IntroductionWe investigated the role of intracellular iron on the capacity of Histoplasma capsulatum (Hc) yeasts to multiply within human macrophages (M+). Coculture of Hc-infected MO with the iron chelator deferoxamine suppressed the growth of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by iron-free transferrin (apotransferrin). Chloroquine, which prevents release of iron from transferrin by raising endocytic and lysosomal pH, induced human MO to kill Hc. The effect of chloroquine was reversed by iron nitriloacetate, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin, which releases iron only in an acidic environment.Chloroquine (40-120 mg/kg) given intraperitoneally for 6 d to Hc-infected C57BL/6 mice significantly reduced the growth of Hc in a dose-dependent manner. At 120 mg/kg there was a 17-and 15-fold reduction (P < 0.01) in CFU in spleens and livers, respectively. The therapeutic effect of chloroquine also correlated with the length of treatment. As little as 2 d of chloroquine therapy (120 mg/kg), when started at day 5 after infection, reduced CFU in the spleen by 50%. Treatment with chloroquine for 10 d after a lethal inoculum of Hc protected six of nine mice; all control mice were dead by day 11 (P = 0.009).This study demonstrates that: (a) iron is of critical importance to the survival and multiplication of Hc yeasts in human MO; (b)

Human neutrophils (PMN) demonstrated potent fungistatic activity against Histoplasma capsulatum (Hc) yeasts in a sensitive microassay that quantifies the growth of yeasts by the incorporation of 13Hjleucine. At a PMN:yeast ratio of 1:2, PMN inhibited the growth of yeasts by 37%. Maximum inhibition of 85% to 95% was achieved at a PMN/yeast ratio of 10:1 to 50:1. Opsonization of the yeasts in fresh or heat-inactivated serum was required for PMN-mediated Previous studies in vitro have generated conflicting results with regard to PMN fungicidal activity against Hc yeasts. Howard (4) reported that in the presence of serum, glycogen-induced, peritoneal neutrophils of guinea pigs killed Hc yeasts after 3 h of incubation. In these experiments, killing was defined by altered stainability ofthe ingested Hc yeasts. However, definition of fungal death by alteration in staining properties has not been accepted widely (5), and these ultrastructural and tinctorial changes were not observed after phagocytosis by human PMN (6). Schaffner et al. (6) found that at a high phagocyte-to-fungus ratio, human PMN killed the mycelial phase, but not the yeast phase of Hc, despite the fact that opsonized yeasts induced the PMN respiratory burst.Most recently, it has been reported that human (7) and murine (8) neutrophils exhibited some fungistatic activity against Hc yeasts. In these experiments, PMN

Previously we demonstrated that human neutrophils mediate potent and long-lasting fungistasis against Histoplasma capsulatum yeasts and that all of the fungistatic activity resides in the azurophil granules. In the present study, specific azurophil granule constituents with fungistatic activity were identified by incubation with H. capsulatum yeasts for 24 h and by quantifying the subsequent growth of yeasts via the incorporation of [ 3 H]leucine. Human neutrophil defensins HNP-1, HNP-2, and HNP-3 inhibited the growth of H. capsulatum yeasts in a concentration-dependent manner with maximum inhibition at 8 g/ml. At a concentration of 4 g/ml, all possible paired combinations of defensins exhibited additive fungistatic activity against H. capsulatum yeasts. Cathepsin G and bactericidal-permeability-increasing protein (BPI) also mediated fungistasis against H. capsulatum in a concentration-dependent manner. The fungistatic activities of combinations of cathepsin G and BPI were additive, as were those of combinations of cathepsin G or BPI with HNP-1, HNP-2, and HNP-3. Lysozyme and elastase exhibited modest antifungal activity, and azurocidin and proteinase 3 exhibited no significant fungistasis against H. capsulatum yeasts. Thus, defensins, cathepsin G, and BPI are the major anti-H. capsulatum effector molecules in the azurophil granules of human neutrophils.Histoplasma capsulatum is a dimorphic fungal pathogen of worldwide importance that causes a broad spectrum of disease activity. The course of H. capsulatum infection is mild in most immunocompetent individuals, but progressive disseminated infections occur in individuals immunocompromised by hematologic malignancies (6,25,30) or cytotoxic therapy (16,32,33) or in individuals infected with human immunodeficiency virus (15,20,31).Infection with H. capsulatum is acquired by inhalation of microconidia into the terminal bronchioles and alveoli of the lung. Inhaled microconidia subsequently convert into yeasts that are responsible for the pathogenesis of histoplasmosis (17). H. capsulatum yeasts are phagocytized by alveolar macrophages (M), within which they multiply (2, 7). Dividing yeasts destroy the alveolar M, and subsequently the yeasts are ingested by other resident alveolar M and by inflammatory phagocytes recruited to the locus of infection. Repetition of this cycle results in spread of infection to hilar lymph nodes and to other organs during the acute phase of primary histoplasmosis. Subsequently, the maturation of specific cell-mediated immunity against H. capsulatum activates M to halt yeast proliferation with gradual resolution of the disease process in immunocompetent hosts (7,22).The role of polymorphonuclear neutrophils (PMNs) in the cell-mediated immune response against H. capsulatum is unclear (7). However, even the earliest studies in a murine model of histoplasmosis described PMNs as being the predominant inflammatory cell type in the lungs during the first 36 h after intranasal inoculation with H. capsulatum macroconidia. In these studies the PMNs ...

