Human T lymphocytes express N-methyl-d -aspartate receptors functionally active in controlling T cell activation

BBRC Biochemical and Biophysical Research Communications 338 (2005) 1875–1883 www.elsevier.com/locate/ybbrc

Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation Gianluca Miglio, Federica Varsaldi, Grazia Lombardi * DiSCAFF Department, Eastern Piedmont University, Via Bovio 6, 28100 Novara, Italy Received 18 October 2005 Available online 9 November 2005

Abstract The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 lg/ml) treatment. D-(
)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10 U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.
2005 Elsevier Inc. All rights reserved. Keywords: Glutamate receptors; NMDA receptors; NMDA receptor antagonists; Human lymphocytes; T cell proliferation; T cell activation; Cell cycle

L-Glutamate (Glu), the main excitatory neurotransmitter in the central nervous system (CNS), acts through two major types of receptors: ionotropic (iGlu) and metabotropic (mGlu) receptors [1]. iGlu receptors are ligand-gated ion channels, which, on the basis of their sequence homology and agonist preference, are classified into N-methyl-D-aspartate (NMDA), a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA), and kainate receptor types [2,3]. mGlu receptors are members of class-C G-protein coupled receptors [4]. They are classified into three groups (I, II, and III) and are linked to several effector systems [5]. NMDA receptors are heterotetramers that are made up of the NR1 subunit and, one or more of the NR2 subunits (NR2A–NR2D). They may also have NR3 subunits [2].

*

Corresponding author. Fax +39 0321 375 821. E-mail address: [email protected] (G. Lombardi).

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.164

The NR1 subunit is ubiquitously expressed in the CNS and provides a binding site for glycine [6], an essential co-agonist of the NMDA receptors. In contrast, the expression of the NR2 subunits is spatially and temporally regulated [7,8]. These subunits provide Glu binding sites [9] and are deputed to the control of channel properties (i.e., current kinetics and channel conductance) [2]. In rats, co-expression of NR1 with one or more NR2 subunits generates NMDA receptor subtypes with distinct functional and pharmacological properties [10–12]. For many years, Glu-signalling was thought to take place primarily in the CNS. However, recent data have demonstrated that it is also functional in the peripheral tissues in non-neuronal cells. The molecular machinery required for Glu-signalling (receptors, plasma membrane transporters, and vesicular transporters) is also expressed in non-neuronal cells, where it plays a role in maintaining cell functionality and integrity [13]. Therefore, Glu acts as both an excitatory neurotransmitter in the CNS and

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may be a more widespread extracellular messenger in the autocrine and/or paracrine system [13–15] than was previously thought. The early observations by Kostanyan et al. [16] on the immune system demonstrated the presence of specific high affinity Glu binding sites on the surface of human T lymphocytes. Several other groups have expanded on their results in studies on both rodent and human cells. In rodents, group-I and -II mGlu receptors are expressed in whole mouse thymus, isolated thymocytes, and the TC1S thymic stromal cell line [17]. Group-III mGlu receptors and NR1 subunits of NMDA iGlu receptors are present in freshly isolated lymphocytes [18]. In humans, GluR3 subunits of AMPA iGlu receptors are expressed in peripheral T cells, the Jurkat leukaemic T cell line, and a CD4+ alloprimed T helper clone [19]. Group-I mGlu receptors are present in resting and activated T lymphocytes, as well as in several lymphoid cell lines [20,21]. To our knowledge, there have been no published results demonstrating the expression of NMDA receptors in human T cells. The aims of our study were to demonstrate the expression of the NMDA receptors in human T cells, to determine the subunit composition of these receptors, and to clarify if there were any receptor composition changes during cell activation. Furthermore, we investigated whether the activation of these receptors can modify cell viability, proliferation, and the cell cycle.

