Invasive pulmonary aspergillosis is acquired through inhalation of conidia. Little is known about early interactions between Aspergillus fumigatus conidia and alveolar epithelial cells, so an in vitro model was developed to study binding between conidia and A549 cells, a line of type II pneumocytes. Conidia rapidly became attached to confluent monolayers of A549 cells in serum-free medium, reaching a plateau within 40 min. Scanning electron microscopy (EM) showed a random pattern of early adherence; viable conidia subsequently became clustered on pneumocyte surfaces. Following germination of pneumocyte-adherent conidia for 12 h, direct penetration of epithelial cells by hyphae could be demonstrated by scanning and transmission EM. These data suggest that an early event following inhalation of A. fumigatus conidia may be binding of conidia to pneumocytes, followed by hyphal penetration of the epithelial cell layer.

IntroductionReceptor-mediated agonists, such as FMLP, induce an early, phospholipase D (PLD)-mediated accumulation of phosphatidic acid (PA) which may play a role in the activation of NADPH oxidase in human PMN. We have determined the effect of changes in PA production on 02 consumption in intact PMN and the level of NADPH oxidase activity measured in a cell-free assay. (13, 14) and others (15-18) have demonstrated that FMLP also activates a phospholipase D (PLD) that hydrolyzes choline-containing phosphoglycerides (PC) such that PA is the first metabolite formed. In this pathway, DG is generated by PA phosphohydrolase (PAP)-catalyzed dephosphorylation of PA (19). The differences in the temporal sequence of formation ofPA and DG via these pathways and the fact that interconversions between these metabolites modulates their accumulation during stimulation, complicate efforts aimed at understanding the individual involvement of PA or DG in the activation of PMN NADPH oxidase.A role for DG (6)(7)(8)(20)(21)(22)

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Aspergillus fumigatus has previously been shown to produce a soluble extracellular inhibitor of the alternative complement pathway, called Aspergillus complement inhibitor, or CI. We now report an efficient method for production of CI which relies on the fact that poorly conidiating cultures yielded CI activity with approximately sevenfold-higher potency than CI produced by conidiating cultures. CI from poorly conidiating cultures provided 50% inhibition of alternative pathway-mediated binding of '25I-labeled complement component C3 to cryptococcal blastoconidia at a mean concentration of 60 ,ug/ml. The ability of crude CI to 3508

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Interleukin 8 (IL-8), a member of the C-X-C branch of the chemokine superfamily, stimulated the breakdown of 1-O-[3H]alkyl-2-acyl-sn- glycero-3-phosphocholine ([3H]EAPC) and the formation of 1-O-[3H]alkyl- 2-acyl-phosphatidic acid ([3H]-EAPA) in human polymorphonuclear leukocytes (PMN) in the presence of cytochalasin B. In addition, the mass of diradyl-PA was increased with similar kinetics. In the presence of ethanol, 1-O-[3H]alkyl-2-acyl-phosphatidylethanol ([3H]EAPEt) was formed at the expense of [3H]EAPA formation, indicating the activation of phospholipase D by the cytokine. The effect was time- and concentration-dependent, reaching a plateau at 30 seconds with the maximally activating concentration of 120 nmol/L IL-8. Preincubation of cells with 1 microgram/mL Bordetella pertussis toxin inhibited the breakdown of [3H]EAPC and [3H]EAPA formation, indicating a role for a pertussis toxin-sensitive guanosine triphosphate-binding protein. Formation of phosphatidic acid (PA) correlated with activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the oxidative burst enzyme, with both events occurring in the same concentration range. Inhibition of PA formation, by the presence of ethanol, also inhibited the oxidative burst stimulation by IL-8. Pretreatment of PMN with 10 nmol/L platelet-activating factor potentiated both [3H]EAPA accumulation and activation of NADPD oxidase by IL-8. Collectively, these data show that IL-8 stimulates the metabolism of choline-containing phosphoglycerides in human PMN and support a role for PA in the signaling mechanisms used by IL-8 to stimulate PMN function.

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