Transendothelial migration is a critical step in the neutrophil recruitment cascade during inflammatory processes. Recently, we could show that TRPC1 and TRPC6 channels play an important role in neutrophil chemotaxis. Here, we investigated whether TRPC1 and TRPC6 are also involved in neutrophil transendothelial migration.

Modified Boyden chamber assays were used to quantify neutrophil transmigration through an endothelial monolayer in vitro. Our assays involved a control group, a group stimulated with the intermediate chemokine KC and a group stimulated with the end target chemoattractant fMLP. The confluency of the endothelial monolayer on the days of our experiments was verified with immunofluorescent stainings of VE-cadherin, FITC-Dextran permeability assays and transendothelial electrical resistance measurements.

Wildtype neutrophils showed a significantly increased transendothelial migration under stimulation with KC and fMLP compared to their unstimulated control group. This effect was also visible when using TRPC1 and TRPC6 knockout neutrophils. However, compared to wildtype cells, transmigration of TRPC6 knockout neutrophils was strongly attenuated. This effect was most prominent in response to KC (~ -40%). Transendothelial migration of TRPC1-/- neutrophils stimulated with KC and fMLP was also inhibited. However, unlike TRPC6-/- neutrophils, TRPC1-/- neutrophils do not differ from wildtype cells in the absence of chemoattractants.

In conclusion, we could show that TRPC1 and TRPC6 channels are required for an efficient transendothelial migration of murine neutrophil granulocytes. Moreover, our results suggest that TRPC6 channels are of special importance for the intermediate chemoattractant induced signaling cascade.