Italy). All other reagents were of analytical grade and obtained from Merk (Darmstadt, Germany). Cell cultures. Heparinised peripheral blood (15–20 ml) was collected from healthy donors after informed consent. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation at 450g for 30 min at room temperature over a Ficoll–Paque PLUS gradient as described by Boyum [22]. After washing, cells were suspended in RPMI 1640 medium, supplemented with heat-inactivated FBS (10% v/v), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 lg/ml), and incubated on dishes for 1 h in a 37 C, humidified 95% air–5% CO2 incubator. Peripheral blood lymphocytes (PBL; non-adherent cells) were collected and maintained in supplemented RPMI 1640 medium in a 37 C, humidified 95% air–5% CO2 incubator. Cell viability, which was always greater than 98%, was evaluated at the end of cell isolation using the Trypan blue dye exclusion test. Activated PBL were obtained by treating freshly isolated PBL with PHA 10 lg/ml for 72 h. To obtain PHA blasts, PBL (1 · 106 cells/ml) were treated with PHA (1 lg/ml) and IL-2 (2 U/ml) for 7 days. Afterwards, cells were washed twice with PBS and seeded in round bottomed, 96-well plates to start proliferation assays [23]. Jurkat cells (clone E6-1) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and cultured in supplemented RPMI 1640 medium in a 37 C, humidified 95% air–5% CO2 incubator. RNA isolation and RT-PCR analysis. Total RNA was extracted from PBL or Jurkat cells by using the GeneElute Mammalian Total RNA Kit (Sigma–Aldrich). Human thalamic RNA was purchased from Clontech (Milan, Italy) and used as the positive internal control. Resulting RNA was reverse-transcribed by using the ThermoScript RT-PCR System (Invitrogen, Milan, Italy) and oligo(dT) primers for 1 h at 50 C. The reaction was terminated by incubating the mixture at 85 C for 5 min. The resulting cDNA was used as a template for PCR amplification. PCR amplification was performed in a 25 ll reaction mixture containing 2.0 lg cDNA, 2.5 ll of 10· buffer, 1.5 ll of 50 mM MgCl2, 0.5 ll of 10 mM dNTPs mix (Invitrogen), 2 U of EuroTaq DNA polymerase (EuroClone, Milan, Italy), and 2.5 ll of each primer (Table 1). PCR amplicons were resolved in a 2% agarose gel by electrophoresis and visualised with ethidium bromide. Signals were quantified using a densitometric analysis software (NIH Image 1.32; National Institutes of Health, Bethesda, MD, USA). Data are expressed as the ratio of the signal obtained for each gene in one sample divided by that obtained for the reference gene (GAPDH) in the same sample. Flow cytometric analysis of protein expression. Cell surface protein expression was evaluated by single-colour cytofluorimetric analysis. Indirect staining for the NR1 protein and direct staining for CD25 were used. Briefly, cells (1 · 106) were collected, washed twice with PBS, and resuspended in 50 ll of staining buffer (PBS supplemented with 1% BSA, 0.05%

Materials and methods Drugs and chemicals. Ficoll–Paque PLUS was obtained from Amersham Bioscience (Uppsala, Sweden). Penicillin, streptomycin, L-glutamine, bovine serum albumin (BSA), propidium iodide (PI), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), RNase, phytohemagglutinin (PHA), and Trypan blue dye were obtained from Sigma–Aldrich (Milan, Italy). D-(
)-2-Amino-5-phosphonopentanoic acid (D-AP5) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)MK 801, dizocilpine] were from Tocris Cookson (Bristol, UK). RPMI 1640 and foetal bovine serum (FBS) were purchased from Gibco (Milan,

