Secretion of S100A8, S100A9, and S100A12 by Neutrophils Involves Reactive Oxygen Species and Potassium Efflux

Abstract

S100A8/A9 (calprotectin) and S100A12 proinflammatory mediators are found at inflammatory sites and in the serum of patients with inflammatory or autoimmune diseases. These cytoplasmic proteins are secreted by neutrophils at sites of inflammation via alternative secretion pathways of which little is known. This study examined the nature of the stimuli leading to S100A8/A9 and S100A12 secretion as well as the mechanism involved in this alternative secretion pathway. Chemotactic agents, cytokines, and particulate molecules were used to stimulate human neutrophils. MSU crystals, PMA, and H2O2 induced the release of S100A8, S100A9, and S100A12 homodimers, as well as S100A8/A9 heterodimer. High concentrations of S100A8/A9 and S100A12 were secreted in response to nanoparticles like MSU, silica, TiO2, fullerene, and single-wall carbon nanotubes as well as in response to microbe-derived molecules, such as zymosan or HKCA. However, neutrophils exposed to the chemotactic factors fMLP failed to secrete S100A8/A9 or S100A12. Secretion of S100A8/A9 was dependent on the production of reactive oxygen species and required K(+) exchanges through the ATP-sensitive K(+) channel. Altogether, these findings suggest that S100A12 and S100A8/A9 are secreted independently either via distinct mechanisms of secretion or following the activation of different signal transduction pathways.

Figure 1

Secretion of S100A8, S100A9, S100A12, and S100A8/A9 by human neutrophils. Neutrophils were stimulated with (a) 10⁻⁷M fMLP, (b) 10 nM PMA, (c) 250 μM H₂O₂, or (d) 1.5 mg/mL MSU crystals for 1 h as described in Section 2. Cells were then centrifuged and supernatants were harvested to perform ELISA for S100A8, S100A9, S100A12, or S100A8/A9. Results represent the means ± SEM of 4 donors. ^∗ P < 0.05; ^∗∗ P < 0.01; ^∗∗∗ P < 0.001.

Figure 2

Secretion of calgranulins by neutrophils stimulated with GM-CSF and TNF. Neutrophils were stimulated with 20 nM GM-CSF or 50 ng/mL TNF for 60 minutes. (a) S100A8/A9 and (b) S100A12 were then quantified by ELISA. Results represent the means ± SEM of 5 donors. ^∗ P < 0.05; ^∗∗ P < 0.01; ^∗∗∗ P < 0.001.

Figure 3

Effects of microbe-derived products and whole yeasts on the release of S100A8/A9 and S100A12. (a) C. albicans (10⁸ cells/mL) enhances the secretion of S100A8/A9 and S100A12, whereas zymosan (1.5 mg/mL) induces only the release of S100A8/A9. (b) Pam3CSK4 (TLR2 ligand, 10 μg/mL) potentiates the release of S100A8/A9 but not S100A12. Neutrophils were incubated with whole microorganisms or microbe-derived product for 60 min at 37°C. The concentrations of secreted S100A8/A9 and S100A12 were determined by ELISA. Results are the mean ± SEM of at least three independent experiments performed on neutrophils from different donors. ^∗ P < 0.05 and ^∗∗ P < 0.01 compared with control by paired t-test.

Figure 4

Phagocytic particles induce the secretion of S100A8/A9 and S100A12. Neutrophils were stimulated with 1.5 mg/mL MSU crystals or fullerenes for increasing periods of time. (a) S100A8/A9 and (b) S100A12 in the supernatants were then quantified by ELISA. (c and d) Neutrophils were stimulated with 1.5 mg/mL MSU crystals, 1.5 mg/mL TiO₂, 3 mg/mL silica, 1.5 mg/mL carbon nanotubes, or 1.5 mg/mL fullerenes for 60 minutes. (c) S100A8/A9 and (d) S100A12 in the supernatants were then quantified by ELISA. Results represent the means ± SEM of 5 donors. ^∗∗ P < 0.01; ^∗∗∗ P < 0.001.

Figure 5

Inhibition of calgranulin secretion by DPI. Neutrophils were preincubated with the NADPH oxidase inhibitor DPI (10 μM) and then stimulated with 1.5 mg/mL MSU crystals or 10 nM PMA for 60 minutes. (a and b) S100A8/A9 and (c and d) S100A12 in the supernatants were then quantified by ELISA. Results represent the means ± SEM of 5 donors. ^∗∗∗ P < 0.001.

Figure 6

Inhibition of calgranulin secretion by glibenclamide. Neutrophils were preincubated with the ATP-sensitive K⁺ channel inhibitor glibenclamide (50 μM) and then stimulated with 1.5 mg/mL MSU crystals or 10 nM PMA for 60 minutes. (a) S100A8/A9 and (b) S100A12 in the supernatants were then quantified by ELISA. Results represent the means ± SEM of 5 donors. ^∗ P < 0.05; ^∗∗∗ P < 0.001.

Figure 7

S100A8, S100A9, S100A12, and calprotectin move toward cytoskeleton/lipid raft fractions following neutrophils stimulation by H₂O₂ or MSU. Neutrophils were stimulated with 1.5 mg/mL MSU crystals or 250 μM H₂O₂ and supernatants harvested before sequential cells lysis as described in Section 2. Fractions were loaded onto SDS-PAGE and (a) β actin, α tubulin, and flotillin-1 or (b) S100A8/A9 or S100A12 was detected by immunoblotting. Results are from one experiment are representative of 2 others. (c) Fractions were subjected to sandwiches ELISA against S100A8, S100A9, S100A12, or S100A8/A9. Results represent the mean ± SEM of 4 donors quantified in duplicate. ^∗ P < 0.05; ^∗∗ P < 0.01; ^∗∗∗ P < 0.001, MSU versus HBSS.

Figure 8

S100A8/A9 and S100A12 are almost absent from microvesicles. Neutrophils were incubated with MSU crystals (1.5 mg/mL) or its diluent for 60 min at 37°C. The MV were isolated after sequential centrifugation. The concentrations of (a) S100A8/A9 and (b) S100A12 were determined by ELISA in the supernatant fractions (S1, S2, S3, and S4) and the pellet fractions (P1, P2, and P3) containing the MV. Data are the mean ± SEM of three separate experiments. (c) The presence of S100A8 and S100A9 in the supernatants and MV was determined by western blot. Results are from one experiment representative of 2 others.