Cutting Edge: Stimulation of Dopamine D₄ Receptors Induce T Cell Quiescence by Up-Regulating Krüppel-Like Factor-2 Expression through Inhibition of ERK1/ERK2 Phosphorylation¹

Neural-immune communication is a well-recognized phenomenon (1, 2, 3). It is now well established that the sympathetic nervous system regulates the immune system by releasing neurotransmitters and neuropeptides from the nerve terminals (3). These neurochemicals interact in turn with their specific receptors, including lymphocytes, that are present in different immune effector cells and thereby influence the functional activities of these cells of the host against disease and other environmental stress (1). Among the several neural mediators regulating immune functions, catecholamines play a critical role; within the catecholamine family, Dopamine (DA)⁴ is of recent interest because different classes of DA receptors (D₁/D₅, D₂, D₃, and D₄) are present in human T lymphocytes (4). We and others had previously demonstrated that pathophysiological concentrations of DA in vitro inhibited the proliferation of activated T cells and their cytokine secretion by modulating the intracellular signaling pathways that regulate these functions of the cells (4, 5, 6, 7, 8). Also, observations from other laboratories have shown that specific physiological concentrations of DA can even drive the resting human T cells into functional activations through the D₂, D₃, and D₁/D₅ classes of DA receptors (9, 10, 11, 12). In addition, the D₄ class of DA receptors is present in normal unstimulated human T cells (13). Although recent reports indicate that the D₄ DA receptors present in the cardiovascular system and the kidney are associated with blood pressure regulation (14), nothing is known about the functional significance of D₄ DA receptors in T lymphocytes. In the present report we have demonstrated that stimulation of the D₄ class of DA receptors in human T cells is associated with the expression of Krüppel-like factor (KLF)-2, a critical regulator of quiescence — a state characterized by decreased cell size and metabolic activity of the T cells (15). Recent reports indicate that quiescence in lymphocytes is an actively maintained state rather than a default pathway in the absence of a signal (15, 16, 17). Transcription factors like KLF2 appear to act as master regulators of gene expression patterns that enforce the quiescent phenotype in T cells, and KLF2 is one of the members of the KLF family that bind to GC-rich DNA elements through the conserved DNA-binding zinc finger domain in the C terminus (15). Furthermore, KLF2 is highly expressed in naive T cells, rapidly lost after T cell activation (15), and reestablished in memory T cells, another quiescent population (16, 17).

T cells, isolated from the blood of healthy donors (approved by the Institutional Review Board of the Chittaranjan National Cancer Institute, Kolkata, India) by using a T cell isolation kit (Miltenyi Biotec), were stimulated with plate-bound anti-CD3 (Sigma-Aldrich) followed by the addition of soluble anti-CD28 (eBioscience) Abs (4 μg/ml each) and 1 × 10⁶ cells together with the specific D₄ DA receptor agonist PD 168,077 maleate salt (1 μM; Sigma-Aldrich) or ABT 724 trihydrochloride (1 μM; Tocris Bioscience) at the onset of 48 h of culture. In other experiments, the above-mentioned agonists were added to the T cell culture alone to assess the effects of the agonists on unstimulated T cells. The dosage of the agonists was determined from the dose-response experiments (described in detail in Results and Discussion). To further determine the specificity of the involvement of the D₄ class of DA receptors, cells were pretreated with the specific D₄ class of DA receptor antagonist U101958 (50 μM; Sigma-Aldrich). Similar experiments were also conducted with the following classes of DA receptor agonists at the specified concentrations as determined from dose-response experiments: D1/D5, SKF38393 (1 μM); D2, quinpirole (2 μM); and D3, PD 128907 (5 μM) (Sigma-Aldrich). In the experiments in which kinase inhibitors were used, T cells were pretreated with vehicle or with the MEK-1/2 inhibitor U0126 (25 μM; Calbiochem), or the p38MAPK inhibitor SB203580 (10 μM; Sigma-Aldrich), or the JNK inhibitor SP600125 (25 μM; Sigma-Aldrich) for 2 h (18, 19) followed by stimulation with anti-CD3 and anti-CD28 in 96-well plates and cultured.

A [³H]thymidine incorporation assay was undertaken to measure cell proliferation following anti-CD3/CD28 stimulation (7).

Cells were stained with FITC- or PE-conjugated Abs of CD69, CD25 (eBioscience), and 7-aminoactinomycin D (7-AAD) (BD Biosciences). After adjustment with isotype controls, positive cells were enumerated by FACS (FACSCalibur; BD Biosciences) (12).

