Light microscopy images of METH treated J774.16 cells incubated in presence of complement or complement + IgM (mAb 12A1, a capsular specific mAb) interacting with AIDS-associated yeast-like fungus C. neoformans. Images show activated macrophage-like cells with phagocytized yeast cells. Black arrows indicate C. neoformans cells outside of macrophages. White arrow heads denote yeast cells inside of macrophages. Scale bar: 10 μm. (B) The phagocytic indices (ratio of number of intracellular yeast cells to the number of macrophages counted) were determined after 2 h. Bars represent the means of four wells (100 cells per well) and error bars denote standard deviations (SDs). Symbols (*, #, ϕ, @) denote P-value significance (P<0.0001) calculated using analysis of variance analysis (ANOVA) and adjusted by use of the Bonferroni correction. *, #, ϕ, and @ indicate significantly higher fungal phagocytosis than in macrophages from untreated, 25 μM cytochalasin D (CytD, an inhibitor of actin polymerization and phagocytosis), 25 and 50 μM METH groups, respectively. Crosses (X) indicate P-value significance (P<0.0001) calculated using student’s t-test analysis. (C) Expression of CR3 (CD11b/CD18) and RhoA were determined by a western blot analysis. GAPDH was used as a control. (D) The levels of expression of CD11b, CD18, and RhoA were measured by determining the relative intensity ratios. Individual band intensities from the western blot in C were quantified using Image J software (US NIH). GAPDH was used to determine the relative intensity ratios shown in C. For panels B and D, symbols (*, #, ϕ) denote P-value significance (P<0.0001) calculated using analysis of ANOVA and adjusted by use of the Bonferroni correction. *, #, and ϕ indicate significantly higher fungal phagocytosis than in macrophages from untreated, 25 μM cytochalasin D (CytD, an inhibitor of actin polymerization and phagocytosis), and 25 μM METH groups. The experiments were performed twice with similar results obtained.

Immunofluorescent images of C. neoformans after co-incubation with METH and either PBS, complement, mAb 12A1 (IgM), or combination of complement and IgM, the cells were washed and incubated with mAb 18B7-FITC-conjugated goat anti-mouse IgG₁ stained to label the capsular polysaccharide. Scale bar, 10 μm. (B) Number of cell aggregates per field. Only clusters of ≥ 3 cryptococci were considered aggregates. (C) Cells per aggregate. Each aggregate was closely examined and the number of cells clustered was counted and recorded. For panels B and C, the number of cell aggregates per field and cells per aggregate was assessed after co-incubation of fungi with METH alone or METH with complement, IgM, or complement + IgM. Bars represent the means of multiple measurements and error bars denote SDs. Symbols (*, #, ϕ, @) denote P-value significance (P<0.0001) calculated using ANOVA and adjusted by use of the Bonferroni correction. *, #, ϕ, and @ indicate significantly higher than control, complement, IgM, and complement + IgM groups, respectively. The experiments were performed twice with similar results obtained.

Macrophages were first allowed to phagocytize C. neoformans H99 cells for 2 h. Each well containing interacting cells was gently washed to get rid of fungal cells that were not phagocytize and incubated with growing medium supplemented with either PBS (untreated), Chlq (25 μM), or METH (25 or 50 μM) for 24 h. (A) For phagosomal pH determinations, the pH of phagosomes of J774.16 macrophage-like cells that contain C. neoformans yeast labeled with pH-sensitive and pH-insensitive probes was measured using a spectrofluorometer after treatment with PBS (untreated), Chlq (25 μM), or METH (25 or 50 μM). Bars represent the means of five measurements and error bars denote SDs. Symbols (*, %) denote P-value significance (P<0.0001) calculated using ANOVA and adjusted by use of the Bonferroni correction. * indicates significantly higher pH than in macrophages from untreated groups whereas % indicates significantly lower than in cells incubated with Chlq. Crosses (X) indicate P-value significance (P<0.01) calculated using student’s t-test analysis. The experiments were performed twice with similar results obtained. (B) For killing assay, phagocytic cells were lysed and fungal cells in the supernatant were plated and CFU were counted. Bars represent the means of four wells (three CFU counts per well) and error bars denote SDs. Bars represent the means of four wells (three CFU counts per well) and error bars denote standard deviations. Symbols (*, &, ≠) denote P-value significance (P<0.01) calculated using ANOVA and adjusted by use of the Bonferroni correction. * indicates significantly higher fungal killing than in macrophages from untreated groups. & and ≠ indicate significantly lower cryptococcal killing than in cells incubated with PBS and 25 μM METH, respectively. Crosses (X) indicate P-value significance (P<0.0001) calculated using student’s t-test analysis. For panels A and B, The experiments were performed twice with similar results obtained.

(A) NO production was quantified using the Griess method after J774.16 cells were treated with aminoguanidine (AG; iNOS inhibitor), PBS (untreated), Chlq (25 μM), or METH (25 or 50 μM). (B) Western blot analysis of inducible nitric oxide synthase (iNOS) of macrophages treated with AG, PBS, Chlq, or METH. (C) Quantitative measurements of individual band intensity in western blot gel in (B) for iNOS. GAPDH was used as a control to determine the relative intensity ratio. For panels A and C, bars represent the means of multiple measurements and error bars denote SDs. Symbols (&, %, ≠, @) denote P-value significance (P<0.0001) calculated using ANOVA and adjusted by use of the Bonferroni correction. &, %, ≠, and @ indicate significantly lower NO production and iNOS expression than in J774.16 cells incubated with PBS, Chlq, 25 and 50 μM METH groups, respectively. Crosses (X) indicate P-value significance (P<0.01) calculated using student’s t-test analysis. The experiments were performed twice with similar results obtained.