Phospholipase cbeta is critical for T cell chemotaxis

Abstract

Chemokines acting through G protein-coupled receptors play an essential role in the immune response. PI3K and phospholipase C (PLC) are distinct signaling molecules that have been proposed in the regulation of chemokine-mediated cell migration. Studies with knockout mice have demonstrated a critical role for PI3K in G(alphai) protein-coupled receptor-mediated neutrophil and lymphocyte chemotaxis. Although PLCbeta is not essential for the chemotactic response of neutrophils, its role in lymphocyte migration has not been clearly defined. We compared the chemotactic response of peripheral T cells derived from wild-type mice with mice containing loss-of-function mutations in both of the two predominant lymphocyte PLCbeta isoforms (PLCbeta2 and PLCbeta3), and demonstrate that loss of PLCbeta2 and PLCbeta3 significantly impaired T cell migration. Because second messengers generated by PLCbeta lead to a rise in intracellular calcium and activation of PKC, we analyzed which of these responses was critical for the PLCbeta-mediated chemotaxis. Intracellular calcium chelation decreased the chemotactic response of wild-type lymphocytes, but pharmacologic inhibition of several PKC isoforms had no effect. Furthermore, calcium efflux induced by stromal cell-derived factor-1alpha was undetectable in PLCbeta2beta3-null lymphocytes, suggesting that the migration defect is due to the impaired ability to increase intracellular calcium. This study demonstrates that, in contrast to neutrophils, phospholipid second messengers generated by PLCbeta play a critical role in T lymphocyte chemotaxis.

Figure 1. PI3Kγ is not essential for T-cell migration

(A) Peripheral T-lymphocytes were isolated from lymph nodes of wild type (WT) and PI3Kγ-null (PI3Kγ −/− mice). Cells were placed into the upper Transwell chamber and 300 ng/ml SDF1α was placed into the lower chamber. The T-cells that migrated toward SDF1α after 3 hours at 37°C, were quantitated by flow cytometry. The number of migrating T-lymphocytes were expressed as the percent migration of the WT cells. The mean, S.E, and paired student’s t-test reflect data from 6 separate experiments. (B) The role of PI3K in SDF1α-mediated T-cell migration was studied using the PI3K inhibitors wortmanin and LY294002. WT and PI3Kγ −/− T-cells were isolated and placed in a Transwell chamber as described in part A. Some of the cells were pre-incubated for 10–15 minutes at 25 °C with the PI3K inhibitors wortmanin (100 nM) or LY294002 (10 µM). The cells were exposed to 300 ng/ml SDF1α, placed below the filter, for 3 hours at 37 °C. The migrating T-lymphocytes were expressed as the percent chemotaxis of the wild type control cells. The mean and S.E. reflect data from 5 separate experiments.

Figure 2. Inflamed skin from PLCβ2β3-null mice demonstrate increased leukocyte infiltration

(A) Ulcerated periorbital skin from PLCβ2β3-null mice demonstrate a chronic hyperplastic epithelial response in areas bordering epithelial denudation (arrows). The dermis is characterized by a mixed inflammatory infiltrate noticeably lacking in lymphocytes, but composed of mononuclear cells, which are predominantly plasma cells, and abundant neutrophils. (B) Skin from wild type mice showed none of the changes described in the PLCβ2β3-null mice. The epithelium has a normal uniform thickness, and the dermis contains normal connective and muscular tissue with very few inflammatory cells present. Sebaceous glands and hair follicles also demonstrate normal architecture. Insets represent higher magnification. Original magnification: A and B, X25; insets, X100.

Figure 3. PLCβ2β3-null lymphocytes are phenotypically similar to wild type lymphocytes

(A) Lymphocytes were isolated from peripheral lymph nodes of WT and PLCβ2β3-null (PLCβ2β3 −/−) mice and submitted to flow cytometric analysis using anti-CD3-APC or anti-B220-Cychrome. Shown is a representative example of 7 separate experiments. Isolated lymphocytes expressing CD3 were gated and cells stained with anti-CD25-FITC (B) or anti-CD69-PE (C) were analyzed. The left panels represent resting lymphocytes and the right panels represent lymphocytes activated with PMA (5 nM) and ionomycin (0.1 µM). Shown is a representative example of 4 separate experiments (D) Data from A and B were expressed as a percent of total lymphocytes isolated from peripheral lymph nodes of WT and PLCβ2β3 −/− mice. (E) Isolated lymphocytes stained with anti-CD3-APC were gated and cells stained with anti-CD44-FITC, -CD62L-Cychrome, and –CD45RB-PE were expressed as a percent of total lymphocytes isolated from peripheral lymph nodes of WT and PLCβ2β3 −/− mice. The mean, S.E., and paired student’s t-test from D and E reflect data from 4–7 separate experiments.

