Galectin-4 induces T cell apoptosis caspase-independently via calpain-mediated pathways.

<p>(A) PBT and LPT were stimulated with anti-CD3/CD28 or -CD2, respectively, in the presence of 0, 50, 100 or 200 µg/ml Gal-4 and apoptosis was determined by annexin-V staining. Data represent mean±SEM of eight individual experiments. *p≤0.05 for increase vs. baseline. (B) PBT were stimulated in the presence of 0, 25, 50, 100 or 200 µg/ml Gal-4. Apoptosis was detected by annexin V/PI staining, necrosis by PI staining followed by flow cytometry. Data represent mean±SEM of six individual experiments. *p≤0.05 for increase vs. baseline. (C) PBT were transfected with Gal-4 siRNA or scrambled control, activated by anti-CD3/CD28 and cultured in the presence of the TNF-α blocker adalimumab for three days. Apoptosis was detected by annexin V/PI staining followed by flow cytometry. Data are representative for three individual experiments. (D) Caspase-3, -8, and -9 activity in anti-CD3/CD28 stimulated T cells cultured in the presence or absence of 100 µg/ml Gal-4. Data are representative for three individual experiments. (E) PBT were activated by anti-CD3/CD28 and cultured for 24 h in the presence or absence of 100 µg/ml Gal-4 and 50 mM Calpain inhibitor z-LLY-fmk, 4 mM EGTA or 30 mM BAPTA-AM. Data are representative for three individual experiments. (F) Disruption of the mitochondrial membrane potential was detected by rhodamine123 staining followed by flow cytometric analysis. T cells were stimulated with anti-CD3/CD28 and incubated for 3 h in the presence or absence of 100 µg/ml Gal-4. Afterwards cells were analysed by flow cytometry. All data are representative for three individual experiments. (G) Disruption of the mitochondrial membrane potential was detected after 12 h by rhodamine123 staining followed by flow cytometric analysis. All data are representative for three individual experiments.</p

Galectin-4 binds to stimulated, but not resting T cells.

<p>(A) Flow cytometric analysis of Gal-4 binding to resting and anti-CD3/CD28 stimulated T cells using Gal-4 detected by an anti-Gal-4 Ab and PE-labeled secondary Ab. Carbohydrate-dependence of the binding was analysed by addition of 50 mM lactose as a pan-galectin inhibitor and 50 mM sucrose as control. Data are representative of three individual experiments. (B) Immunoprecipitation of Gal-4 binding complexes. BSA served as negative, Gal-1 as positive control. Data are representative for four individual experiments.</p

Galectin-4 and its domains distinctively reduce secretion of pro-inflammatory cytokines.

<p>(A) Cytokine release was tested in PBT stimulated with anti-CD3/CD28 for 72 hours. Data represent mean±SEM of six individual experiments. *p≤0.01 for decrease vs. baseline. (B) IL-17 secretion of LPT and PBT was determined by an IL-17 specific ELISA. LPT and PBT were activated by anti-CD2 or -CD3/CD28 mAb, respectively and cultured in the presence or absence of 100 µg/ml Gal-4. Data represent mean±SEM of three individual experiments.</p

Identification of Galectin-4 expression.

<p>(A) Gal-4 expression was detected in cryosections of the GI tract of healthy volunteers. The results are representative for four individuals (original magnifications: ×100). (B) PCR analysis of Gal-4 mRNA expression in resting and anti-CD3/CD28 stimulated PBT. (C) Flow cytometric analysis of intra- and extracellular Gal-4 content in resting and anti-CD3/CD28 stimulated PBT. Data are representative of three individual experiments.</p

Galectin-4 ameliorates experimental colitis by inducing apoptosis and reduction of pro-inflammatory cytokine secretion.

<p>(A) Disease activity index in acute DSS-induced colitis, treated with 0.9% sterile saline (control) or 1 mg/kg BW Gal-4 i.p. three times daily. *p≤0.01 for change vs. control. (B) Detection of apoptotic cells by TUNEL staining in cryosections of colonic tissue from mice with experimental colitis treated with 0.9% sterile saline (control) or 1 mg/kg BW Gal-4 i.p. three times daily. (C) Apoptotic cells after TUNEL staining were counted in three power fields on four slices of different animals by an investigator blinded to the protocol. *p≤0.05 for change vs. control. (D) Detection of proliferating cells by BrdU staining in cryosections of colonic tissue from mice with experimental colitis treated with 0.9% sterile saline (control) or 1 mg/kg BW Gal-4 i.p. three times daily. (E) BrdU positive cells were counted in three power fields on four slices of different animals by an investigator blinded to the protocol. *p≤0.01 for change vs. control. (F) Cytokine secretion was determined by CBA analysis from colonic cultures from mice with experimental colitis treated with 0.9% sterile saline (control) or 1 mg/kg BW Gal-4 i.p. three times daily. All data represent mean±SEM of ten individual experiments. *p≤0.05 for change vs. control.</p