Norepinephrine, dopamine and dexamethasone modulate discrete leukocyte subpopulations and cytokine profiles from human PBMC

Journal of Neuroimmunology 166 (2005) 144 – 157 www.elsevier.com/locate/jneuroim

Norepinephrine, dopamine and dexamethasone modulate discrete leukocyte subpopulations and cytokine profiles from human PBMC Karen C.L. Torres a, Lis R.V. Antonelli a, Adriano L.S. Souza b, Mauro M. Teixeira b, Walderez O. Dutra c, Kenneth J. Gollob a,* a

Laboratory of Lymphocyte Biology, Department of Biochemistry-Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Av. Antonio Carlos, 6627, C.P. 486, Belo Horizonte, MG 30161-970, Brazil b Laboratory of Immunopharmacology, Department of Biochemistry-Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil c Laboratory of Biology of Cell Interactions, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil Received 28 February 2005; accepted 6 June 2005

Abstract The interplay between the immune and neuroendocrine systems is intense, with the cross-talk between these two systems increasing during stress circumstances. Stress events culminate with hormonal pathway activation elevating the plasma levels of glucocorticoids and catecholamines. The majority of the works evaluating the effects of stress hormones on immune cells have utilized in vivo animal models or clinical studies. This work evaluates the effects of norepinephrine, dopamine, dexamethasone, and the combination of norepinephrine and dexamethasone on cellular activation and expression of immunoregulatory cytokines and chemokines by human PBMC in vitro. Norepinephrine and dopamine increased lymphocyte activation accompanied by augmented Th1 and Th2 type cytokine production. Dexamethasone reduced cell activation and decreased frequencies of cytokine producing cells and chemokine production. The action of norepinephrine together with dexamethasone resulted in immunosupression. The observed effects of hormones and neurotransmitters on leukocyte subsets likely underlie their immunomodulatory action in vivo. D 2005 Elsevier B.V. All rights reserved. Keywords: Norepinephrine; Dopamine; Dexamethasone; PBMC; Cytokines; T cell

1. Introduction The immune and neuroendocrine systems are intimately conjugated to maintain an organism’s homeostasis. This interplay is commonly associated with the pronounced effects of stress on immune function (Haddad et al., 2002) through the activation of the HPA axis (hypothalamic – pituitary – adrenal) and the SNS (Sympathetic Nervous System) (Webster et al., 2002). Physical, physiological or psychological stress induces changes in the hormonal pathway activation, releasing hormones and neurotransmit-

* Corresponding author. Tel./fax: +55 31 3499 2655. E-mail address: [email protected] (K.J. Gollob). 0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2005.06.006

ters that may alter immune functions in vivo and in vitro. Specifically, catecholamines and glucocorticosteroids are altered upon exposure to stress (Moynihan, 2003), and thus, are important in such immune alterations. One important class of neuroendocrine mediators for regulation of the human immune response is catecholamines (Roupe van der Voort et al., 2000). Norepinephrine (NE) is the main neurotransmitter of the SNS and has been shown to alter lymphoid functions (Sanders and Straub, 2002). The presence of adrenergic receptors, particularly of h-adrenergic receptors, on lymphoid cells has been demonstrated, and the stimulation of these receptors can affect the immune response (Kohm and Sanders, 2000). While different experimental systems have yielded contradictory results concerning NE, showing an enhancement or a suppression

