Figure 3. PSMα4 stimulates HBP release from PMNs via FPR2 and PI3K signaling pathway.

(A) FPR2 antagonist WRW4 significantly reduced HBP release from whole blood. Whole blood was pre-incubated with WRW4 (50 μg/mL) or the scramble all d-amino acid control wwrw3 (50 μg/mL) at 37 °C for 15 min³⁰ and then treated with PSMα4 (10 μg/mL) at 37 °C for 30 min. HBP in the supernatant was analyzed by ELISA. (B) Formyl PSMα4 peptide induced HBP release dose-dependently. Human whole blood was incubated with 10, 20, or 30 μg/mL non-formyl or formyl PSMα4 at 37 °C for 30 min. PBS was used as the negative control. (C) The critical amino acid residues of PSMα4 to induce HBP release were screened by alanine substitution. The concentration of the peptides was 10 μg/mL. Replacement of I3, V4, G5, T6, I11, I15, I17, or F18 with alanine significantly abolished HBP release. (D) PI3K-specific inhibitor wortmannin completely abolished HBP release. (E) Rac-specific inhibitor NSC23766 completely abolished HBP release. Whole blood was pre-incubated with 1 μM wortmannin or 50 μM NSC23766 at 37 °C for 1 h and then treated with PSMα4 (10 μg/mL) for 30 min. HBP in the supernatant was analyzed by ELISA. (F) EGTA in culture media significantly reduced HBP release. Human whole blood was pre-treated with 20 mM EGTA at 37 °C for 15 min and then stimulated with PSMα4 (10 μg/mL). (G) PSMα4 induced Ca²⁺ influx into PMNs. Fluo-3/AM was loaded to PMNs before stimulation with PSMα4. fMLP (1 μM) was used as a positive control for Ca²⁺ influx.