Human galectin-3 promotes Trypanosoma cruzi adhesion to human coronary artery smooth muscle cells

Abstract

Human galectin-3 binds to the surface of Trypanosoma cruzi trypomastigotes and human coronary artery smooth muscle (CASM) cells. CASM cells express galectin-3 on their surface and secrete it. Exogenous galectin-3 increased the binding of T. cruzi to CASM cells. Trypanosome binding to CASM cells was enhanced when either T. cruzi or CASM cells were preincubated with galectin-3. Cells stably transfected with galectin-3 antisense show a dramatic decrease in galectin-3 expression and very little T. cruzi adhesion to cells. The addition of galectin-3 to these cells restores their initial capacity to bind to trypanosomes. Thus, host galectin-3 expression is required for T. cruzi adhesion to human cells and exogenous galectin-3 enhances this process, leading to parasite entry.

FIG. 1.

Human galectin-3 binds to the surface of human CASM cells in a lectin-like manner. Human CASM cell monolayers at 30% density were washed with Hanks balanced salt solution (HBSS), fixed with 1% paraformaldehyde, and incubated with FITC-labeled galectin-3 (2 μg/ml) (A), 100× unlabeled galectin-3 plus FITC-labeled galectin-3 (B), 5 mM lactose plus FITC-labeled galectin-3 (C), or 5 mM sucrose plus FITC-labeled galectin-3 (D) for 1 h at 37°C. Cells were washed and examined under fluorescence microscopy. The results shown are from a representative experiment of three experiments performed with the same results.

FIG. 2.

Human galectin-3 binds to the surface of T. cruzi trypomastigotes in a lectin-like manner. Culture trypomastigotes (2 × 10⁶) were washed with HBSS, fixed with 1% paraformaldehyde, and incubated with FITC-labeled galectin-3 (2 μg/ml) (A), 100× unlabeled galectin-3 plus FITC-labeled galectin-3 (B), 5 mM lactose plus FITC-labeled galectin-3 (C), or 5 mM sucrose plus FITC-labeled galectin-3 (D) for 1 h at 37°C. Parasites were washed and examined under fluorescence microscopy. The results shown are from a representative experiment of three experiments performed with similar results.

FIG. 3.

Galectin-3 is expressed on the surface of human CASM cells and is secreted, and exogenous galectin-3 enhances T. cruzi trypomastigote binding to human CASM cells. (A) Galectin-3 is expressed on the surface of human CASM cells and is secreted. Left panel, biotinylated surface proteins of human CASM cells were immunoprecipitated with anti-galectin-3 antibodies (abs) (lane 1) or with preimmune antibodies (lane 2), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and developed by enhanced chemiluminescence (ECL). Right panel, immunoblots of culture supernatants of human CASM cells incubated in DMEM for 6 h (lane 1) or medium alone (lane 2), probed with anti-galectin-3 antibodies, and developed by ECL. (B) Galectin-3 enhances T. cruzi trypomastigote binding to human CASM cells. Triplicate monolayers of human CASM cells were exposed or not exposed to several concentrations of human galectin-3 free of endotoxin and to T. cruzi trypomastigotes at a ratio of 20 parasites per cell for 2 h at 37°C. Unbound parasites were washed out, and trypanosome binding was evaluated by using an immunofluorescence assay as described above. (C) Lactose, but not sucrose, inhibits T. cruzi trypomastigote binding to human CASM cells. Trypomastigote binding assays were performed as described for panel B in the presence of HBSS, with several concentrations of lactose or sucrose, at the ratio of 20 parasites per cell. Unbound trypanosomes were washed out, and binding was evaluated by immunofluorescence as for panel B. The results presented in panels A to C are from one respective representative experiment of three independent experiments performed with similar results. Each point in panels B and C is the mean of results for triplicate samples in one representative experiment (± 1 standard deviation). For panel B, the P value was <0.05 for differences between 0 (control) and ≥1 μg of galectin-3/ml. For panel C, the P value was <0.05 with Student's t test for differences between lactose and sucrose at all points except 0 μg of sugar/ml.

FIG. 4.

Trypanosome binding to CASM cells was enhanced when T. cruzi trypomastigotes or CASM cells were preincubated with galectin-3. Monolayers of human CASM cells or trypomastigotes were washed with HBSS and preincubated separately in PBS with 4 μg of galectin-3/ml. Parasites and cells were then incubated together at a ratio of 10 parasites per cell for 2 h at 37°C. Unbound trypanosomes were washed out, and trypanosome binding was evaluated by using an immunofluorescence assay as for Fig. 3B and C. Other controls included human CASM cells incubated with trypanosomes exposed or not exposed to galectin-3. Bars represent the means of results from triplicate samples in one representative experiment (± 1 standard deviation) selected from three experiments with similar results. *, significant difference compared to control values (P < 0.05).

FIG. 5.

Reduced expression of galectin-3 inhibits the attachment of T. cruzi to human cells, but the addition of exogenous galectin-3 restores the initial capacity of these cells to bind to trypanosomes. (A) Blocking galectin-3 expression in human cells by transfection with galectin-3 antisense caused a reduction in T. cruzi adhesion to cells, and adding exogenous galectin-3 restores the initial capacity of these cells to bind to trypanosomes. Monolayers of human 435 cells transfected with vector alone or galectin-3 antisense were washed with HBSS and incubated separately with PBS or with 4 μg of galectin-3/ml in PBS and trypomastigotes at the ratio of 10 parasites per cell for 2 h at 37°C. Unbound trypanosomes were washed off, and trypanosome binding was evaluated by using an immunofluorescence assay as described for Fig. 3B. Bars represent the means of results for triplicate samples in one representative experiment (± 1 standard deviation) selected from three experiments with similar results. The difference between results labeled * and ** and the difference between results labeled 0 and 00 are significant (P < 0.05). (B) Human 435 cells transfected with galectin-3 antisense show significantly reduced expression of galectin-3 as evaluated by immunoblotting. Three μg of 435 cells transfected with either vector alone (lane 1) or galectin-3 antisense (lane 2) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, probed with anti-galectin-3 polyclonal antibodies, and developed by ECL. Loading controls were evaluated by stripping the same blots, probing them with anti-β-actin antibodies, and developing them by ECL. The results shown are from a representative experiment of three experiments performed with the same results. 435/vector alone, 435 cells transfected with pCNC10 vector alone; 435/galectin-3 antisense, 435 cells transfected with pCNC10-galectin-3 antisense.