Figure 5
PSMα4 induces vascular leakage in vivo.
(A,B) The supernatants of the wild-type USA300 and α4compΔα strains induced vascular leakage to a significantly higher extent than the supernatant of the Δα strain. Supernatant (0.1 mL) was injected intradermally into mice. TSB (0.1 mL) was injected as a negative control. (C,D) Synthetic PSMα4 induced vascular leakage via FPR2. PSMα4 peptide induced vascular leakage in mice via FPR2. The FPR2 antagonist WRW4 and the control wwrw3 were intravenously injected into mice (7.5 mg/kg) 30 min before intradermal injection of 100 μL PSMα4 peptide (10 μg/mL). PBS was injected as a negative control. The mice were intravenously injected with 100 μL Evans blue dye solution (1–2.5% in PBS) 3 h after intradermal injection of stimuli. The mice were scarified 30 min after Evans blue injection. Evans blue in mouse tissue was extracted and quantified. (E,F) The supernatant of the wild-type USA300 and Δα strains did not induce vascular leakage in neutropenic mice. Cyclophosphamide (1250 mg/kg) was administered intraperitoneally to mice to induce neutropenia 72 h before intradermal injection of the supernatant. The PMN count in neutropenic mice was <200 × 106/L. Supernatant (0.1 mL) was injected intradermally into mice. TSB (0.1 mL) was injected as a negative control (G,H) Synthetic PSMα4 did not induce vascular leakage in neutropenic mice. Neutropenic mice were injected intradermally with 100 μL of PSMα4 peptide (10 μg/mL) and control peptide (KAFIDIIAKIIKIITGVIAM) (10 μg/mL). (I,J) The supernatant of mouse PMNs stimulated by PSMα4 induced vascular leakage in neutropenic mice. Mouse PMNs (purity >90%, 106/mL) were incubated with HBSS, PSMα4 (10 μg/mL) or control peptide (KAFIDIIAKIIKIITGVIAM) (10 μg/mL) at 37 °C for 30 min and then 0.1 mL of the respective supernatants was injected into neutropenic mice intradermally. (K) Synthetic PSMα4 peptide stimulated MPO release from mouse PMNs. Mouse PMNs (106/mL) were incubated with PSMα4 (10 μg/mL) at 37 °C for 30 min. MPO release was evaluated by ELISA.