K.C.L. Torres et al. / Journal of Neuroimmunology 166 (2005) 144 – 157

of the immune responses, most evidence points to a down modulation of pro-inflammatory/Th1 cytokines, and an increase in the Th2 type cytokine secretion profile (Kalinichenko et al., 1999; Elenkov et al., 2000; Kohm and Sanders, 2001). Since conflicting data exist concerning the effects of NE on immune cells, in vitro experiments with peripheral blood mononuclear cells (PBMC) from healthy human donors could, under controlled conditions, be used to simulate an in vivo stress situation, and might reflect the in vivo role of NE on molecules important for immunoregulation such as cytokines and chemokines. Given that dopamine (DA), a neurotransmitter of both the central and peripheral nervous systems, is a precursor of NE and lymphocytes express the h-hydroxylase, the enzyme that converts DA into NE (Musso et al., 1996), we sought to investigate the effects of DA on immune cells. The presence in leukocytes of DA receptors, a specific endogenous transport system, as well as an endogenous synthesis pathway of this catecholamine, all demonstrate DA’s functional significance in the immune system (Basu and Dasgupta, 2000). Moreover, DA can interact with immune cells via the h-adrenoreceptor (Hasko et al., 2002). Further studies have shown that DA enhances the production of IL-10 and suppresses the production of IL-12p40 (Hasko et al., 2002). Also, DA inhibits the release of IL-2, IL-4 and IFN-g from T cells by D2 and D3 receptors activation (Ghosh et al., 2003), and induces a down regulation of splenic IFN-g-producing cells (Carr et al., 2003) in animal models. Most of the literature concerning the effects of DA on immune cells were performed using murine models and point in general toward DA acting as a suppressor of pro-inflammatory cytokines. Thus, it is necessary to evaluate DA effects on immune cells of healthy humans to understand its effects on cell activation and cytokine production by specific cell subpopulations. Glucocorticoids are believed to be immunoregulatory (Galon et al., 2002). Some studies have shown that glucocorticoids presented in levels mimicking physiologic stress or supraphysiologic plasma levels, cause a shift in the type 1 (IFN-g, TNF-a)/type 2 (IL-4, IL-5, IL-13) cytokine balance produced by human PBMC toward a predominantly type 2 response (Agarwal and Marshall, 1998). Dexamethasone (DEX) is a synthetic glucocorticoid widely administered in human inflammatory pathologies (Galon et al., 2002), and it is known to affect the immune response at several stages including the suppression of various cytokines (Bessler et al., 1999). Dexamethasone binds to glucocorticoid receptor (GR) with high affinity and is potent in vivo (Ashwell et al., 2000; Webster et al., 2002). DEX enters in the cell by passive diffusion and binds to the GR which then translocates to the nucleus and binds to glucocorticoid response elements (GRE). Down regulation can occur via glucocorticoid element or via GRE interaction with other co-factors like AP-1, NF-nB or by stimulating de novo synthesis of the protein InBa that leads to the rapid turnover of InBa – NF-nB complexes (Ashwell et al., 2000;

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Webster et al., 2002). As glucocorticoids are the key players in stress responses, an in vitro approach with human immune cells treated with DEX is important to determine a detailed picture of the glucocorticoid effects on immune cell subpopulations to understand better how glucocorticoid affects immune functions. The aim of the current work was to study the effects of the main stress released hormones, glucocorticoids and catecholamines, to well characterize the actions of each one in vitro, and also determine their combined actions on cells of the immune system. Through the use of PBMC from healthy individuals we evaluate the individual actions of DEX, NE and DA, and also the combined effects of both NE and DEX, on the activation state, cytokine, and chemokine production by anti-CD3/CD28 stimulated cells. This analysis was performed using flow cytometry analysis of the frequency of cells producing key pro- or antiinflammatory cytokines, accompanied by the analysis of overall cytokine and chemokine production by PCR and/or ELISA. These studies point to distinct immunoregulatory effects of catecholamines and the glucocorticoid on lymphocyte activation, overall cytokine production, and on the frequency of cytokine producing cells providing a detailed analysis of human leukocyte subpopulations modulated by these important molecules of the stress response.

