Figure 2.

Neutrophil migration to Neisseria gonorrhoeae (GC) requires leukotriene B₄ (LTB₄) but not epithelial 5-lipoxygenase activity. A, Supernatants from apical and basolateral compartments following GC infection and/or neutrophil transepithelial migration were collected at the indicated time points and conditions and passed through a 0.2-μm filter, and LTB₄ was quantified by enzyme-linked immunosorbent assay. B, Polarized human End1/E6E7 (End1) monolayers were infected with GC at a multiplicity of infection (MOI) = 10 for 1 hour. During neutrophil transepithelial migration, LY223982, an antagonist to the high-affinity LTB₄ receptor BLT₁, was added to the apical reservoir immediately prior to addition of neutrophils at the indicated concentrations (μg/mL). Gray bars represent neutrophil transepithelial migration to an imposed apical gradient of LTB₄ and black bars represent neutrophil migration to GC. C, Polarized End1 monolayers were pretreated with 5-lipoxygenase inhibitor Zileuton (25 μM), caffeic acid (50 μM), or an equivalent concentration of the vehicle control (dimethyl sulfoxide) diluted in Hanks’ Balanced Salt Solution with calcium and magnesium, 10mM HEPES, and 5mM NaHCO₃ for 1 hour prior to GC infection, thoroughly washed, and infected at an MOI = 10 and neutrophil transepithelial migration. There was no significant difference in neutrophil migration. D, Neutrophil transepithelial migration was assayed across End1 monolayers infected with GC at an MOI = 10 for 1 hour, with End1 cells either stably transformed with vector short hairpin RNA (shRNA) or shRNA against alox5. For all panels, results are expressed as a mean ± standard error of the mean for at least 3 independent experiments per condition. Statistics were calculated using a 2-tailed, paired (A) or unpaired (B and C) Student t test. *P < .05. Abbreviations: Ap., apical; Ba., basolateral; DMSO, dimethyl sulfoxide; LTB₄, leukotriene B₄; LY, LY223982; PMN, polymorphonuclear cell/neutrophil; shRNA, short hairpin RNA.