Cutting edge: Vascular endothelial growth factor-mediated signaling in human CD45RO+ CD4+ T cells promotes Akt and ERK activation and costimulates IFN-gamma production

Abstract

In this study, we find that CD45RO+ memory populations of CD4+ T lymphocytes express the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 at both the mRNA and protein levels. Furthermore, by Western blot analysis, we find that VEGF increases the phosphorylation and activation of ERK and Akt within CD4+CD45RO+ T cells. These VEGF-mediated signaling responses were inhibited by a KDR-specific small interfering RNA in a VEGF receptor-expressing Jurkat T cell line and by SU5416, a pharmacological KDR inhibitor, in CD4+CD45RO+ T cells. We also find that VEGF augments mitogen-induced production of IFN-gamma in a dose-dependent manner (p < 0.001) and significantly (p < 0.05) increases directed chemotaxis of this T cell subset. Collectively, our results for the first time define a novel function for VEGF and KDR in CD45RO+ memory T cell responses that are likely of great pathophysiological importance in immunity.

FIGURE 1

Expression of VEGFRs on Jurkat T cells and CD4⁺CD45RO⁺ human memory T cells. The expression of NRP-1, Flt-1, and KDR was examined by FACS on Jurkat T cells (A) and on CD4⁺CD45RO⁺ T cells (B and C). B illustrates the purity of CD4⁺CD45RO⁺ T cells following negative isolation from PBMCs. A and C, The difference (Δ) in mean fluorescence staining (experimental minus isotype control) is shown within each FACS plot. D, Western blot analysis for VEGFR expression. M, memory CD4⁺ CD45RO⁺ T cells; J, Jurkat T cells. E, RT-PCR for KDR and Flt-1 in Jurkat T cells (lane 2) and CD4⁺CD45RO⁺ T cells (lane 3). Lane 1 represents RT-PCR of non-reverse-transcribed RNA from the CD4⁺CD45RO⁺ T cells as a negative control. F, The mRNA expression of KDR was evaluated by quantitative real-time PCR in pooled populations of CD4⁺ cells, either unactivated or following mitogenic activation (anti-CD3, 1 μg/ml). A–C are representative of at least 10 experiments, and each PCR is representative of two with similar findings.

FIGURE 2

VEGF-inducible signaling in human CD4⁺CD45RO⁺ T cells. Jurkat T cells or purified populations of CD4⁺CD45RO⁺ T cells (negatively isolated from PBMCs) were stimulated with VEGF (10 ng/ml) for 2, 10, and 15 min and were subsequently subjected to Western blot analysis. A and B illustrate the expression of pERK and pAkt or total ERK and total Akt in Jurkat T cells transfected either with KDR siRNA or control siRNA, prior to stimulation with VEGF. Densitometry illustrating the relative expression of pERK/ERK (A) and pAkt/Akt (B) is illustrated above each Blot. In C, CD4⁺CD45RO⁺ T cells were treated with VEGF alone, and in D and E, CD4⁺CD45RO⁺ T cells were treated with VEGF in combination with SU5416, a pharmacological KDR signal inhibitor (1 μM). Following treatment, the expression of pERK/ERK and/or pAkt/Akt was evaluated by Western blot analysis. F, Western blot analysis of pERK/ERK expression in Jurkat T cells stimulated with VEGF alone, anti-CD3 (0.3 μg/ml) alone or both in combination as indicated. Densitometry is illustrated above the blot. All illustrated blots are representative of at least three with similar results.

FIGURE 3

VEGF-induced costimulation of IFN-γ production in human CD4⁺CD45RO⁺ T cells. A, CD45RO⁺ populations of CD4⁺ T cells were negatively isolated from human PMBCs and were treated with anti-CD3 (0.1 and 0.3 μg/ml) and VEGF (10 and 20 ng/ml) as indicated. After 24 h, ELISPOT was performed to evaluate IFN-γ production. B shows representative wells in triplicate from the experiment shown in A. The spot count in each well is illustrated. C, T cells were cultured with anti-CD3 (0.3 μg/ml), VEGF (20 ng/ml), and the pharmacological KDR signal inhibitor SU5416 (SU1, 1 μM) and SU5 (5 μM) as illustrated. ELISPOT was again performed for IFN-γ production after 24 h. Each illustrated experiment is representative of at least four with similar results. p Values were calculated using the Student t test.

FIGURE 4

VEGF mediates chemotaxis of human CD4⁺CD45RO⁺ memory T cells. A, Cell culture medium was added into the lower chamber of 3-μm pore FluoroBlok Transwells in the absence or presence of VEGF (50 ng/ml) or IP-10 (50 ng/ml, as a positive control). Subsequently, CFSE-labeled CD4⁺CD45RO⁺ T cells (1 × 10⁵ cells) were placed in the upper chamber of each Transwell (in duplicate wells), and migration into the lower chamber was monitored via the assessment of fluorescence. T₀ represents the time in between plate setup and the first fluorescence reading (~5–7 min). T_15–90 represent automated fluorescence readings in the lower chamber every 15 min for a total of 90 min. B and C, CD4⁺CD45RO⁺ T cells (1 × 10⁵ cells) were placed in the upper chamber of a microchemotaxis Boyden chamber and migration into the lower chamber was assessed after 3 h, as described in Materials and Methods. T cells were used untreated or following 2-h pretreatment with the Akt inhibitor LY294002, the Erk inhibitor PD98059, or pertussis toxin. VEGF or IP-10 was added into the lower chambers, as indicated. The illustrated experiments are representative of at least three performed in duplicate, all with similar results. p Values were calculated using the Student t test (*p < 0.05).