2. Material and methods 2.1. Selection of subjects Healthy volunteers (n = 9) of both sexes (mean age 26 T 3 years) who had not taken any medications at the time of blood collection donated whole blood via venous puncture. The blood was taken between 9:00 – 10:30 AM to control for diurnal variation. This project was approved by the Ethics Committee of the Universidade Federal de Minas Gerais-Brazil, number 310/03. 2.2. Cultures PBMC were obtained using a Ficoll/Hypaque (Sigma) gradient. Cells (2  105) were stimulated with anti-CD3 monoclonal antibodies (1 Ag/mL) (PharMingen-Becton Dickinson) and anti-CD28 monoclonal antibodies (0.5 Ag/ mL) (PharMingen-Becton Dickinson) in RPMI 1640 (Sigma) supplemented with 5% heat-inactivated human serum (Sigma), 1 mM of L-glutamine, antibiotics 200 U of penicillin (Sigma). The PBMC were cultured in the presence of media alone (control), 1 10 6 M of Dexamethasone phosphate (DEX—HypoFarma), or 1 10 7 M of Norepinephrine bitartarate (NE—HypoFarma), or 5  10 7 M of Dopamine chloridrate (DA—Ariston). The activity of endogenous glucocorticoids and catecholamines present in the 5% heat-inactivated human serum would be expected to be minimal due to the heat inactivation, and the control

K.C.L. Torres et al. / Journal of Neuroimmunology 166 (2005) 144 – 157

cultures would reflect any such possible activity. The dose of the reagents utilized in the experiments were previously determined through titrations and chosen to approximate the highest concentrations on in vivo stress situations or supraphysiologic levels. For DEX both physiologic levels mimicking stress situations, or supraphysiologic levels have similar effects on immune cells with respect to cytokine production (Franchimont et al., 2000; Agarwal and Marshall, 1998). These concentrations were tested and confirmed not to induce apoptosis or cell death at the dose used (data not shown). Cultures were harvested following 18 h of stimulation. 2.3. Flow cytometry Flow cytometric analysis was performed as described in Bottrel et al. (2001) with the following modifications. PBMC (2  105) stimulated in 200 AL of culture media in 96 well plates for 18 h in the presence of NE, DA or DEX were cultured with brefeldin A (1 AL/mL) (Sigma) during the last 4 h of culture to impair protein secretion by the Golgi complex. Cells were then stained with fluorescein isothiocyanate (FITC), phycoerytrin (PE)-labeled or biotinylated (Biot) antibody solutions for 20 min at 4 -C. Next, the preparations were washed and those stained with biotinylated antibodies, were incubated with streptavidin (1 : 100) conjugated with Cychrome (CY) for 20 min at 4 -C. Then, PBMC were washed with 0.1% sodium azide PBS (Sigma), and fixed with 2% formaldehyde in PBS. The antibodies used for extracellular staining were anti-CD8-FITC (Pharmingen), anti-CD14-FITC, anti-CD69-FITC, anti-CD62L-FITC (Cal-

DA 1.0

50.7

36.3

43.9

CD8

CD4

DEX 21.0

11.7

44.7

CD8

0.1

57.3

DA 16.1

% of CD69 expression

37.8

% of CD69 expression

11.7

0.1

CD4

NE 6.2

DEX . 1.0

1.6

CD4

Control % of CD69 expression

RNA extraction was done with TRI reagent (Sigma). The RNA samples were subjected to first-strand cDNA synthesis using MMLV reverse transcriptase (200 U/AL) (Amershan) primed with oligo dT12 – 18 (Amershan). PCR primers were

% of IFN-γ expression

1.7

CD4

CD8

2.4. RT-PCR analysis

% of IFN-γ expression

54.6

7.2

2.3.1. Analysis of FACS data Leucocytes were analyzed for their frequencies of surface markers and intracellular cytokine expression using the program Cell Quest (Becton&Dickinson) as shown in Fig. 1. The frequency of positive cells was analyzed in two regions for each staining: lymphocyte gate and the monocyte/ macrophage gate. Limits for the quadrant markers were always set based on negative populations and isotype controls.

