Formyl methionyl leucyl phenylalanine (fmlp)

Eugenol, Hank’s Balanced Salt Solution (HBSS), Phosphate Buffered Saline (PBS), Phorbolmyrisate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLF), mouse monoclonal anti-β-actin antibody, phosphatase and proteases inhibitors were from Sigma Aldrich (Saint Quentin Falavier, France). Dextran T500 and Ficoll were purchased from GE healthcare (Orsay, France). SDS-PAGE and western blot reagents were from Bio-Rad Laboratories (Hercules, CA, USA). Anti-phospho-Raf, Anti-phospho-MEK1/2, Anti-phospho-p38 and Anti-phospho-ERK 1/2 antibodies were from cell signaling Technology (Boston, MA, USA). The anti-phospho-p47phox antibodies (Ser-328 and Ser-345) were generated by our lab as previously described^{35 (link)}. HRP-conjugated goat anti-rabbit, HRP-conjugated goat anti-mouse, AP-conjugated goat anti-rabbit antibodies and ECL (enhanced chemiluminescence) reagent were from Santa Cruz Biotechnology Inc (Heidelberg, Germany).

Due to the cornea harboring very low numbers of stromal cells and neutrophils, these cells were isolated from bone marrow for our in vitro experiments. Neutrophils were isolated from bone marrow of C57BL/6 mice using a neutrophil isolation kit (purity ≥ 95%) (MACS; Miltenyi Biotec, Inc., San Diego, CA, USA).^{17 (link),18 (link)} Purified neutrophils were cultured alone or stimulated with fMLP (formyl-methionyl-leucyl-phenylalanine, 1 μM; Sigma-Aldrich Corp.) for 1 hour.^{19 ,20 (link)} Bone marrow–derived mesenchymal stromal cells (stromal cells) were generated by culturing bone marrow cells using the plastic adherence method and characterized as described previously.^{11 (link),12 (link)} Stromal cells were passaged every 3 to 5 days and were used for experiments at passage three. Stromal cells were stimulated with IL-1β (100 ng/mL; Biolegend, San Diego, CA, USA) for 24 hours.^{12 (link)} For coculture assays, neutrophils were cultured alone or on stromal cell monolayer at the ratio of 1:1 for 1 hour. For TSG-6 neutralization experiments, cocultures were pretreated with a standard maximal concentration (10 μg/mL) of anti-TSG-6 antibody (AF2326; R&D Systems, Minneapolis, MN, USA) for 1 hour and were then stimulated with fMLP for an additional 1 hour. Two mice were used in each experiment, and each experiment was repeated three times.

The BAT was performed, as previously described.¹⁴ (link) Heparinized whole blood was stimulated for 30 minutes at 37°C with PE (ALK-Abelló) diluted in RPMI medium at serial 10-fold dilutions from 10 μg/mL to 0.1 ng/mL. Polyclonal goat anti-human IgE (Sigma-Aldrich, St Louis, Mo), monoclonal mouse anti-human FcεRI (eBioscience, San Diego, Calif), formyl-methionyl-leucyl-phenylalanine (Sigma-Aldrich), or RPMI alone were used as controls. Cells were stained with CD123–fluorescein isothiocyanate (eBioscience), CD203c-phycoerythrin, HLA-DR–peridinin-chlorophyll-protein complex, and CD63-allophycocyanin (BioLegend, San Diego, Calif), and erythrocytes were lysed. Basophils were gated as low side scatter/CD203c⁺/CD123⁺/HLA-DR⁻. Basophil expression of CD63 and CD203c was evaluated with the FACSCanto II with FACSDiva software (BD Biosciences, San Jose, Calif). Data were analyzed with FlowJo software, version 7.6.1 (TreeStar, Ashland, Ore).

WT and RP105^−/− bone marrow-derived monocytes (10⁵ cells per well) were applied in quadruplicate to the upper chamber of a transwell system (24 wells, 8 µm pore size, PAA) in RPMI supplemented with 0.05% BSA (Sigma-Aldrich) and PBS or LPS in PBS (10 ng/ml) was added. Monocyte chemotaxis was assessed towards 100 nM FMLP (formyl-methionyl-leucyl-phenylalanine, Sigma-Aldrich) in the basolateral chamber. After 2 hours of incubation the number of monocytes migrated to the basolateral chamber was counted manually.

Chemotaxis was measured using a transwell chamber with 6.5 mm diameter polycarbonate filters (3 µm pore size; Corning, Tewksbury, MA). Immediately after isolation, cells were incubated in RPMI 1640 supplemented with FCS in the absence or presence of EtOH (6.25 and 12.5 mM correspond to 3 and 6% of EtOH in human blood, respectively) for 2 h. Then, cells were transferred to RPMI 1640 supplemented with FCS, cultivated on filters and allowed to migrate toward the chemo attractant fMLF (formyl-methionyl-leucyl-phenylalanine, 10⁻⁶ M; Sigma) or medium alone at 37°C, 5% CO₂. After 1 h, the filters were removed, and the cells that migrated through the membrane were fixed, stained and counted using light microscopy.