Culture supernatants were collected from 24-well plates following the stimulation of T cells (1 × 10⁶/ml) with anti-CD3/anti-CD28 in the presence or absence of the D₄ agonist PD 168,077 (1 μM) for 24 h at 37°C. Thereafter, the concentration of IL-2 was measured by ELISA using Quantikine ELISA kits (R&D Systems) (7).

Proteins from lysed cells were immunoblotted with primary Abs (D₄ DA receptor, phospho-ERK1/2, p38 MAPK, and JNK; Santa Cruz Biotechnology) and KLF2 (Imgenex) (7).

Total RNA was extracted from isolated T cells following the manufacturer’s protocol (Ambion). Two micrograms of total RNA was reverse transcribed into first strand cDNA by using poly(dT) priming (7) following amplification using a PCR kit (Ambion). The sequences of PCR primers were as follows: D₄ DA receptor, 5′-TGCTGTGCTGGACGCCCTTCTTCG-3′ (forward) and 5′-CGTTGCGGAACTCGGCGTTGAAGA-3′ (reverse) (20); KLF2, 5′-TGCCGTCCTTCTCCACTTTC-3′ (forward) and 5′-CTCTTGGTGTAGGTCTTGCC-3′ (reverse) (21); 15S rRNA (internal control), 5′-TTCCGCAAGTTCACCTACC-3′ (forward) and 5′-CGGGCCGGCCATGCTTTACG-3′ (reverse) (7).

All data are expressed as the mean ± SEM. Statistical comparisons were performed using Student’s t test. p < 0.05 was considered significant (7).

RT-PCR and Western blot analysis demonstrated the presence of the D₄ class of DA receptors in T cells isolated from normal volunteers (Fig. 1, a and b). Thereafter, when these T cells were treated with the specific D₄ DA agonist PD 168,077 (1 μM) at the onset of 48 h of TCR stimulation with anti-CD3/CD28 so that the D₄ DA agonist remained throughout this period, these cells showed significant inhibition of T cell proliferation as evident from [³H]thymidine incorporation (Fig. 1,c). Treatment with another D₄ DA agonist, ABT 724 trihydrochloride (1 μM), also showed similar results (Fig. 1,c). Furthermore, to confirm the specificity of the D₄ DA receptor-mediated abrogation of anti-CD3/CD28-induced cell proliferation, when these cells were pretreated with U101958, a D₄ receptor antagonist, the inhibitory effect of the D₄ receptor agonist on T cell proliferation was lost (Fig. 1,c). The dosage of D₄ receptor agonists used in the present study was determined from the different dose-response (1 nM to 1 mM) effects of the agonists on anti-CD3/CD28-induced T cell proliferation inhibition. From these dose-response studies it was demonstrated that for both of the D₄ agonists a concentration of 1 μM was the most effective in inhibiting cell proliferation with negligible cell death (Fig. 1 c). In contrast, significant cell death was observed when these agonists were used at concentrations of >6 μM (data not shown). Furthermore because both of the D₄ agonists demonstrated a similar inhibition of anti-CD3/CD28-induced cell proliferation, only one D₄ agonist, PD 168,077 (1 μM) was used for additional experiments.

It should also be noted here that the treatment of unstimulated human T cells with different concentrations of the D₄ agonist PD 168,077 (1 nM to 6 μM) alone and without any TCR stimulation had no effect on the proliferation of these cells (Fig. 1,c). This result therefore indicates that the dosage of the D₄ agonist, which inhibits anti-CD3/CD28-mediated proliferation of stimulated T cells and induces quiescence of these cells, has no effect on unstimulated T cells. Next, to rule out the possibility that the abrogation of anti-CD3/CD28-induced cell proliferation following D₄ receptor stimulation by its agonist PD 168,077 was due to cell death, 7-AAD-positive cells were enumerated by flow cytometry. No significant differences were observed between agonist-treated and untreated groups, indicating that the D₄ receptor-mediated inhibition of proliferation was not due to the death of the agonist-treated T cells (Fig. 1, d and e).

Because proliferation was inhibited without any increase in cell death, the quiescence of these cells was a possibility. Therefore, we first enumerated the surface expression of the early activation markers CD69 and CD25, which, although insignificantly expressed in quiescent T cells, are highly expressed in the activated cells (22). The results demonstrated that T cells stimulated with anti-CD3/CD28 for 24 h robustly expressed surface CD69 and CD25. In contrast, when the anti-CD3/anti-CD28-stimulated T cells were treated with the D₄ DA receptor agonist PD 168,077, the expression pattern of these activation markers was similar to that of the resting cells (Fig. 1 f).

