PMC

Migration of primary cells to CCL21 on fibronectin. Primary naive T lymphocytes were allowed to migrate to CCL21 (10 nm to 2 μm) through 5-μm pore membranes that had been preincubated in serum-free RPMI 1640 medium (n = 3) (A) or 10 μg/ml fibronectin (n = 4) (B) in the presence (□) or absence (■) of 10 μg/ml β1 integrin function-blocking antibodies.

Depletion of PLCγ1 reduces migration to CCL21. Cells were pretreated with PLCγ1 shRNA and assayed for knockdown (KD) of PLCγ1 (A); induced to migrate through a 5-μm pore fibronectin-coated membranes to 400 nm CCL21 (n = 3; p = 0.002 for migration of cells pretreated with PLCγ1 shRNA versus control shRNA) (B); stimulated with 400 nm CCL21 and assayed for levels of activated β1 integrins (12G10) by flow cytometry (C); or lysed and assayed for phospho-PLCγ and re-probed for total PLCγ or for phospho-ERK1/2 and re-probed for total ERK1/2 (D). E, graphical representation of densitometric measurements of changes in levels of ERK1/2 phosphorylation of blots (n = 3).

PLC mediates migration to CCL21. Primary naive T lymphocytes were preincubated with 2 μm U73122 or U73343 for 20 min and induced to migrate to (10 nm to 2 μm) CCL21 through 5-μm pore fibronectin-coated membranes (n = 3; p = 0.0312 for U73122 versus U73343 pretreated cells) (A) or stimulated with 400 nm CCL21 and assayed for phospho-PLCγ, stripped, and re-probed for total PLCγ or for phospho-ERK1/2, stripped, and re-probed for total ERK1/2 (n = 3) (B). Densitometric analysis was used to analyze the blots.

Gα_i mediates cell migration to CCL21 and β1 integrin activation. Primary naive T lymphocytes were pretreated with pertussis toxin. After 72 h, cells were (A) assayed for PLCγ expression and migration through 5-μm pore fibronectin (10 μg/ml)-coated membranes to 400 nm CCL21 (n = 3; p = 0.0331 for pertussis toxin (PTX) versus vehicle control cells), (B) Western-blotted for phospho-PLCγ, stripped, and re-probed for total PLCγ or for phospho-ERK1/2, stripped, and re-probed for total ERK1/2 (n = 3), or (C and D) stimulated with 400 nm CCL21 for the indicated times and assayed for levels of activated β1 integrins (12G10) by flow cytometry.

Inhibition of ERK1/2 decreases migration of primary naive T lymphocytes to CCL21. Primary human naive T lymphocytes were treated with 1 μm U0126 or an equivalent dilution of dimethyl sulfoxide (control) for 90 min. Then, the cells were induced to migrate to CCL21 (10 nm to 2 μm) through a 5-μm pore membrane that had been preincubated in 10 μg/ml fibronectin (n = 3; p = 0.031 for plot of migration of cells pretreated with vehicle compared with lymphocytes pretreated with U0126) (A); stimulated lysates were assayed for phospho-PLCγ, stripped, and re-probed for total PLCγ or for phospho-ERK1/2, stripped, and re-probed for total ERK1/2 (n = 3) (B); or U0126-treated cells were stimulated with 400 nm CCL21, stained for activated β1 integrins, and analyzed by flow cytometry (n = 3; p = 0.41 for integrin activation of control lymphocytes compared with lymphocytes pretreated with U0126) (C).