NE 0.4

% of IFN-γ expression

% of IFN-γ expression

Control 0.5

tag), anti-CD5-FITC (Immunotech), anti-CD25-PE (Caltag), anti-CD19-PE (Caltag), anti-CD8-Biot-SA-CY (Pharmingen), and anti-CD4-Biot-SA-CY (Caltag). After extracellular staining, the cells were permeabilized with a solution of 0.5% saponin and stained for cytoplasmic cytokines, for 30 min at room temperature, using anti-cytokine monoclonal antibodies directly conjugated with PE (Caltag). For single-cell cytoplasmatic cytokine staining the monoclonal antibodies used were anti-IL-6, anti-IL-10, anti-IFN-g and anti-TNFa.FITC and PE-labeled immunoglobulin isotype control antibodies were included in all experiments. The stained cells were acquired and analyzed using a FACSVANTAGE or FACScan (Becton&Dickinson) and the CellQuest software program.

% of CD69 expression

146

4.4

10.7

24.0

CD8

Fig. 1. Representative contour-plots from PBMC. Shown is the frequency of IFN-g producing CD4+ T cells or the frequency of CD69 expressing CD8+ T cells after 18 h of culture with anti-CD3/CD28 in media alone (control) or with, NE, DA or DEX. The contour-plots demonstrate the frequencies of cells expressing the indicated molecules as detected using antibodies conjugated with either PE or FITC or SA-CY as described in Materials and methods.

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intron-spanning and included: HPRT (hypoxanthine-guanine phosphoribosyl transferase) sense 5V-CCT GCT GGA TTA CAT CAA AGC ACT G-3V, anti-sense 5V-TCC TGG GGT GCT TCA CAA CCT-3V; IL-2 sense 5V-ACT CAC

147

CAG GAT GCT CAC AT-3V, anti-sense 5V-AGA CTT GTC TAC CTA ATG GA-3V; IL-4 sense 5V-TCC ACG GAC ACA AGT GCG ATA TCA CCT-3V, anti-sense 5V-GAC GTG TCG TCA AGG TGT CCG TGT TCG-3V; IL-5 sense 5V-

% expression of CD62L

% expression of CD69

90 80 70 60 50 40

85 80 75 70 65 60 55 50

control

NE

control

total lymphocyte

A

NE

in CD4

CONTROL

NE in CD8

B

% expression of TNF-α

% expression of IFN-γ

6 5 4 3 2 1 0 NE

control

total lymphocyte

NE

control

in CD8

0,5

control

NE

control

total lymphocyte

in CD4

D

CD8

NE

control NE in CD4

1

% expression of IL-10

% expression of IL-6

E

1

N

4

3

2

1

0

1,5

0

control

C

2

control

NE

total lymphocyte

control in CD4

0,8 0,6 0,4 0,2 0

NE

F

control NE total lymphocyte

control

NE

in CD4

Fig. 2. Norepinephrine induces an increase in the frequency of activated cells and cytokine producing cell subpopulations. PBMC were stimulated with antiCD3/CD28 in the presence of NE 1 10 7 M. After 18 h in culture the cells were stained with anti-surface markers and anti-cytokines mAbs and analyzed by flow cytometry. NE increased the frequency of CD69 expressing lymphocytes and CD4+ T cells expressing CD69 (A) and decreased the expression of CD62L in CD8+ T cells (B). The expression of pro-inflammatory cytokines IFN-g (C) and TNF-a (D) were increased by NE in total lymphocytes, in CD4+ and CD8+ T cells; also IL-6 (E) in lymphocytes and in CD4+ T cells. Moreover, NE increased the expression of the immunomodulatory cytokine, IL-10, in lymphocytes and in CD4+ T cells (F). Values, analyzed by paired T test when data fit a normal distribution or Wilcoxon test when results did not fit a normal distribution, are significantly different from control. P-values are less than 0.05 in all presented graphs. N = 9.

