Aspergillus fumigatus stimulates leukocyte adhesion molecules and cytokine production by endothelial cells in vitro and during invasive pulmonary disease

Abstract

Invasive aspergillosis is characterized by hyphal invasion of the blood vessels, which contributes to the pathogenesis of this disease. During this angioinvasion, Aspergillus fumigatus interacts with the endothelial cell lining of the blood vessels. We investigated the response of vascular endothelial cells to A. fumigatus infection in vitro and in mouse models of invasive pulmonary aspergillosis. Infection with hyphae, but not with conidia, stimulated endothelial cells to synthesize E-selectin, vascular cell adhesion molecule 1 (VCAM-1), interleukin 8, and tumor necrosis factor alpha (TNF-alpha) in vitro. Killed hyphae induced approximately 40% less stimulation than did live hyphae. Endothelial cell stimulation required contact between the hyphae and endothelial cells but not endocytosis of the organisms. Studies with DeltagliP and DeltastuA null mutants of A. fumigatus indicated that the extent of endothelial cell stimulation was not influenced by gliotoxin or other StuA-dependent factors synthesized by A. fumigatus. In neutropenic mice infected with wild-type A. fumigatus, increased pulmonary expression of E-selectin, cytokine-induced neutrophil chemoattractant (KC), and TNF-alpha occurred only when neutropenia had resolved. In nonneutropenic mice immunosuppressed with corticosteroids, A. fumigatus stimulated earlier pulmonary expression of E-selectin, VCAM-1, and KC, while expression of intercellular adhesion molecule 1 and TNF-alpha was suppressed. In both mouse models, expression of E-selectin and KC was associated with high pulmonary fungal burden, angioinvasion, and neutrophil adherence to endothelial cells. Therefore, the expression of leukocyte adhesion molecules and secretion of proinflammatory cytokines by endothelial cells in response to A. fumigatus could enhance the host defense against this organism by contributing to the recruitment of activated leukocytes to sites of angioinvasion.

FIG. 1.

A. fumigatus hyphae stimulate endothelial cells. Endothelial cells were exposed to the indicated conditions for 8 h, after which the surface expression of E-selectin (A) and VCAM-1 (C) was measured and the accumulation of IL-8 (B) and TNF-α (D) in the culture medium was determined. Results are the means ± standard deviations from three experiments, each performed in duplicate or triplicate. *, P < 0.0001 versus endothelial cells exposed to M199 or conidia; †, P < 0.009 versus live hyphae and P < 0.0001 versus M199; ‡, P < 0.0005 versus M199. OD, optical density.

FIG. 2.

A. fumigatus stimulation of cell E-selectin expression requires direct endothelial cell contact but not endocytosis of the organism and is not influenced by gliotoxin or other stuA-dependent secondary metabolites. Endothelial cell expression of E-selectin was measured when the organisms were suspended above the endothelial cells (ECs) in filter inserts with 0.2-μm pores (A), after infection with wild-type A. fumigatus hyphae in the presence (+CD) or absence of cytochalasin D (B), and after infection with the indicated A. fumigatus mutants (C). Results are the means ± standard deviations from three experiments, each performed in triplicate. *, P < 0.035 compared to endothelial cells exposed to hyphae without cytochalasin D. OD, optical density; WT, wild type.

FIG. 3.

Time course of pulmonary fungal burden in mice with invasive pulmonary aspergillosis. Mice were immunosuppressed either with cortisone acetate and cyclophosphamide (A) or with high-dose cortisone acetate alone (B). They were infected with A. fumigatus conidia in an aerosol chamber. Control mice were immunosuppressed but not infected. At the indicated time points, the mice were sacrificed, after which the galactomannan content of their lungs was determined. Results are the medians ± interquartile ranges for six to nine mice per group. *, P < 0.009 compared to uninfected mice at the same time point.

FIG. 4.

Histopathology of the lungs of neutropenic mice with invasive aspergillosis. (A) Day 4 results showing a small focus of hyphae surrounded by a mononuclear infiltrate. (B) Day 6 results showing a larger focus of infection with more hyphae surrounded by leukocytes with apoptotic nuclei. (C and D) Day 8 results showing extensive hyphae surrounded by numerous neutrophils (C) and angioinvasion, disruption of endothelial cells, and adjacent neutrophil margination (D). In panels A to C, GMS staining is shown on the left and PAS staining on the right, while the tops are shown at a magnification of ×200 and the bottoms at ×400. (D) PAS stain; top-panel magnification, ×100; bottom-panel magnification, ×400. Single white arrow, fungi; black arrows, leukocytes; arrowheads, blood vessel walls; double white arrows, hyphae within the blood vessel lumen; double black arrows, neutrophils adherent to endothelial cells.

FIG. 5.

Time course of pulmonary leukocyte adhesion molecule expression during invasive aspergillosis of neutropenic and nonneutropenic immunosuppressed mice. Concentrations of sE-selectin (A and B), sVCAM-1 (C and D), and sICAM-1 (E and F) in lung homogenates at the indicated time points. Results are the medians ± interquartile ranges of at least six mice per time point. *, P < 0.03 compared to uninfected mice at the same time point.

FIG. 6.

Time course for pulmonary cytokine expression during invasive aspergillosis in neutropenic and nonneutropenic immunosuppressed mice. Concentrations of KC (A and B) and TNF-α (C and D) in lung homogenates at the indicated time points. Results are the medians ± interquartile ranges of at least six mice per time point. *, P < 0.01 compared to uninfected mice at the same time point.

FIG. 7.

Histopathology of the lungs of nonneutropenic mice with invasive aspergillosis. (A) Day 2 results showing a focus of hyphae in the bronchial epithelium that is invading into the adjacent lung tissue and is surrounded by a neutrophilic infiltrate. (B) Day 4 results showing a larger focus of infection with more hyphae surrounded by neutrophils. The hyphae have invaded into a blood vessel. Another lesion shows neutrophil margination. (C) Day 6 results showing extensive hyphae surrounded by numerous neutrophils, with invasion of three blood vessels and adjacent neutrophil margination. GMS staining is shown in the upper left panels and PAS staining in the other panels; top-panel magnification, ×100 (note the difference in magnification from that in Fig. 4.); bottom-panel magnification, ×400. Single white arrow, fungi; black arrows, leukocytes; arrowheads, blood vessel walls; double white arrows, hyphae within the blood vessel lumen; double black arrows, neutrophils adherent to endothelial cells; single blue arrow, bronchial epithelium.