Table 1 Oligonucleotides and PCR conditions used in this study PCR primers

Denaturation

Annealing

Extension

Cycles

NR1

F: 5 0 -GATGTCTTCCAAGTATGCGGA-3 0 R: 5 0 -GGGAATCTCCTTCTTGACCAG-3 0

Amplicon size (bp) 667

94 C, 30 s

58 C, 30 s

72 C, 30 s

35

NR2A

F: 5 0 -CCGGCCTGGGTTGCTCTTC-3 0 R: 5 0 -AGTTCGCTTTGGATTCTGTGCTCA-3 0

458

94 C, 10 s

66 C, 10 s

72 C, 60 s

35

NR2B

F: 5 0 -CTGCCGGACATCACCACCACAACA-3 0 R: 5 0 -CATCACGCGACCCACAGCCTTACC-3 0

442

94 C, 10 s

70 C, 10 s

72 C, 60 s

35

NR2C

F: 5 0 -GAACGGCATGATTGGGGAGGTGTA-3 0 R: 5 0 -CGTGTAGCTGGCGAGGAAGATGAC-3 0

460

94 C, 10 s

67 C, 10 s

72 C, 60 s

35

NR2D

F: 5 0 -CCGCCGTGTGGGTGATGATGTTCG-3 0 R: 5 0 -ACGCGGGGCTGGTTGTAG-3 0

457

94 C, 10 s

69 C, 10 s

72 C, 60 s

35

GAPDH

F: 5 0 -GGTCGGAGTCAACGGATTTGG-3 0 R: 5 0 -ACCACCCTGTTGCTGTAGCCA-3 0

1000

96 C, 30 s

60 C, 30 s

72 C, 45 s

25

G. Miglio et al. / Biochemical and Biophysical Research Communications 338 (2005) 1875–1883 NaN3). Goat IgG (200 lg/ml final concentration; Caltag Laboratories, An Der Grub, Austria) was added for 15 min at 4 C to prevent non-specific binding. Blocked cells were washed with staining buffer, incubated with an unconjugated anti-NR1 monoclonal antibody (mAb) (Chemicon International, Temecula, CA, USA), or with an FITC-conjugated anti-CD25 mAb (clone 3G10; Caltag Laboratories) for 1 h at 4 C. Cells labelled with anti-NR1 mAb were washed again with staining buffer and incubated with a phycoerythrin (PE)-conjugated goat anti-mouse IgG F(ab 0 )2 (Dacko Cytomation, Milan, Italy) for 30 min at 4 C in the dark. Finally, cells were washed and analysed. A modification of the above-described protocol was used for the simultaneous analysis of the surface and intracellular protein expression at single-cell level. Briefly, washed cells were fixed (fixation solution; eBioscience, San Diego, CA, USA) and permeabilised (permeabilisation solution; eBioscience) to allow mAb to intracellularly stain. Emitted fluorescence was detected on a FACSVantage flow cytometer (BD Bioscience, Milan, Italy) and analysed by using Cell Quest-PRO software (BD Bioscience). Cell proliferation assay. Freshly isolated PBL and PHA blasts were cultured in a 96-well plate in 0.1 ml in FBS (5% v/v) supplemented RPMI 1640 medium and incubated in a 37 C, humidified 95% air–5% CO2 incubator. All the substances used were added to the culture medium at the beginning of the experiments. Cell proliferation was determined by the MTT colorimetric method [24] after 72 h of culture. Absorbance was measured at 570–630 nm using an Ultramark microplate reader (Bio-Rad Laboratories, Milan, Italy). Percent inhibition of cell proliferation (inhibition %) was calculated as 100
[100 · (x
y)/(z
y)], where x, y, and z were the absorbance read in drug-treated, resting, and drug-untreated cells, respectively. Flow cytometric analysis of cell cycle. The amount of cells in the different phases of the cell cycle was measured by flow cytometry. Briefly, after culturing, cells were harvested and centrifuged. The pellets were then gently resuspended in 2 ml of ice-cold 70% ethanol and incubated for 16 h at
20 C. After incubation, the cells were washed twice with PBS, resuspended in PBS that contained RNase (final concentration, 0.5 mg/ ml), and incubated for 1 h at 37 C. Finally, PI (final concentration, 50 lg/ ml) was added. The PI fluorescence of individual nuclei was measured on a FACSVantage flow cytometer (BD Bioscience). Cell cycle analysis was performed by using ModFit LT 3.0 software (Verity Software House, Topsham, ME, USA). Data analysis. Results were expressed as means ± SE mean of n experiments Significance was assessed with StudentÕs t test for paired varieties with p 6 0.05 as the cut-off. Data were fitted as sigmoidal concentration–response curves and analysed with a four-parameter logistic equation. The IC50 values were determined with a non-linear regression

A

1

2

3

4

1877

model using the software, Origin 6.0 (Microcal Software, Northampton, MA, USA).