148

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Table 1 Norepinephrine induces an increased message expression for interleukins in activated PBMC

IL-2b IL-4b IL-5b IL-13b IL-8b MIP-1ab MCP-1b

Control (mean T S.D.)a

NE (mean T S.D.)a

P-value

Fold change

32.87 T 20.16c 43.53 T 30.41c 33.78 T 7.07c 30.92 T 16.96c 33.85 T 18.71c 71.96 T 47.47c 29.4 T 27.20c

61.77 T 29.70c 80.70 T 50.02c 93.30 T 41.57c 49.20 T 19.40c 61.58 T 16.98c 70.53 T 25.65c 17.89 T 25.10c

0.05 <0.01 <0.01 <0.01 <0.01 N.S. N.S.

1.90 1.80 2.70 1.60 1.81

N.S. — difference not significant ( P-value > 0.05). a Arbitrary units of mRNA as determined using semi-quantitative RTPCR as described in Materials and methods. b Cytokine/chemokine message expression was determined at 18 h following stimulation with anti-CD3/CD28 as described in Materials and methods. c The values represent the mean T S.D. for 9 individuals.

CTT GGA GCT GCC TAC GTG TAT GC-3V, anti-sense 5VCCA CAT TAC TTG TGG CTC ACC-3V; IL-8/CXCL-8 sense 5V-AAG CTG GCC GTG GCT CTC TTG-3V, antisense 5V-AGC CCT CTT CAA AAA CTT CTC-3V; IL-13 sense 5V-CCC AGA ACC AGA AGG CTC CGC TCT G-3V, anti-sense 5V-AAG CGC TCC CTG CCA AGT TG-3V; MIP1a/CCL3 sense 5V-CGC CTG CTG CTT CAG CTA CAC CTC CCG GCA GA-3V, anti-sense 5V-TGG ACC CCT CAG GCA CTC AGC TCC AGG TCG CT-3V; MCP-1/CCL2 sense 5V-AGG AAG ATC TCA GTG-3V, anti-sense 5V-AGT CTT CGG AGT TTG CCT TTG-3V. Amplification was performed for 35 cycles, according to Gollob et al. (1996). An initial denaturation of 2 min at 92 -C was performed. PCR cycles consisted of 40 s at 94 -C, annealing temperature for HPRT, for IL-4, IL-5, IL-13 was 60 -C, for IL-2 55 -C, for IL-8 and MCP-1/CCL-2 was 65 -C and MIP-1a/CCL-3 was 75 -C followed by an extension for 40 s at 72 -C. A final extension of 10 min at 72 -C was included. PCR products were visualized in 1% agarose gels with ethidium bromide, and digitalized using the Nucleo Vision image system. The image analysis was performed using the Gel Expert software (NucleoTech). HPRT was used to normalize the quantity of cDNA reverse-transcribed. For each sample a correction factor was used based on HPRT intensity (Torres et al., 2004). Results are expressed as arbitrary units based on normalized band intensity. Positive controls were performed using PBMC stimulated with SEB (staphylococcal enterotoxin B) in culture for 24 h. 2.5. Enzyme-linked immunosorbent assay (ELISA) After 18 h of PBMC cultures activation by anti-CD3/ CD28, in the presence of NE, DA or DEX, supernatants were collected and stored at 20 -C. IFN-g, MCP-1/ CCL2, eotaxin-1/CCL-11 and RANTES/CCL5 levels were quantified using the ELISA sandwich technique, utilizing commercially available antibodies, according to the protocol provided by the supplier (duo-set R&D systems); MIG/

CXCL-9 and IP-10/CXCL-10 (Pharmingen) and MIP-1a/ CCL-3 matched antibody pairs (Pharmingen). The reactions were read using an ELISA reader E-max (Molecular Devices) at 492 nm. The analysis was done with the software Softmaxpro. The concentrations of cytokines and chemokines in the supernatants were determined based on a seven-point standard curve multiplied by the samples’ dilution factor. 2.6. Statistics Statistical analysis were performed using the software JMP from SAS. The paired T test was used for comparison between the control and each specific condition for data with normal distribution. For results not respecting a normal distribution the Wilcoxon test was used for comparison between the control and each specific condition. In all cases the P-value less than 0.05 was taken to be significant.

