Fig. 4.

CD14 can act as a co-receptor for SES. (A) HPMC were immunostained with a PE-conjugated CD14 monoclonal antibody in the presence or absence of 10% serum and human PBMC were used as a positive control for CD14 expression (isotype control antibody shaded in gray and CD14 expression outlined in black). Data presented are a representative result of six experiments performed with primary cell lines derived from separate donors. (B) HPMC were stimulated in medium with SES (1/10 dilution) in the presence or absence of anti-CD14-blocking antibody (10 μg/mL) for 1 h 30 min. CXCL8 levels were quantified in cell-free culture supernatants by ELISA. Data presented are representative of three experiments performed in triplicate with HPMC derived from three independent donors. (C) PBMC were isolated from healthy human volunteers and were stimulated in medium containing 0.5% human AB serum with LPS (50 ng/mL) or SES (1/80 dilution) in the presence or absence of anti-CD14-blocking antibody (10 μg/mL) for 1 h. CXCL8 levels were quantified in cell-free culture supernatants by ELISA. Data presented are representative of three experiments performed in triplicate with PBMC derived from three independent donors (**P < 0.01). (D) HPMC were stimulated with Pam₃Cys (2 μg/mL) in the presence or absence of 0.1% FCS (as a source of sCD14) for 24 h. CXCL8 levels were quantified in cell-free culture supernatants by ELISA. Data presented are representative of three experiments performed in triplicate with primary cell lines derived from three independent donors (*P < 0.05). (E) HPMC were stimulated with SES (Neat) in the presence or absence of 0.1% FCS (as a source of sCD14) for 24 h. CXCL8 levels were quantified in cell-free culture supernatants by ELISA. Data presented are representative of three experiments performed in triplicate with primary cell lines derived from three independent donors (*P < 0.05). (F) HPMC were stimulated with increasing doses of SES in medium without serum in the absence or presence of sCD14 (500 ng/mL) for 24 h. CXCL8 levels were quantified in cell-free culture supernatants by ELISA. Data presented are representative of three experiments performed in triplicate with HPMC derived from three independent donors. (*P < 0.05 between unstimulated cells and the indicated doses of SES).