Galectin-9 enhances cytokine secretion, but suppresses survival and degranulation, in human mast cell line.

Galectin-9 (Gal-9), a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells

Galectin-9 induces phosphorylation of Erk1/2, but not p38 MAPK, in HMC-1 cells.

<p>HMC-1 cells were cultured in the presence of 1 µM recombinant human galectin-9 (rhGal-9) for the indicated times. Then the levels of phosphorylation of Erk1/2 and p38 MAPK in the cells were determined by western blot analysis. Data show a representative result of three independent experiments.</p

Galectin-9 inhibits PMA- and ionomycin-dependent degranulation of HMC-1 cells.

<p>(a, b) HMC-1 cells were treated with 0, 0.25, 0.5 or 1 µM (a) and 0 or 0.5 µM (b) recombinant human galectin-9 (rhGal-9) for 30 min. The cells were then stimulated with 0.1 µg/ml PMA +1 µg/ml ionomycin for 30 min. The level of degranulation was assessed from the activity of β-hexosaminidase in the culture supernatant and plotted as the percent release. (c) The number of viable cells in (b) was determined by trypan blue staining. (d) The proportion of propidium iodide-negative and annexin V-positive apoptotic cells in (b) was assessed by flow cytometry. (e) The relative level of degranulation per live HMC-1 cells was determined as (b)/(c). Data show the mean ± SD of triplicate samples and are a representative result of three (a) or two (b–e) independent experiments. *p<0.05, **p<0.01 versus PMA+ionomycin alone.</p

Gal-9 induces cytokine and chemokine production by HMC-1 cells.

<p>ELISA was performed to determine the levels of IL-6, IL-8 and MCP-1 in the culture supernatants of HMC-1 cells (<b>a</b>), HMC-1 cells pre-treated with 20 mM lactose or sucrose (<b>b</b>), HMC-1 cells pre-treated with recombinant human TIM-3/Fc (rhTIM-3/Fc) or control human IgG (human IgG) (<b>c</b>) and HMC-1 cells pre-treated with ERK inhibitor (PD98059) or its control (SB202474) (<b>d</b>) after 18 hours’ stimulation with 0, 0.25, 0.5 or 1 µM recombinant human Galectin-9 (rhGal-9). Data show the mean ± SD of triplicate samples and are a representative result of three independent experiments. *p<0.05 and/or **p<0.01 versus 0 µM rhGal-9 (<b>a–d</b>), and †p<0.05 and/or ††p<0.01 versus sucrose (<b>b</b>), control human IgG (<b>c</b>) or ERK inhibitor control (<b>d</b>).</p