Activation of porcine NADPH-oxidase in homologous neutrophils

FINK, N. E.; ELOLA, M. T.; CHIESA, M. E.

BLOOD, THE JOURNAL OF THE AMERICAN SOCIETY OF HEMATOLOGY - PRINT

Springer

Lugar: WASHINGTON DC; Año: 2001 vol. 98 p. 26 - 27

0006-4971

Galectins are lectins which exhibit beta.galactoside-binding activity in a Ca++-independent fashion and share a consensus sequence in the carbohydrate binding domain. Relative to biological functions of galectins in immune response regulation, galectin-3 is known to activate mast cells, neutrophils and eosinophils in an IgE-mediated fashion, in allergic inflammation and host defense; galectin-3 also potentiates interleukin-1 production by human blood monocytes. In regards to superoxide (O2-) production, neutrophils, macrophages and eosinophils destroy invading microorganisms through the generation of oxidizing radicals derived from partial reduction of oxygen to superoxide anion mediated by the enzyme NADPH-oxidase. Human galectin-3 induces the formation of this oxygen radical by neutrophils and monocytes in a dose-dependent manner. The objective of this work was to evaluate the superoxide production by human neutrophils Ander the stimulation of the human splenic galectin-1 (HSG-1), in comparison to the effects obtained with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), a well-known neutrophil activator. Peripheral human blood PMN were isolated by Histopaque-1077 density gradient centrifugation, followed by erythrocyte sedimentation in 6% dextran and erythrocyte lysis through hypotonic shock. Superoxide forming activity was monitored using the cytochrome c assay in which O2- reduces ferric cytochrome c to its ferrous form, a change which can be sensitively measured in a double-beam spectrophotomer at 550 nm and 37ªC. For each, stimulant, 2x106 cells/cuvette were incubated with cytochrome c and cytochalasin B at final concentrations of 75 µM and 5 µg/ml, respectively, with and without 60 µg/ml of the enzyme superoxide dismutase. The absorbance change was recorded at 4-sec intervals for 4 min to determine V max which was expressed as nmoles O2-/min/107 PMN. HSG-1 at a concentration of 1 µM induced the production of O2- with a rate of 4.51 ± 3.82. Higher concentrations of HSG-1 produced the following significantly higher rates in a dose-response manner: 8.47 ± 3.31 and 12.52 ± 4.69 O2-/min/107 PMN for lectin concentrations of 2 and 4 µM, respectively (n=4). fMLP at a concentration of 1 µM, which was included for comparison, produced a maximal rate about 6-fold higher (26.76 ± 12.17 O2-/min/107 PMN; n=11) than HSG-1 at the same concentration. The effect of the galñectin could be significantly inhibited by 25 mM lactose, which is a specific saccharide ligand for these molecules, therefore the results obtained were dependent on the lectin function. We conclude that HSG-1 is a mild neutrophil activator for superoxide production.