CCR7/CCL21 migration on fibronectin is mediated by phospholipase Cgamma1 and ERK1/2 in primary T lymphocytes

Abstract

CCR7 binds to its cognate ligand, CCL21, to mediate the migration of circulating naive T lymphocytes to the lymph nodes. T lymphocytes can bind to fibronectin, a constituent of lymph nodes, via their β1 integrins, which is a primary mechanism of T lymphocyte migration; however, the signaling pathways involved are unclear. We report that rapid (within 2 min) and transient phosphorylation of ERK1/2 is required for T cell migration on fibronectin in response to CCL21. Conversely, prevention of ERK1/2 phosphorylation by inhibition of its kinase, MAPK/MEK, prevented T lymphocyte migration. Previous studies have suggested that phospholipase Cγ1 (PLCγ1) can mediate phosphorylation of ERK1/2, which is required for β1 integrin activation. Paradoxically, we found that inhibition of PLCγ1 phosphorylation by the general PLC inhibitor U73122 was associated with a delayed and reduced phosphorylation of ERK1/2 and reduced migration of T lymphocytes on fibronectin. To further characterize the relationship between ERK1/2 and PLCγ1, we reduced PLCγ1 levels by 85% using shRNA and observed a reduced phosphorylation of ERK1/2 and a significant loss of CCR7-mediated migration of T lymphocytes on fibronectin. In addition, we found that inhibition of ERK1/2 phosphorylation by U0126 resulted in a decreased phosphorylation of PLCγ1, suggesting a feedback loop between ERK1/2 and PLCγ1. Overall, these results suggest that the CCR7 signaling pathway leading to T lymphocyte migration on fibronectin is a β1 integrin-dependent pathway involving transient ERK1/2 phosphorylation, which is modulated by PLCγ1.

FIGURE 1.

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.

FIGURE 2.

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).

FIGURE 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.

FIGURE 4.

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).

FIGURE 5.

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.