Figure 3.

In vitro and in vivo comparative studies of transendothelial migration of polymorphonuclear neutrophil (PMN) induced by E44, ZD1 and complemented strains. (A) Induction of PMN transmigration with YGF beads coated with IbeA protein. PMN transmigration assays were performed with stimulation of YGF beads coated with IbeA, heat‐inactivated IbeA, or BSA (2 × 10⁶ beads/mL). (B) Induction of PMN transmigration with ZD1 coated with IbeA protein or BSA. E44 and ZD1 [(1 ×  10⁶ cfu/mL = 25 multiplicities of infection (MOIs)] were included as the positive and negative controls, respectively. In vitro assays were performed in triplicates (A,B). C. IbeA protein induced PMN transmigration in vivo. Meningitis was induced by injecting 10 µL of PBS with or without 0.8 µg IbeA or BSA protein into the cisterna magna of 10‐day‐old mice. After 5 h, CSF was harvested and PMN numbers were counted under microscope. (D) PMN recruitment into the CNS of mice in response to the wildtype (E44/pUC13), mutant (ZD1/pUC13) and complemented mutant (ZD1/pUC23A) strains. Meningitis was induced by injection of different E. coli strains into the cisterna magna of 10‐day‐old mice. After 12 h, CSF was harvested and PMN numbers were counted in a blinded‐fashion under microscope. Results are expressed as PMN numbers per microliters of CSF. *P < 0.05; **P < 0.01. Eight animals were used for each group.