3. Results 3.1. Norepinephrine increases cellular activation and induces an up modulation of pro- and anti-inflammatory cytokine expression, as well as the frequency of cytokine producing cells To test the effects of NE on cell subpopulation activation and function, the frequency of the recent activation markers CD25 and CD69 expression, as well as CD62L, was evaluated by flow cytometry (Fig. 2). An increase in the total frequency of CD69 expressing lymphocytes, as well as the commitment of CD4+ T cells to the expression of CD69 (Fig. 2A) were observed. The frequency of CD25 expression by lymphocytes did not differ from control cultures (data not shown). A decreased frequency of CD62L expression in CD8+ T cells was seen following culture with NE as compared to control cultures (Fig. 2B). Table 2 Norepinephrine induces an increased production of IFN-g and a decreased production of chemokines in activated PBMC

IFN-gb MIP-1ab MCP-1b MIGb RANTESb IP-10b Eotaxin-1b

Control (mean T S.D.)a

NE (mean T S.D.)a

P-value

Fold change

4.16 T 1.36c 16.53 T 3.80c 6.66 T 0.63c 16.16 T 11.09c 3.68 T 0.81c 15.62 T 13.93c 0.05 T 0.02c

6.70 T 1.10c 18.97 T 4.87c 6.38 T 0.81c 12.44 T 9.20c 3.78 T 0.55c 13.09 T 1.35c 0.03 T 0.01c

<0.01 N.S. N.S. <0.01 N.S. N.S. 0.04

1.60

0.76

0.60

N.S. — difference not significant ( P-value > 0.05). a Nanogram per milliliter as determined using ELISA as described in Materials and methods. b Cytokine/chemokine production was determined at 18 h following stimulation with anti-CD3/CD28 as described in Materials and methods. c The values represent the mean T S.D. for 9 people.

K.C.L. Torres et al. / Journal of Neuroimmunology 166 (2005) 144 – 157

To determine which cytokine producing cells were affected by treatment with NE individual cell subpopulations were analyzed in conjunction with specific pro-inflammatory

149

cytokines (IFN-g, TNF-a and IL-6) and the immunomodulatory cytokine, IL-10. This catecholamine induced an increase in the frequency of the IFN-g expressing lympho-

40

% expression of CD69

% expression of CD5 +

2,5

2

1,5

1

35 30 25

0,5

20 CONTROL control

DA DA

CONTROL control

CD19 + GROUPS EXPERIMENTAL

A

DA DA

CD4 EXPERIMENTAL GROUPS

B

8

IFN-G

3

% expression of TNF-α

% expression of IFN-γ

10

6 4 2

2

1

0

0 controlO DA control DA CONTROL control DA DA O DA CONTR DA CONTR total EX ROUPS lymphocyte

C

in CD8 EXP ROUPS

in CD4 ROUPS EXPE

D

control DA CONTR control CONTR O DA O total EXP ROUPS lymphocyte

DA CONTR control DADA DA

in CD8 EXP ROUPS

in CD4 EX OUPS

1,5

% expression of IL-10

% expression of IL-6

5 4 3 2 1

1

0,5

0

E

control DA control DA DA CONTROL CONTROL total in CD4 ROUPS GROUPS EXPER EXPER lymphocyte

