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. 2017 Mar 7;114(10):2693-2698.
doi: 10.1073/pnas.1612900114. Epub 2017 Feb 17.

PLCε1 Regulates SDF-1α-induced Lymphocyte Adhesion and Migration to Sites of Inflammation

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PLCε1 Regulates SDF-1α-induced Lymphocyte Adhesion and Migration to Sites of Inflammation

Marianne Strazza et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Regulation of integrins is critical for lymphocyte adhesion to endothelium and migration throughout the body. Inside-out signaling to integrins is mediated by the small GTPase Ras-proximate-1 (Rap1). Using an RNA-mediated interference screen, we identified phospholipase Cε 1 (PLCε1) as a crucial regulator of stromal cell-derived factor 1 alpha (SDF-1α)-induced Rap1 activation. We have shown that SDF-1α-induced activation of Rap1 is transient in comparison with the sustained level following cross-linking of the antigen receptor. We identified that PLCε1 was necessary for SDF-1α-induced adhesion using shear stress, cell morphology alterations, and crawling on intercellular adhesion molecule 1 (ICAM-1)-expressing cells. Structure-function experiments to separate the dual-enzymatic function of PLCε1 uncover necessary contributions of the CDC25, Pleckstrin homology, and Ras-associating domains, but not phospholipase activity, to this pathway. In the mouse model of delayed type hypersensitivity, we have shown an essential role for PLCε1 in T-cell migration to inflamed skin, but not for cytokine secretion and proliferation in regional lymph nodes. Our results reveal a signaling pathway where SDF-1α induces T-cell adhesion through activation of PLCε1, suggesting that PLCε1 is a specific potential target in treating conditions involving migration of T cells to inflamed organs.

