Effect of S. suis and TNFα on PMN transmigration through PCPEC and barrier function. (A, B, C) PCPECs were infected with S. suis strain 10 and the non-encapsulated mutant strain 10 Δcps (MOI 10) or stimulated with TNFα (10 ng/ml) as described in Methods. PMNs were applied after 24 h of stimulation to the basolateral side of PCPEC. Effects on barrier function were measured by TEER (A) and TR-dextran flux (B) of PCPEC monolayers. The TEER results are expressed as absolute TEER values (Ω × cm2). (A) For simplification only TEER values of IL-8-treated PCPECs are shown because they did not differ from TEER values of PCPEC cultures without IL-8. TEER was measured before stimulation, 24 h later that was directly before PMN transmigration and after 4 h of PMN transmigration. #, P value of < 0.001, compared to time point before.(B) The TR-dextran flux was measured in the basolateral-to-apical direction (upper to lower compartment) with or without S. suis infection, respectively, and TNFα stimulation and is expressed as percentage of tracer in the basolateral (upper) compartment. TR-dextran flux was determined after 4 h of PMN transmigration. In indicated samples IL-8 (10 ng/ml) was used as chemoattractant. Data are shown are mean ± SD for four independent experiments, each performed in triplicate. *, P value of < 0.05 compared to corresponding control PCPECs without IL-8 stimulation. §, P value of < 0.05 compared to corresponding control PCPECs with IL-8 stimulation. (C) BCECF-loaded PMNs were applied after 24 h of stimulation to the basolateral side of inverted PCPEC in a PCPECs:PMNs ratio of 1:10. In indicated samples IL-8 (10 ng/ml) was used as chemoattractant. The number of PMNs was fluorometrically measured after 4 h of transmigration. PMN transmigration is expressed% PMN transmigration from totally applied PMNs. Data are shown as mean ± SD for four independent experiments, each performed in triplicate. *, P value of < 0.0001 compared to PCPEC without IL-8; #, P value of < 0.0001, compared to corresponding control PCPEC +/- IL-8; §, P value of < 0.0001 compared between different stimuli.