Results NMDA receptor subunit gene expression in human T cells To determine the expression of the NMDA receptor subunit genes in human T cells, we first carried out a RT-PCR analysis. In the first series of experiments, a PCR primer pair directed against the human NR1 subunit was used: products of appropriate size (667 bp) were detected in both Jurkat T cells and resting or PHA-activated PBL (Fig. 1A). Since functional NMDA receptors are heteromeric complexes containing NR1 and NR2 subunits [2], we then investigated whether NR2 receptor subunits are expressed in human T cells by using specific PCR primers directed against human NR2A–D. NR2B transcripts were detected in both Jurkat T cells and resting or PHA-activated PBL, whereas the messengers for NR2A and NR2D were detected only in PHA-activated PBL (Fig. 1A, lane 4). No NR2C transcripts were found in any of the cells tested. Products of the appropriate size were obtained with all primer pairs from human thalamic RNA (Fig. 1A, lane 1). The identity of the amplified fragments was confirmed by direct sequencing (data not shown). The intensity of the bands generated from T cell cDNA appeared weaker than that from the human brain. Thicker bands than in resting T cells were obtained in PHA-activated cells (Fig. 1A, lane 4). These results suggest that PHA-induced activation is able to up-regulate NMDA receptor subunit gene expression in T cells. NR1 subunit protein expression in human T cells To examine NR1 subunit protein expression, PBL were cultured in the absence or presence of PHA

B

5

80 70

NR2A

458 bp

60

NR2B

442 bp

NR2C

460 bp

NR2D

475 bp

GAPDH

1000 bp

density (A.U.)

NR1

667 bp

resting PHA-activated

50 40 30 20 10 0

NR1

NR2A NR2B NR2C NR2D

Fig. 1. Expression of NMDA receptor subunit genes in human T cells. Semiquantitative RT-PCR was performed on total RNA isolated from human thalamic nucleus (lane 1), Jurkat T cells (lane 2), resting PBL (lane 3) or activated PBL (lane 4) using specific primer pairs directed against NR1 or NR2 (A–D) subunits. In the negative control (lane 5) reverse transcriptase was omitted (A). The signals obtained from resting- or activated PBL were analysed densitometrically (B). Data, calculated as means ± SE mean of at least four determinations, are expressed as the ratio of the signal obtained for each sample divided by that obtained for GAPDH in the same sample to permit sample comparisons of RNA species. A.U. arbitrary units.

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(10 lg/ml, 0–72 h) and analysed by flow cytometry using a specific mAb against an extracellular epitope of the NR1 protein. When permeabilised cells were incubated with anti-NR1 mAb, a distinct population of NR1+ cells emerged (Fig. 2, upper panels). The percentage of NR1+ cells increased upon PHA treatment in a timedependent manner: from 24.6% to 89.4% at 0 and 72 h, respectively. Functional NMDA receptors in neurons are assembled in the endoplasmic reticulum from the essential NR1 subunit and different NR2 and/or NR3 subunits; thereafter the assembled receptors are targeted to the synaptic membranes [7,8]. For this reason, cell surface expression of the NR1 subunit protein reflects the expression of assembled NMDA receptors. To determine cell surface expression of the NR1 subunits, we performed experiments on nonpermeabilised PBL. The percentage of NR1+ cells was less than that obtained from the permeabilised cells but it increased upon PHA treatment in a time-dependent manner, from 5.6% to 50.0% at 0 and 72 h, respectively (Fig. 2, lower panels). These results clearly demonstrate that T cell activation increases NMDA receptor expression by mechanisms operating at different levels. NMDA receptor antagonists inhibit T cell proliferation To evaluate the contribution of NMDA receptor stimulation on T cell function, we studied the effect of cell exposure to specific NMDA receptor antagonists on T cell proliferation.

0h

The effects of (+)-MK 801, a non-competitive NMDA receptor antagonist, and D-AP5, a competitive NMDA receptor antagonist, were studied on T cell proliferation induced by 10 lg/ml PHA. Previous experiments at our laboratory have shown that this concentration is able to evoke maximal responses in our system [14]. (+)-MK 801 (1–500 lM) significantly (p<0.05; n = 4) inhibited PHA-induced T cell proliferation in a concentration-dependent manner (IC50 calculated was 56.5 lM). The maximum effect (98.9 ± 11.4% of inhibition) was measured at 200 lM of (+)-MK 801 (Fig. 3A). D-AP5 (10–5000 lM) was less potent and effective than (+)-MK 801 in inhibiting T cell proliferation. The IC50 calculated was 1.2 mM; the maximum effect (69.6 ± 7.6% of inhibition) was measured at 2 mM of D-AP5 (Fig. 3B). Microscopic examination of resting (Fig. 4A) and PHA-treated (Fig. 4B) cells confirmed that NMDA receptor antagonists inhibit T cell proliferation (Figs. 4C and D). Neither (+)-MK 801 nor D-AP5 modified cell viability in either resting or PHA-activated T cells (Trypan blue exclusion tests) (data not shown). The effects of NMDA receptor antagonists on IL-2 (10 U/ml)-induced T cell proliferation of PHA blasts were then studied. Results showed that neither (+)-MK 801 (1– 500 lM) nor D-AP5 (10–5000 lM) significantly inhibited cell proliferation of PHA blasts at any of the concentrations tested (Fig. 3B). These results suggest that NMDA receptor antagonists inhibit mitogen-induced proliferation of resting T cells but not that of the T blasts. This suggests some inhibitory effect on the early events of T cell activation.