0

F

control DA CONTROL DA total GROUPS EXPE lymphocyte

control CONTROL

DA DA

in CD4 EXPEROUPS

Fig. 3. Dopamine induces an enhancement of cell activation, and an increase of the frequency of cytokine producing lymphocytes. PBMC were stimulated with anti-CD3/CD28 in the presence of DA 5  10 7 M. The cells were stained with anti-surface markers and anti-cytokines mAbs, after 18 h in culture, and analyzed by flow cytometry. DA increased the expression of CD5+D19+ cells (A), and the frequency of CD4+ positive to the CD69 expression (B). The frequency of pro-inflammatory cytokines IFN-g (C) and TNF-a (D) expression were increased by DA in total lymphocytes, in CD4+ and in CD8+ T cells; also frequency of IL-6 was increased (E) in total lymphocytes and in CD4+ T cells. Moreover, DA increased the expression of the immunomodulatory cytokine, IL10, in lymphocytes and in CD4+ T cells (F). Values, analyzed by paired T test when data fit a normal distribution or Wilcoxon test when results did not fit a normal distribution, are significantly different from control. P-values are less than 0.05 in all presented graphs. N = 9.

150

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Table 3 Dopamine induces an increased message expression for interleukins and for MCP-1 in activated PBMC

IL-2b IL-4b IL-5b IL-13b IL-8b MIP-1ab MCP-1b

Control (mean T S.D.)a

DA (mean T S.D.)a

P-value

32.87 T 20.16c 43.53 T 30.41c 33.78 T 7.07c 30.92 T 16.96c 33.85 T 18.71c 71.96 T 47.47c 32.60 T 26.70c

36.58 T 27.23c 77.08 T 34.50c 81.23 T 18.97c 40.38 T 23.62c 90.87 T 45.70c 88.65 T 57.49c 59.20 T 39.07c

N.S. <0.01 <0.01 N.S. <0.01 N.S. 0.04

Table 4 Dopamine induces an increased production of IFN-g and IP-10 and a decreased production of other several chemokines in activated PBMC

Fold change 1.77 2.40 2.68 1.81

N.S. — difference not significant ( P-value > 0.05). a Arbitrary units of mRNA as determined using semi-quantitative RTPCR as described in Materials and methods. b Cytokine/chemokine message expression was determined at 18 h following stimulation with anti-CD3/CD28 as described in Materials and methods. c The values represent the mean T S.D. for 9 people.

cytes that was reflected by an increase in both IFN-g expressing CD8+ and CD4+ T cells (Fig. 2C). The same pattern of enhanced frequencies was detected when TNFa (Fig. 2D) and IL-6 producing lymphocytes were analyzed (Fig. 2E). In addition to NE enhancing the frequency of proinflammatory cytokine producing cells, this catecholamine also enhanced the frequency of IL-10 producing lymphocytes reflected within CD4+ T cells (Fig. 2F). The effects of NE on the message expression of cytokines and chemokines were also evaluated by RTPCR. NE showed a capacity to increase mRNA expression of activated PBMC for Th1 interleukin, IL-2, approximately by two fold compared to control (Table 1). The message expression of the Th2 related cytokines IL-4, IL-5 and IL-13 was also enhanced by NE compared to control (Table 1). Lastly, IL-8/CXCL-8 mRNA expression was increased by almost two fold (Table 1). The level of IFN-g, detected in the supernatant of PBMC activated by NE was also increased (Table 2). Interestingly, the production of the chemokines MIG/CXCL-9 and eotaxin1/CCL-11 was decreased by NE, as compared to the control (Table 2). 3.2. Dopamine, like norepinephrine, increases the frequency of cytokine producing cells, pro- and anti-inflammatory cytokine expression, and cellular activation DA’s effects on cell subpopulations were investigated. DA induced an increase of CD19+ B cells frequency