Keywords: PLCε1; Rap1; SDF-1α; T cells; adhesion.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
PLCε1 is required for SDF-1α–induced Rap1 activation. (A) Jurkat T cells transfected with GFP–Ral–GDS–RBD; stimulation with soluble anti-CD3 (5 µg/mL) or SDF-1α (100 ng/mL). (B) GTP–Rap1 in primary human T cells following SDF-1α treatment for 2 min quantified by pull-down using the GST–Ral–GDS–RBD. (C) Primary human T cells treated with soluble anti-CD3 or SDF-1α for indicated amounts of time before GST pull-down assay; n = 3. (D) Western blot of PLCε1 in Jurkat T cells, shScr, or shPLCε1. (E) RT-PCR of cDNA from primary human and Jurkat T cells expressing scramble (si/shScr) or PLCε1 (si/shPLCε1) interfering RNA. Two shPLCε1 constructs were used (#1 and #2). (F) Jurkat T cells stimulated with soluble anti-CD3 or SDF-1α followed by GST pull-down. **P ≤ 0.01; ***P ≤ 0.001; n = 3.
Fig. S1.
Fig. S1.
Activated Rap1 is at the plasma membrane following anti-CD3 and SDF-1α stimulation. (A) Jurkat T cells were transfected with GFP–Ral–GDS–RBD. Representative cells demonstrating inactive and active probe localization with fluorescent line analysis. An inactive cell has a plateau of fluorescent intensity; an active cell has two peaks of fluorescent intensity. (B) Jurkat T cells were transfected with GFP–Rap1 and cherry–RIAM RAPH then left unstimulated or stimulated with anti-CD3 antibody (5 µg/mL) or SDF-1α (100 ng/mL). Cell membrane localization of Cherry–RIAM RAPH indicates Rap1 activation.
Fig. S2.
Fig. S2.
Kinetics of Rap1 activation by SDF-1α. (A) Primary human T cells were treated with SDF-1α (100 ng/mL) for indicated amounts of time before GTP–Rap1 was quantified by the GST pull-down assay; n = 3. (B) Jurkat T cells were transduced with shRNA constructs yielding individual cell lines with stable knockdown of one GEF or control cells expressing nontargeting, scramble shRNA. Cells were then treated with soluble anti-CD3 or SDF-1α for 2 min before GST pull-down assay. Fold change in GTP–Rap1 was relative to untreated cells. (C) Jurkat T cells transfected with GFP–Ral–GDS–RBD; stimulation with soluble anti-CD3 or SDF-1α.
Fig. 2.
Fig. 2.
Firm adhesion using shear stress downstream of SDF-1α requires PLCε1. (A) Carboxyfluorescin succinimidyl ester (CFSE)-labeled Jurkat T cells expressing shScr or shPLCε1 plated onto a CHO–ICAM-1 monolayer. A series of 30 images was captured over 10 min following SDF-1α stimulation, analyzed by Volocity 3D Image Analysis Software. Each vector indicates an individual cell. Inset number is average vector length (in micrometers). (B and D) Flow adhesion assay with CFSE-labeled T cells expressing shScr or shPLCε1, plated onto a CHO–ICAM-1 monolayer on µ-flow VI chambers. Images taken before shear force and after 5 min. Quantification of Jurkat T cells (B) and primary human T cells (D) using ImageJ. (C) Circularity index of individual cells was calculated using ImageJ. Experimental conditions as described. (E) Jurkat T cells expressing shScr or shPLCε1 were stained for surface expression of CXCR4 and analyzed by flow cytometry. **P ≤ 0.01; ***P ≤ 0.001; ****P < 0.0001; n = 3.
Fig. S3.
Fig. S3.
SDF-1α–induced morphological changes are dependent on PLCε1. (A) Flow adhesion assay with CFSE-labeled T cells expressing shScr or shPLCε1, plated onto a CHO–ICAM-1 monolayer on µ-flow VI chambers. Images were taken before shear force and after 5 min. Images shown are representative. (B) Morphological analysis of CFSE-labeled Jurkat T cells shows that cellular elongation, demonstrated by an increase in the ratio of Ferets, occurs following SDF-1α stimulation in cells expressing nontargeting, scramble shRNA (shScr), but not shRNA specific for PLCε1 (shPLCε1).
Fig. 3.
Fig. 3.
The lipase function of PLCε1 is not required to activate Rap1 downstream of SDF-1α. (A) Jurkat T cells transfected with GFP–C1A–C1A. Images represent majority; live cells were imaged 20 min following PMA (10 ng/mL), anti-CD3, or SDF-1α. Inset percentage from 20 cells counted over two independent experiments. (B) Intracellular calcium by fura-2 fluorescence in primary human T cells. (C) Percent of cells counted exhibiting calcium flux in response to stimulation. (D) Adhesion under static conditions (Left), primary T cells were treated with BAPTA-AM (50 µM) before anti-CD3. Adhesion using flow conditions (Right), primary human T cells were treated with BAPTA-AM before SDF-1α. (E) Jurkat T cells deficient in PLCγ were treated with U73122 (1 µM) before SDF-1α followed by GST pull-down assay. Quantification at Right. (F) Schematic of PLCε1 containing the H1436L point mutation. (G) HEK 293T cells transfected with GFP alone, C3G, full-length PLCε1–GFP (FL), or the lipase dead mutant PLCε1–GFP H1436L. GTP–Rap1 levels were quantified by GST pull-down assay. Quantification at Right.