24 h

48 h

47.9%

5.6%

28.9%

88.8%

89.4%

cell number

24.6%

72 h

36.9%

50.0%

NR1-PE Fig. 2. Time-course of the effect of PHA-induced T cell activation on NR1 expression. PBL (1 · 106 cells/ml) were activated with PHA (10 lg/ml) and analysed by flow cytometry (see Materials and methods). Upper panels represent the histograms obtained from permeabilised cells (whole cell expression); lower panels represent the histograms obtained from non-permeabilised cells (plasma membrane expression). An isotype-matched Ab was used in all experiments as control (thin open histograms), whereas overlay histograms (bold open histograms) illustrate the up-regulation of the NR1 subunit. Representative results from three independently performed experiments are shown.

G. Miglio et al. / Biochemical and Biophysical Research Communications 338 (2005) 1875–1883

**

(+)-MK 801 D-AP5

**

inhibition (%)

80

*

60

(+)-MK 801 D-AP5

100

** **

80

inhibition (%)

100

**

40 20 0

1879

60 40 20 0

1

10

100

1000

10000

1

10

100

1000

10000

[drug] (µM) Fig. 3. Concentration–response curves of NMDA receptor antagonists on T cell proliferation. PBL (1 · 106 cells/ml) were untreated or treated with PHA (10 lg/ml) in the absence or presence of increasing concentrations of (+)-MK 801 (1–500 lM) or D-AP5 (10–5000 lM) (A). PHA blast (0.2 · 106 cells/ml) were untreated or treated with IL-2 (10 U/ml) in the absence or presence of increasing concentrations of (+)-MK 801 (1–500 lM) or D-AP5 (10–5000 lM) (B). Cell proliferation was measured by MTT colorimetric assays after 72 h (see Materials and methods). The IC50 values for (+)-MK 801 and D-AP5 were 56.5 lM and 1.2 mM, respectively. Values are means ± SE means of at least five experiments run in triplicate. *p<0.05; **p<0.01, vs cell treated with PHA (A) or IL-2 (B) alone.

Fig. 4. Effects of NMDA receptor antagonists on PHA-induced cellular aggregate formation. PBL (1 · 106 cells) were untreated (A) or treated with PHA (10 lg/ml) in the absence (B) or presence of (+)-MK 801 (100 lM) (C) or D-AP5 (1 mM) (D). After 72 h, microscopic examination of cell culture was performed. Representative images from four independently performed experiments.

NMDA receptor antagonists inhibit the expression of the activation marker CD25 To confirm that T cell exposure to specific NMDA receptor antagonists affect cell activation, we analysed the

expression of CD25 (IL-2 receptor a chain), a plasma membrane marker of T cell activation. The expression of CD25 (flow cytometry using an FITC-conjugated anti-human CD25 mAb) was not present in resting PBL (mean fluorescence intensity = 2.81), whereas it was observed in PHA