Figure 4. Loss-of-function mutations in PLCβ2β3 impair T cell migration to SDF1α

(A) To determine the optimal concentration at which to measure T-cell chemotaxis, peripheral T-lymphocytes were isolated from lymph nodes of WT and PLCβ2β3 −/− mice and placed into the upper Transwell chamber. Varying concentrations of SDF1α were placed into the lower chamber. The T-cells that migrated toward the resultant SDF1α gradient, after 3 hours at 37°C, were quantitated by flow cytometry. For this dose response, the mean, S.E., and paired student’s t-test reflect data from 3 separate experiments.* p<0.05. (B) The migrating T-lymphocytes to 300 ng/ml SDF1α were expressed as the percent chemotaxis of the wild type cells. The mean, S.E., and paired student’s t-test reflect data from 12 separate experiments. (C) In order to evaluate whether SDF1α-induced T-cell movement in the Transwell assay is chemotactic or chemokinetic, WT and PLCβ2β3 −/− T-cells were isolated and placed in a Transwell chamber. The cells were incubated with 150 ng/ml SDF1α placed above and below the filter to abolish the chemotactic gradient. After 3 hours at 37 °C, the cells were recovered from both the upper and lower chambers and counted by flow cytometry. The data are reflective of 3 separate experiments.

Figure 5. SDF1α -induced T-cell migration requires intracellular calcium release, but not PKC activation

(A) To determine whether specific second messengers generated by PLCβ contribute to cell migration, wild type T-cells were placed in a Transwell chamber. Some of the cells were pre-incubated for 10–15 minutes at 25 °C with the PKC inhibitor GF109203x (10 µM) or the intracellular calcium chelators, either BAPTA-AM (20 nM) or Quin-2 AM (25µM). All but control cells were exposed to 300 ng/ml SDF1α for 3 hours at 37 °C. The migrating T-lymphocytes were expressed as the percent chemotaxis of the WT cells. The mean and S.E. reflect data from 4 separate experiments. (B) To determine whether PLCβ2β3 −/− T-cells have impaired changes in the concentrations of cytosolic calcium in response to SDF1α, peripheral node T-lymphocytes of WT and PLCβ2β3 −/− mice were loaded with 5 µM Fura-2 and placed in a fluorescence spectrophotometer under continuous stirring. SDF1α, 300 ng/ml, was added at the indicated time (arrow), and the ratio of emitted light at 510 nm was recorded when the cells were illuminated by 340 nm and 380 nm excitation light, respectively. The data is a representative example of 3 separate experiments. (C) To confirm that GF109203x decreased SDF1α-induced PKC activation, wild type murine T-cells were pre-incubated with GF109203x for 15 minutes at 25 °C. After the addition of 300 ng/ml SDF1α for 1 minute, cells were lysed in boiling 1% SDS with β-mercaptoethanol and immunoblotted with a phospho-pan PKC antibody. PKC activity was inferred by its phosphorylation status.

Figure 6. PLCβ2β3 T-cells are able to assemble actin normally in response to SDF1α

Peripheral T-lymphocytes were isolated from lymph nodes of WT and PLCβ2β3 −/−mice. The T-cells were suspended in RPMI 1640 with 0.5% BSA (1 × 10⁶/ml) and treated with 300 ng/ml SDF1α at various time points. The assembly of actin monomers into filaments, or F-actin, was quantitated by staining cells with fluorescently labeled phalloidin followed by flow cytometry. Data are expressed as percent of WT baseline (without SDF1α). The mean and S.E. reflect data from 5 separate experiments.

Figure 7. Model of chemokine-stimulated T-cell chemotaxis

In T-lymphocytes, PLCβ and PI3Kγ are activated by Gαi-protein coupled chemokine receptors. Together, they modify phospholipids produced by PIP2. Hydrolysis of PIP2 and generation of IP3 with resultant increase of intracellular calcium has a significant effect on T-cell migration. In contrast, there appears to be little role for the generation of DAG and activation of PKC in this process. Phosphorylation of PIP2 to PIP3 by PI3K is also essential for T-cell migration by a mechanism that is yet unknown.