Confluent MDCK cell cultures were exposed to either TNF or IFN for 24 hours before TER was measured. selleck bio Exposure to TNF induced a dose dependent elevation in TER, whereas expo sure to IFN induced no significant effect on MDCK cell TER. A time course was performed for up to 72 hours on con fluent MDCK cell cultures exposed to TNF IFN while TER was monitored at regular intervals. Figure 3A repre sents the changes in TER under the following conditions media only control, TNF IFN, 3/6 ng/ml, TNF IFN, 10/20 ng/ml, and TNF IFN, 30/60 ng/ml. In the first six hours of cytokine exposure TER values are relatively sta ble. Between 12 and 24 hours, a significant dose depend ent elevation of TER is observed. MDCK cells have 70. 3 0. 6 ? cm2 of resistance, at 24 hours cells treated with 3/6 ng/ml of TNF IFN developed 99.
9 0. 8 ? cm2 and 115. 7 1. 9 ? cm2 when treated with 30/60 ng/ml of TNF IFN. This represents a 65% increase in TER at 24 hr in the presence of the highest concentration of cytokine. Interestingly, between 24 and 72 hours there is a return toward baseline in MDCK cells treated with the lower doses of cytokine, whereas cells treated with the highest dose show a 104% increase in TER. These studies imply that treatment with TNF IFN in MDCK cells positively regulates factors that contribute to TER. To investigate the contribution of the MAP kinase signal ing pathway we employed several potent and specific pharmacological agents. MDCK cell grown to confluence on Transwell inserts were treated with TNF IFN for 24 hr in the presence and absence of a panel of inhibitors, U0126, SB202190 and a SP600125.
Treatment with TNF IFN resulted in a 95% increase in TER compared to control cells, the addition of U0126 to cells treated with cytokine resulted in a significant dose dependent decrease in TER. However, the treatment with SB202190, a p38 inhibitor produced a significant eleva tion of TER compared to cytokine alone, resulting Batimastat in a dose dependent increase of 33% and 80%. The combina tion of low doses of ERK1/2 and p38 inhibition in the presence of cytokine produced minimal effect on the cytokine treated cells, due to their opposing action on TER. The addition of SP600125, a JNK inhibitor did lower TER values a modest 22% in the presence of cytokine. MAP kinase activation and signaling pathways differen tially regulate TER in this model of cytokine exposure in MDCK cells. Proinflammatory cytokines elevate flux The effect of flux assay temperature in confluent MDCK cell cultures was determined, cells were placed into one of two treatment groups for 24 hours. control or TNF IFN. The paracellular flux tracer 4 kD FITC dextran was added to the apical chamber and recovery was determined from the basolateral chamber at given intervals.