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(10 lg/ml, 48 h)-treated PBL (mean fluorescence intensity; 68.85) (Figs. 5A and B). When the cells were treated with PHA in the presence of (+)-MK 801 (100 lM) (Fig. 5A) or D-AP5 (1 mM) (Fig. 5B), a reduction in PHA-induced CD25 expression was observed (mean fluorescence intensity; 8.67 and 13.95, respectively). These results confirm that NMDA receptor antagonists reduce cell responses to mitogens by inhibited T cell activation. Effects of NMDA receptor antagonists on cell cycle After stimulation with PHA, T lymphocytes enter the G1 phase in 2–4 h, the S phase after approximately 18–24 h, and reach the G2/M phase by 36–48 h [25]. We tried to determine if NMDA receptor antagonists arrest the cell cycle of T cells (flow cytometry under PI staining). Resting PBL existed almost exclusively in the G0/G1 phase (Fig. 6A). When the cells were induced to cycle with PHA (10 lg/ml; 72 h), fluorescence intensity increased from that of the G0/G1 phase to the S and G2/M phases (Fig. 6B). Similar results were obtained in cells activated with PHA in the presence of (+)-MK 801 (100 lM) (Fig. 6C) or D-AP5 (1 mM) (Fig. 6D). A computer software was then used to determine the percentage of cells in the sub-G1 (hypodiploid cells), G0/ G1, S, and G2/M phase in the presence or absence of NMDA receptor antagonists. No significant differences were observed in the percentage of cells in any of the cycle phases (Table 2). These results indicate that the inhibitory effects of NMDA receptor antagonists on T cell proliferation are not due to cell cycle arrest or cell death.

A

Discussion Results here reported demonstrate that human T cells express NMDA receptors that are similar to those expressed into the CNS. Pharmacological blockade of these receptors by specific receptor antagonists reduced T cell proliferation through an inhibition of the activation, but it did not affect cell cycle progression or death. The overall data confirm that Glu-signalling is also functionally active in human immune cells. From our data, the molecular composition of NMDA receptors, expressed on T cells, is dynamically regulated. In resting PBL and Jurkat T cells, these receptors are heterotetramers made up of NR1 and NR2B subunits, whereas they are also assembled with NR2A and NR2D subunits in activated T cells. Similar occurrences take place in the CNS at the synaptic level during physiological development [26,27], and as a consequence of neuronal activity [28]. At the level of the CNS, the synaptic composition of the NMDA receptor changes from initially containing NR2B to that which contains NR2A [26–28]. NR2B-containing NMDA receptors are expressed at the synaptic level in immature neurons, whereas they are mainly localised at the extra-synaptic site in mature ones [29,30]. NR2A-containing NMDA receptors are, on the contrary, expressed later during development [26,27]. In the CNS, Glu-signalling partially depends on the number and the subunit composition of the NMDA receptors in the plasma membrane, which are dynamically regulated by neuronal activity [2,7,8,28]. A wide-variety of mechanisms operating at different levels (transcriptional, translational, post-translational, and intracellular trafficking) assure a fine regulation of NMDA receptor subunit expression [7,8,28]. Our results suggest that similar regulations may occur also in

B

CD25-FITC

CD25-FITC

Fig. 5. Effects of NMDA receptor antagonists on PHA-induced CD25 expression. PBL (1 · 106 cells) were untreated (thin open histograms) or treated with PHA (10 lg/ml) in the absence (bold open histograms) or presence (filled grey histograms) of (+)-MK 801 (100 lM) (A) or D-AP5 (1 mM) (B). After 48 h, cells were stained with an FITC-conjugated anti-CD25 mAb and analysed by flow cytometry (see Materials and methods). Representative results from four independently performed experiments.

G. Miglio et al. / Biochemical and Biophysical Research Communications 338 (2005) 1875–1883

A

B

C

D

sub-G1

G0/G1

S

1881

G2/M

Fig. 6. Effects of NMDA receptor antagonist on cell cycle. PBL (1 · 106 cells) were untreated (A) or treated with PHA (10 lg/ml) in the absence (B) or presence of (+)-MK 801 (100 lM) (C) or D-AP5 (1 mM) (D). After 72 h, cells were stained with PI and DNA content of the cells was analysed by flow cytometry (see Materials and methods). The figure shows representative results from four independently performed experiments.

