Alveolar macrophages and lung surfactant in the defense against Cryptococcus neoformans

Abstract

Cryptococcus neoformans causes disease mainly in immunosuppressed patients, especially those with AIDS, and on corticosteroids. The yeast is normally inhaled and the lung is the primary site of infection, where the alveolar macrophages (AM) provide a first line of host defense. The aim of this thesis was to study immune responses of AM and immunomodulatory functions of lung surfactant phospholipids in the defense against C. neoformans, using the rat or rabbit as animal models. AM were obtained by lung lavage. Phagocytosis was assessed with a fluorescence method that distinguishes between attached and ingested organisms. The production of nitric oxide and superoxide anions by AM was evaluated by the Griess reaction and the nitroblue tetrazolium (NBT) test, respectively. The pH of the phagolysosomes was measured using a cytofluorometric technique. Killing was studied by a colony-forming assay and lipid peroxidation (LPO) by the LPO-586 assay. Fresh rat serum (FRS) or anticryptococcal IgG (anti-CnIgG) was required for phagocytosis of the yeast by AM in vitro. The attachment of FRS-opsonized, but not of unopsonized C. neoformans was enhanced by interferon (IFN)-gamma. The ingestion of unopsonized and opsonized yeast with FRS or anti-CnIgG was increased by surfactant. AM, obtained from rats treated with cortisone acetate (CA), showed lower attachment and ingestion after 3 and 4 weeks of treatment, respectively, than those from control animals.

In vivo studies showed a good attachment and ingestion of C. neoformans by AM. Most of the yeast cells were located in phagolysosomes in vivo and in vitro at pH<5.5 after a 24 h challenge. AM stimulated in vitro by FRS-opsonized C. neoformans showed hardly any NBT reduction during 1h incubation, but reached high levels after 3 and 24 h phagocytosis. Opsonization with anti-CnIgG caused a greater NBT response than did FRS. Surfactant decreased the NBT reduction by AM incubated with the yeast. AM from rats treated with CA for 4 weeks and challenged with cryptococci in vitro showed impaired NBT reduction. We found a markedly increased NBT reduction by AM 24 h after instillation of the yeast in our in vivo studies. C. neoformans induced a small but significant increase in nitric oxide (NO) production by AM in vitro. This response was synergistically enhanced by IFN-gamma. AM treated with IFN-gamma showed higher killing activity against the yeast than untreated AM, a finding that correlated with increased NO production by AM. AM stimulated by C. neoformans induced cellular LPO. Addition of lung surfactant caused an increase in LPO, accompanied by greater detachment of AM.

Our findings indicate that AM probably play an important role in confining C. neoformans infections to the lungs of an immunocompetent host. Impairment of AM functions by steroid therapy constitutes a risk for dissemination of the yeast to other organs. In vitro studies suggest that surfactant affects AM defense against C. neoformans by enhancing its ingestion and diminishing the superoxide anions produced by AM. In the lungs, AM exposed to C. neoformans might promote peroxidation of surfactant lipids.