Fig. 4.
Fig. 4.
The RA1/2 domains of PLCε1 contribute to SDF-1α signaling. (A) Jurkat T cells (shScr or shPLCε1) treated with anti-CD3 or SDF-1α. GTP–Ras levels were quantified by GST-Raf1 pull-down assay. (B) Western blot of Ras in Jurkat T cells shScr or N-Ras and K-Ras targeting shRNA (shRas). (C) Jurkat T cells (shScr or shRas) treated with SDF-1α followed by GST pull-down assay. (D) Jurkat T cells treated with wortmannin before SDF-1α stimulation followed by GST pull-down assay. (E) Schematic of PLCε1–NtermRA constructs. (F) Jurkat T cells (shScr or shPLCε1) transfected with GFP–Ral–GDS–RBD ± PLCε1–NtermRA or K2150E/K2152E (K2150/52E) or PLCε1NRAΔPH mutant constructs, and SDF-1α stimulated. Images represent majority phenotype for each condition. (G) Percent of cells imaged displaying an activated Rap1 phenotype based on probe recruitment to the plasma membrane and fluorescent line analysis; n = 3; more than 200 cells counted per condition. (H) Flow adhesion assay with Jurkat T cells (shScr or shPLCε1) transfected with GFP alone or GFP–Rap1–V12, on a CHO–ICAM-1 monolayer on µ-flow VI chambers. *P ≤ 0.05; **P ≤ 0.01; n = 3.
Fig. 5.
Fig. 5.
PLCε1 is required for recruitment of sensitized T cells to site of inflammation. (A) Histology of the ears of mice DNFB challenge, collected on day 7; sections were stained with H&E and imaged. (B) Grading of mononuclear cell infiltration following DNFB; (−) no infiltration, (+) mild infiltration, (++) major infiltration. (C and D) Immunohistochemistry of tissue sections with anti-CD3, anti-CD4, or anti-CD8 antibodies were imaged. Quantification per high-power field (HPF) views. (E) Flow adhesion assay with CFSE-labeled T cells isolated from spleens. Quantification was performed using ImageJ.
Fig. S4.
Fig. S4.
Schematic of the mouse model of contact sensitivity.
Fig. 6.
Fig. 6.
PLCε1 does not contribute to T-cell sensitization and priming in the lymph node. (A) Macroscopic assessment of inguinal lymph nodes collected from naïve or DNFB-sensitized mice on day 5. Images are representative. (B) Flow cytometry of cells isolated from lymph nodes following staining for CD3, CD4, and CD8. Scatters show a gated CD3+ population. (C) Cell titer proliferation assay on cells isolated from lymph nodes following stimulation with Con A (10 µg/mL) for 48 h. (D) Secreted IL-2 levels from cells isolated from lymph nodes following stimulation with Con A were quantified by ELISA. (E) Secreted IFN-γ levels from cells isolated from lymph nodes following coculture with DNBS-loaded primary antigen presenting cells for 48 h were quantified by ELISA. *P ≤ 0.05; **P ≤ 0.01. (F) Adoptive transfer of T cells from wild-type or PLCε1 knockout mice into PLCε1 knockout mice before sensitization. Ear histology of mice after treatments; sections were prepared and analyzed as in B. c, cartilage; white arrow, necrosis; black arrow, cellular infiltrate; asterisk, spongiosis necrosis. (G) Grading of mononuclear cell infiltration following DNFB; (−) no infiltration, (+) mild infiltration, and (++) major infiltration.
Fig. S5.
Fig. S5.
T-cell adhesion downstream of CXCR4 signaling requires the GEF activity of PLCε1. Presentation of SDF-1α on an endothelial surface signals through CXCR4 on the surface of rolling T cells. The increase in Ras–GTP at the plasma membrane acts as a binding partner for PLCε1; this interaction is at least partially responsible for localization of PLCε1 at the plasma membrane. Though the lipase domain is not playing a role in this cascade, the CDC25 domain acts as a Rap1 GEF leading to an accumulation of Rap1–GTP, and consequently activation of LFA-1 and increased T-cell adhesion to ICAM-1.
Fig. S6.
Fig. S6.
RANTES induces Rap1 activation. (A) GTP–Rap1 in CCR5-expressing Molt4 T cells following RANTES (100 ng/mL) treatment for 2 min was quantified by GST pull-down using the Ral–GDS–RBD. (B) Flow adhesion assay with CFSE-labeled CCR5 expressing Molt4 T cells plated onto a CHO–ICAM-1 monolayer on µ-flow VI chambers. Quantification was performed using ImageJ.

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