Salival glycans induce NETosis mediated by sialyl LewisX – l-selectin-mediated signaling. Neutrophils were stimulated to NETosis and examined by immunofluorescence microscopy. NET formation was quantified as described in “Materials and methods.” Quantification data are shown from 3 independent experiments, except in the experiments with l-selectin blocking antibodies in which the results represent 4 independent experiments. Representative images are shown in supplementary Figure 2. (A) Neutrophils were stimulated both with saliva and salival mucins in saliva buffer with the ionic composition of saliva or in saliva buffer with adjustment of the NaCl concentration to 140 mM. (B) Saliva and mucins were treated with PGNase F to remove N-linked glycancs. Neutrophils were subsequently stimulated with the PGNase F-treated saliva and mucin after removal of the cleaved-off glycans and with these glycans. Lectin blot for N-linked glycans (supplementary Figure 2) demonstrated that not all N-linked glycans were removed by PGNase F treatment. (C) Before stimulation of neutrophils, saliva was treated with sialidase (neuraminidase). (D) Neutrophils were stimulated with isolated salivary glycans (SG) (removed from by PGNase F treatment) in the presence of sialyl LewisX blocking IgM (KM93) or negative control IgM. The glycans were resuspended in RPMI 1640 before incubation with antibodies. (E) Neutrophils were stimulated with either sialyl LewisX tetrasaccharide (SleX) or glucose tetrasaccharide (GTS). Scale bars, 100 μm. (F) Neutrophils were incubated with blocking antibodies to l-selectin antibodies or control antibodies prior to stimulation with saliva. (G) Western blot of l-selectin in the medium of neutrophils adhering to coverslips or incubated on ice. Columns denote average values and error bars standard deviations. *P < .05; **P < .01; ***P < .01, refer to nominal significance of induction or inhibition of NET formation.
