Neutrophils as part of the innate immune system play a major role in the first host defence. During an inflammation neutrophils are guided by chemoattractant-gradients from the blood vessels into the affected tissue.Thischemotaxis involves chemoattractant-receptor activation leading to the initialization of signaling pathways that involve intracellular Ca²⁺ transients. On our way to identify the potentially involved Ca²⁺ entry channels we investigated the role of TRPC6 channels in chemotaxis of murine neutrophils.

We studied the impact of TRPC6-deficiency on chemotaxis of murine neutrophils in 3-dimensional matrices with time-lapse videomicroscopy. In chemotaxis assays fMLP was used as end-target chemoattractant and a murine ex-vivo chemokine-cocktail or KC/CXCL1 were applied as intermediary chemoattractants.The chemokine cocktailwas a product of a mouse peritonitis induced with casein and consisted of the peritoneal exudates after casein treatment. Receptor-signaling was studied via western blot and Ca²⁺-influx was analyzed by Fura-2 Ca²⁺-measurements.

Chemotaxis of TRPC6^-/- neutrophils with the chemokine-cocktail and KC was strongly impaired while chemotaxis towards fMLP was unaffected. Blocking of chemokine receptor CXCR2 led to a diminished chemotaxis of WT cells but had no effect on TRPC6^-/- cells. Western blot analysis indicated that CXCR2 expression was not influenced by TRPC6 but CXCR2 signaling via phosphorylation of Akt/PKB was impaired in TRPC6^-/- cells. Intracellular Ca²⁺-imaging showed no difference in Ca²⁺-mobilization after fMLP stimulation but reduced Ca²⁺-transients after intermediary chemokine stimulation.

Our findings indicate that TRPC6 is involved in CXCR2-mediated chemotaxis of murine neutrophils. TRPC6 is a component of intermediary chemoattractant signaling but does not influence end-target chemotaxis.