Histoplasma capsulatum (Hc) is a facultative intracellular fungus that modulates the intraphagosomal environment to survive within macrophages (Mφ). In the present study, we sought to quantify the intraphagosomal pH under conditions in which Hc yeasts replicated or were killed. Human Mφ that had ingested both viable and heat-killed or fixed yeasts maintained an intraphagosomal pH of ∼6.4–6.5 over a period of several hours. These results were obtained using a fluorescent ratio technique and by electron microscopy using the 3-(2,4-dinitroanilo)-3′-amino-N-methyldipropylamine reagent. Mφ that had ingested Saccharomyces cerevisae, a nonpathogenic yeast that is rapidly killed and degraded by Mφ, also maintained an intraphagosomal pH of ∼6.5 over a period of several hours. Stimulation of human Mφ fungicidal activity by coculture with chloroquine or by adherence to type 1 collagen matrices was not reversed by bafilomycin, an inhibitor of the vacuolar ATPase. Human Mφ cultured in the presence of bafilomycin also completely degraded heat-killed Hc yeasts, whereas mouse peritoneal Mφ digestion of yeasts was completely reversed in the presence of bafilomycin. However, bafilomycin did not inhibit mouse Mφ fungistatic activity induced by IFN-γ. Thus, human Mφ do not require phagosomal acidification to kill and degrade Hc yeasts, whereas mouse Mφ do require acidification for fungicidal but not fungistatic activity.

Recombinant cytokines and colony-stimulating factors (CSFs) were tested for their abilities to activate human monocytes/macrophages (M4) to inhibit the intracellular growth of or kill Histoplasma capsulatum yeasts. None of the cytokines or CSFs or combinations of cytokines and CSFs activated M+ fungistatic activity when they were added to M+ monolayers concurrently with yeasts. In contrast, culture of monocytes for 7 days in the presence of interleukin 3, granulocyte-M+ CSF, or M+ CSF stimulated M4 fungistatic (but not fungicidal) activity against H. capsulatum yeasts in a concentration-dependent manner. Optimal activation of M+ by CSFs required 5 days of coculture, and the cultures had to be initiated with freshly isolated peripheral blood monocytes. Culture of monocytes with combinations of CSFs or addition of CSFs during the 24 h of coculture with the yeasts did not further enhance M4 fungistatic activity for H. capsulatum. Addition of gamma interferon or tumor necrosis factor alpha to CSF-activated M+ also did not enhance M4 fungistatic activity. These results suggest that interleukin 3, granulocyte-M4 CSF, and M4 CSF may play a role in the cell-mediated immune response to H. capsulatum by enhancing monocyte/M+ fungistatic activity. Human monocytes, cultured monocyte-derived macrophages (MO), alveolar M4, and polymorphonuclear neutrophils recognize unopsonized Histoplasma capsulatum yeasts and conidia via the CD18 family of adhesion-promoting glycoproteins (3, 16, 26). Attachment of yeasts and conidia to M4 is followed rapidly by ingestion (16). Phagocytosis of the yeasts stimulates the M4 respiratory burst (3) and phagolysosomal fusion (19). Despite exposure of yeasts to the M4 antifungal armamentarium, ingested yeasts multiply readily within human monocytes/M4 (5, 18). Recently, we demonstrated that human monocytes and