact with Candida. Quantitative studies revealed only a minimal increase in specific release of lysosomal enzymes from azurophil granules, but much greater release of lysozyme from specific granules. Candida activated neutrophil oxidative microbicidal mechanisms, as shown by iodination of Candida by neutrophils, and chemiluminescence from neutrophils interacting with Candida. Unlike live Candida, killed Candida did not induce chemiluminescence, were not iodinated, and did not attach to neutrophils by microscopy. Like Candida pseudohyphae, contact between neutrophils and hyphal forms of Aspergillus and Rhizopus occurred in the absence of serum. This did not occur with Cryptococcus neoformans, an encapsulated yeast, and was low with Candida yeasts. These findings indicate that neutrophils can recognize and attach to Candida pseudohyphae, then damage the Candida. This may represent a general reaction between neutrophils and large forms of fungi. Though the size of the organisms precludes complete ingestion, neutrophil oxidative microbicidal mechanisms are activated, and preferential release of contents of specific granules appears to occur.

Authors

Richard D. Diamond, Raymond Krzesicki, Wellington Jao