Table 2 Effects of NMDA receptor antagonists on cell cycle Condition

Cell (%) sub-G1

G0/G1

S

G2/M

Resting PHA (10 lg/ml) PHA (10 lg/ml) + (+)-MK 801 (100 lM) PHA (10 lg/ml) + D-AP5 (1 mM)

8.4 ± 2.2 9.7 ± 1.3 7.8 ± 1.9 8.0 ± 1.2

93.3 ± 2.5 63.8 ± 3.4 58.7 ± 2.1 56.3 ± 2.3

4.1 ± 0.7 31.1 ± 1.2 34.0 ± 1.2 37.4 ± 1.8

2.6 ± 0.8 5.1 ± 0.7 7.3 ± 1.1 6.3 ± 0.9

PBL (1 · 106 cells) were untreated or treated with PHA in the absence or presence of (+)-MK 801 or D-AP5 as indicated. After 72 h cells were collected for cell cycle analysis (see Materials and methods). Data are representative of three independently performed experiments.

the immune system. T cell activation changed the number and the subunit composition of the NMDA receptors expressed on the T cell surface. This may have been due to the up-regulation of the gene transcription and/or the expression of subunits in activated T cells (NR2A, NR2D) that were undetectable in resting T cells; and/or enhanced targeting of NMDA receptors to the cell membrane. Previous data from Boldyrev et al. have reported the expression of the NR1 subunit in resting rodent T cells [18]. Our results confirm these observations in resting and

activated human T lymphocytes, and add new evidences showing also the expression of NR2 subunits in these cells. Cell culture media, commonly used for T lymphocyte growth, usually contain micromolar concentrations of NMDA receptor agonists (e.g., Glu and L-aspartate) and L-glutamine, a substrate for the synthesis of Glu from cells during culture [31]. This should be taken into account when studying in vitro Glu-induced effects: the presence of NMDA receptor agonists in the culture media may tonically stimulate cells, cause Glu receptor desensitisation [2,3], and

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mask possible Glu-mediated effects. Moreover under these experimental conditions, it may be difficult to discriminate between the receptor-dependent and receptor-independent effects of Glu. To investigate possible functional effects of NMDA receptor activation, we exposed the cells to competitive and non-competitive NMDA receptor antagonists. Both (+)-MK 801, an NMDA channel blocker, and DAP5, a competitive Glu antagonist, inhibited PHA-induced proliferation of T cells: they did not affect IL-2-induced proliferation of PHA blasts. Flow cytometry analysis showed that NMDA receptor activation most probably does not influence cell cycle progression or death, since cell exposure to both receptor antagonists did not cause cell cycle arrest or death. However, optic microscopic evaluation suggested that NMDA receptor signalling may modulate the early phases of cell activation (the so-called Ôcommitment periodÕ). Cell surface analysis of CD25 expression, a marker of T cell activation, confirmed this hypothesis. Other non-competitive NMDA receptor antagonists (e.g., ketamine, phencyclidine, and amantadine) have been shown to determine inhibitory effects on T cell proliferation [32–36]. The mechanisms underlying these effects remain largely elusive and several explanations have been proposed at different sites (i.e., K+ channels, r receptors). Our findings suggest other new mechanisms showing that these drugs may antagonise the function of the NMDA receptors expressed on human T cells. In the CNS, NMDA ion channel receptors are inactive in the absence of sufficient membrane depolarisation to remove the Mg2+ block. Under depolarisation (i.e., by intense activation of co-localised post-synaptic AMPA receptors), the voltage-dependent Mg2+ block is partially relieved, allowing ion flux (mainly Na+ and Ca2+) through the activated receptors [2]. In the periphery, the membrane potential of resting T lymphocytes is around
60 mV, owing to the basal activity of K+ channels [37], so that NMDA receptors are mainly inactive (closed-state). However, during T cell activation, oscillations in the membrane potential have been observed [37,38]. These oscillations may cause cell membrane depolarisation. Launay et al. [39] have recently proposed that T cell activation causes the [Ca2+]i-dependent activation of TRPM4, non-selective cation channels, which leads to an increase in the membrane potential. Cell depolarisation allows for the activation of the NMDA receptors, leading to an intracellular Ca2+ concentration rise, significantly contributing to the Ca2+ signalling essential for T cell proliferation [40]. NMDA receptor antagonists by blocking these receptors prevent Ca2+ entry [2], and this action may translate into the interference with cell proliferation we showed. Moreover, Glu itself, which is physiologically present in the blood at a concentration range of 10–50 lM, may act at the level of the AMPA-type receptor [19] and may directly trigger membrane depolarisation. Another possibility is that Glu indirectly affects cell membrane depolarisation by increasing the number of Kv1.3 potassium channels in the inactivated-state. Kv1.3 potassium channels are the

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