Figure 1.

Contact-dependent Neisseria gonorrhoeae (GC) infection of polarized endocervical cell monolayers stimulates neutrophil transepithelial migration. Human End1/E6E7 (End1) monolayers were grown on the underside of collagen-coated Transwell inserts (6.5 mm diameter, 3.0-μm pores). Neutrophil transepithelial migration was quantified by myeloperoxidase (MPO) assay. A, Schematic of in vitro 3-component system depicting apical infection and neutrophil transepithelial migration. Polarized End1 monolayers were inverted in a humidified chamber and apically infected with GC. Following infection, infected monolayers were thoroughly washed in Hanks’ balanced salt solution with calcium and magnesium, 10mM HEPES, and 5mM NaHCO₃ (HBSS⁺) to remove nonadherent bacteria and reverted into a 24-well tissue culture well containing 1 mL HBSS⁺. Primary human neutrophils (1 × 10⁶) were added to the basolateral reservoir and transmigration was allowed to proceed for 2 hours unless otherwise noted. Monolayers were then removed and the number of neutrophils in the apical reservoir quantified by MPO assay. B, Polarized End1 monolayers were infected at various multiplicities of infection (MOI) for 1 hour and then assayed for neutrophil transepithelial migration. C, Polarized End1 monolayers were apically infected with GC at an MOI = 10 for 1 hour, and then neutrophil transmigration was assayed after 60, 75, 90, and 120 minutes. D, End1 monolayers were apically infected with GC at MOI = 10, an equivalent number of GC was added to the apical reservoir without interacting with End1 cells, or an equivalent number of GC was added to the apical reservoir an acellular Transwell insert and incubated for 1 hour before assaying for neutrophil transmigration. B–D, White bars represent neutrophil transepithelial migration to buffer alone with no apical stimulus (–), gray bars represent neutrophil transepithelial migration to an imposed apical gradient of N-formyl-l-methionyl-l-leucyl-l-phenylalanine, and black bars represent neutrophil migration to GC. Results are expressed as a mean ± standard error of the mean for at least 3 independent experiments per condition. Statistics for (B) were calculated using a 1-way analysis of variance with Tukey post hoc test, and statistics for (C) and (D) were calculated using a 2-tailed, unpaired Student t test. *P < .05. Abbreviations: Ap., apical; Ba., basolateral; End1, End1 E6/E7 cells; fMLP, fMLP, N-formyl-l-methionyl-l-leucyl-l-phenylalanine; GC, Neisseria gonorrhoeae; MOI, multiplicity of infection; ns, not significant; PMN, polymorphonuclear cell/neutrophil.