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. 2005 Feb;73(2):770-7.
doi: 10.1128/IAI.73.2.770-777.2005.

Enhanced killing of Candida albicans by human macrophages adherent to type 1 collagen matrices via induction of phagolysosomal fusion

Simon L Newman  1 , Bindu Bhugra, Angela Holly, Randal E Morris
Affiliations

Enhanced killing of Candida albicans by human macrophages adherent to type 1 collagen matrices via induction of phagolysosomal fusion

Simon L Newman et al. Infect Immun. 2005 Feb.

Abstract

Candida albicans, a component of the normal flora of the alimentary tract and mucocutaneous membranes, is the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients and of oropharyngeal disease in AIDS patients. As little is known about the regulation of monocyte/macrophage anti-Candida activity, we sought to determine if fungicidal activity might be regulated by extracellular matrix proteins to which monocytes/macrophages are adherent in vivo. Compared to monocyte/macrophages that adhered to plastic, human monocytes and monocyte-derived macrophages that adhered to type 1 collagen matrices, but not to fibronectin, vitronectin, or laminin, demonstrated a significant increase in candidacidal activity. The enhancement of monocyte fungicidal activity was maintained over a 4-h period, whereas macrophage fungicidal activity was maximum at 1 h. Although adherence of monocytes and macrophages to collagen matrices concomitantly enhanced the production of superoxide anion, only the fungicidal activity of collagen-adherent monocytes was partially blocked by superoxide dismutase and catalase. Remarkably, we found that only 10% of the phagosomes in C. albicans-infected macrophages that adhered to plastic fused with lysosomes. In contrast, 80% of yeast-containing phagosomes of collagen-adherent macrophages fused with lysosomes. These data suggest that nonoxidative mechanisms are critical for human macrophage anti-Candida activity and that C. albicans pathogenicity is mediated, in part, by its ability to inhibit phagolysosomal fusion in macrophages.

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Figures

FIG. 1.
FIG. 1.
Collagen-adherent monocytes and Mφ have enhanced fungicidal activity against C. albicans. Freshly isolated monocytes and suspension-cultured Mφ were allowed to adhere to plastic (PL), type 1 collagen matrices (COL), fibronectin (FN), vitronectin (VIT), or laminin (LAM) for 1 h at 37°C. Alternatively, monocytes were cultured for 7 days on the various ECM components (adherent Mφ). After the monolayers were washed, the cells were incubated with 2 × 104 C. albicans yeasts in the presence of 10% pooled human serum for 1 h at 37°C. At the end of the incubation period, the remaining CFU were quantified as described in Materials and Methods. The data are the means ± SEM of the remaining CFU from five experiments with monocytes and suspension-cultured Mφ and seven experiments with adherently cultured Mφ. *, P < 0.05 compared to plastic (t test); **, P < 0.05 compared to plastic (Mann-Whitney rank sum test).
FIG. 2.
FIG. 2.
Time course of monocyte/Mφ fungicidal activity on plastic versus collagen matrices. Freshly isolated monocytes and suspension-cultured Mφ were allowed to adhere to plastic or type 1 collagen matrices for 1 h at 37°C, or monocytes were cultured for 7 days on plastic or collagen. After the monolayers were washed, the cells were incubated with 2 × 104 C. albicans yeasts in the presence of 10% pooled human serum for 1, 2, and 4 h at 37°C. Remaining CFU then were quantified as described in Materials and Methods. The data are the means ± SEM of the remaining CFU from eight experiments with monocytes and adherently cultured Mφ and five experiments with suspension-cultured Mφ. * and **, P < 0.01 and < 0.05, respectively, compared to plastic (t test).
FIG. 3.
FIG. 3.
Adherence of monocytes/Mφ to collagen matrices enhances the production of superoxide anion. Monocytes (A) or Mφ (B) adherent to plastic, collagen, or collagen gels (Col Gel) were incubated in buffer alone (Co) or with opsonized C. albicans (Ca) (107 yeasts) for 1 h at 37°C in the absence or presence of SOD (40 μg/ml), and the production of superoxide anion was quantified by measuring the reduction of cytochrome c. The data are the means ± SEM of three experiments with monocytes (A) and six experiments with Mφ (B). *, P < 0.05, compared to plastic (t test).
FIG. 4.
FIG. 4.
Reversal of monocyte fungicidal activity by SOD and catalase. Monocytes were allowed to adhere to plastic or type 1 collagen matrices for 1 h at 37°C. After being washed, the monocytes were incubated with 2 × 104 C. albicans yeasts for 1 h at 37°C in the presence or absence of SOD or catalase. Remaining CFU then were quantified as described in the legend to Fig. 1. The data are the means ± SEM of five experiments. *, P < 0.05 compared to plastic control (Mann-Whitney rank sum test).
FIG. 5.
FIG. 5.
Adherence of Mφ to collagen matrices enhances PL fusion. Glass- or collagen-adherent Mφ were loaded with Lysotracker red and then incubated for 1 h with viable (V) or HK C. albicans (Ca) or H. capsulatum (Hc) or viable S. cerevisiae (V Sc). The percentage of lysosomal fusion was quantified as described in Materials and Methods. The data are the means ± SEM of four to nine experiments with C. albicans and H. capsulatum and seven experiments with S. cerevisiae. (A) Data comparing levels of PL fusion for viable and HK C. albicans and H. capsulatum and viable S. cerevisiae when the Mφ were allowed to adhere to glass coverslips. *, P < 0.001 compared to viable fungi (t test). (B) Data comparing levels of PL fusion with viable fungi for Mφ adherent to glass coverslips versus collagen gels. *, P < 0.001 compared to glass-adherent Mφ (t test). (C) Plastic- or collagen-adherent Mφ were labeled with HRP-Au18 and then incubated with C. albicans for 1 h at 37°C. After fixation and processing for electron microscopy, the number of gold particles in yeast-containing phagosomes was quantified. The data are the means ± SEM of the numbers of gold particles per phagosome. The number above each bar on the graph denotes the number of phagosomes counted for each fungus. *, P < 0.001 compared to plastic (Mann-Whitney rank sum test).
FIG. 6.
FIG. 6.
Adherence of Mφ to collagen matrices enhances PL fusion. Plastic- or collagen-adherent Mφ were labeled with HRP-Au18 and then incubated with C. albicans for 1 h at 37°C and then fixed and processed for electron microscopy. (Top) Electron micrograph showing a C. albicans yeast within the phagosome of a Mφ adherent to plastic. Note that there are numerous gold particles in the surrounding cytoplasm but none in the phagosome. (Bottom) Collagen-adherent Mφ with numerous gold particles within the yeast-containing phagosome. Bars, 1 μm.
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