To further confirm that the stimulation of D₄ DA receptors in T cells inhibits the induction of T cell activation via a TCR, we next investigated the effect of D₄ receptor stimulation on IL-2 secretion by T cells stimulated with anti-CD3/CD28. It was observed that human T cells, when stimulated with anti-CD3/CD28, released IL-2 in the culture. In contrast, when these CD3/CD28-stimulated cells were simultaneously treated with D₄ receptor agonist PD 168,077 (1 μM), the IL-2 concentration was similar to that of the unstimulated cells (Fig. 2).

We next investigated whether activation of the D₄ class of DA receptors was associated with the expression of KLF2, the transcription factor that regulates quiescence in T cells (16). Unstimulated T cells showed considerable expression of KLF2. In contrast, when these cells were stimulated with anti-CD3/CD28 Abs, the KLF2 expression was undetectable (Fig. 3, a and b) after 24 h of stimulation as previously reported (23). Furthermore when these cells were costimulated with the specific D₄ DA receptor agonist PD 168,077 and with anti-CD3/CD28, the expression of KLF2 remained intact as shown by RT-PCR and Western blot analyses (Fig. 3, c and d). However, the D₄ DA receptor agonist PD 168,077 alone, without any further stimulation of a TCR, had no effect on KLF2 expression (Fig. 3, c and d).

Because the stimulation of ERK1/ERK2 is a critical signaling step for cell activation (24), we investigated the expression of ERK1/ERK2 following the activation of D₄ DA receptors by PD 168,077 and its correlation with KLF2 expression in T cells. We observed that T cell activation by anti-CD3/CD28 was associated with the phosphorylation of ERK1/ERK2 (Fig. 3,e). However, when these cells were treated with the specific D₄ receptor agonist PD 168,077 in presence of anti-CD3/CD28 Abs, there was significant inhibition of ERK1/ERK2 phosphorylation (Fig. 3,e), but KLF2 was strongly expressed in these cells (Fig 3, c and d). In contrast, the D₄ DA receptor agonist PD 168,077 (1 μM) had no effect on ERK1/ERK2 phosphorylation in T cells not stimulated by anti-CD3/CD28 (Fig. 3,e). Similarly, when these cells were pretreated with a MEK1/2 inhibitor (U0126), KLF2 remained expressed in them even after treatment with anti-CD3/CD28 (Fig. 3,f), thereby indicating the involvement of the ERK1/ERK2 pathway in D₄ DA receptor-mediated regulation of KLF2 expression in T cells. In contrast, the inhibition of p38MAPK by its specific inhibitor (SB203580) (Fig. 3,f) or that of JNK activity (Fig. 3 f) by its specific inhibitor (SP600125) failed to show similar effects on KLF2 expression.

The above experiments were also repeated with another specific D₄ DA receptor agonist, ABT724 trihydrochloride, and similar results were observed (data not shown).

The dose responses (1 nM to 1 mM) of DA receptor agonists (D₁/D₅, D₂, and D₃) in inhibiting anti-CD3/CD28-induced T cell proliferation were investigated. It was observed that although the specified doses of the D₁/D₅ agonist SKF38393 (1 μM), the D₂ agonist quinpirole (2 μM), and the D₃ agonist, PD 128907 (5 μM) significantly inhibited anti-CD3/CD28-induced T cell proliferation with negligible cell death (data not shown), these doses did not induce KLF2 expression in anti-CD3/CD28-stimulated T cells (Fig. 4). Also, the inhibitory mechanisms of these DA receptors were different from those of the D₄ DA receptors (7, 8). The present results therefore indicate that the induction of KLF2 in T cells is specific for the D₄ class of DA receptors expressed in T cells.

Taken together, our results indicate for the first time that stimulation of the D₄ class of DA receptors in T cells is associated with its quiescent by expressing KLF2 via the ERK1/ERK2 pathway. Because quiescent plays a critical role in T cell survival (15, 16) and KLF2 is a dominant factor in regulating the quiescence state of the T cells (15, 16, 17, 18, 25, 26, 27), the present results demonstrate that the correlation between D₄ DA receptor activation and the expression of KLF2 in human T cells may be one of the regulatory mechanisms of neurotransmitter-mediated T cell homeostasis. Thus, investigating the molecular switches that regulate lymphocyte quiescence will enable us to gain a better understanding of cell cycle control in T lymphocytes. In addition, strategies for inducing quiescence by using specific D4 DA receptor agonists might be useful in the treatment of diseases in which uncontrolled T cell proliferation plays an important pathogenic role (15).

The authors have no financial conflict of interest.