IFN-gb MIP-1ab MCP-1b MIGb RANTESb IP-10b Eotaxin-1b

Control (mean T S.D.)a

DA (mean T S.D.)a

P-value

Fold change

5.89 T 4.40c 16.53 T 3.80c 6.66 T 0.63c 16.16 T 11.10c 3.68 T 0.81c 15.62 T 13.93c 0.05 T 0.02c

8.54 T 4.40c 15.12 T 2.87c 6.06 T 1.06c 11.25 T 8.81c 3.76 T 0.47c 12.67 T 11.61c 0.03 T 0.01c

<0.01 N.S. 0.03 <0.01 N.S. 0.02 0.03

1.45 0.90 0.70 0.81 0.60

N.S. — difference not significant ( P-value > 0.05). a Nanogram per milliliter as determined using ELISA as described in Materials and methods. b Cytokine/chemokine production was determined at 18 h following stimulation with anti-CD3/CD28 as described in Materials and methods. c The values represent the mean T S.D. for 9 people.

positive for CD5 expression (Fig. 3A). Moreover, DA enhanced the frequency of CD4+ T cells positive for CD69 (Fig. 3B). However, the frequency of CD25 and CD62L expressing lymphocytes, cultured in the presence of DA, did not differ from control culture cells (data not shown). The effect of DA on the frequency of cytokine expressing cells was evaluated for IFN-g, TNF-a, IL-6, and IL-10 expression in individual cell subpopulations. DA induced a 100% increase in IFN-g producing lymphocytes that was reflected by a parallel increase in both CD8+ and CD4+ T cells producing IFN-g (Fig. 3C). The same pattern of increases was detected for the frequencies of TNF-a and of IL-6 producing cells (Fig. 3D and E). Moreover DA induced an increase in the frequency of IL-10 producing lymphocytes that was reflected within the CD4+ T cell population (Fig. 3F). The effects of DA on cytokine and chemokine message expression were also evaluated. This catecholamine induced an increased mRNA expression of activated PBMC for Th2 interleukins, IL-4 and IL-5 compared to control (Table 3). In addition, DA was able to increase the mRNA expression for IL-8/CXCL-8 and MCP-1/CCL-2 compared to control (Table 3). DA also induced an increased IFN-g secretion by activated PBMC and interestingly a decreased production of MIG/CXCL-9, IP-10/CXCL-10 and eotaxin-1/CCL-11 (Table 4). Despite DA increased mRNA expression for MCP-1/CCL-2 (Table 3), the production of this chemokine was decreased by DA (Table 4), bringing the possibility of protein consumption by the cells in culture or increased

Fig. 4. Dexamethasone induces an inhibition of the cell activation, and frequency of cytokine producing cells in lymphocytes and monocytes subpopulations. PBMC were stimulated with anti-CD3/CD28 in the presence of DEX 1 10 6 M. After 18 h in culture the cells were stained with antisurface markers and anti-cytokines mAbs and analyzed by flow cytometry. DEX decreased the expression of CD5 in CD19 cell subpopulation (A) and increased the expression of CD14 in monocytes (B). The expression of CD25 (C) and CD69 (D) was decreased in total lymphocytes, in CD4+ and in CD8+ T cells. DEX also decreased the frequency of IFN-g producing lymphocytes and within CD4+ and CD8+ T cells (E), and TNF-a in CD4+ T cells (F). The frequency of the IL-10 expression was decreased in CD14 cells, while total monocytes increased the frequency of IL-10 expression (G). Values, analyzed by paired T test when data fit a normal distribution or Wilcoxon test when results did not fit a normal distribution, are significantly different from control. P-values are less than 0.05 in all presented graphs. N = 9.

K.C.L. Torres et al. / Journal of Neuroimmunology 166 (2005) 144 – 157

90

% expression of CD14

% expression of CD5

40 35 30 25 20 15 10

80 70 60 50 40 30 20

5

10 control

DEX

control

in CD19

A

% expression of CD69

50 40 30 20 10

% expression of IFN-γ

control DEX control DEX ONTR control DEX ONTRO DEX ONTRO LDEX O DEX total in CD8 in CD4 ROUPS EX ROUPS EXP ROUPS EX lymphocyte

3

2

1

0

in CD8 EXP OUPS

in CD4 EXPEOUPS

% expression of IL-10

8 7 6 5 4 3 2 1

G

60 50 40 30

control DEX CONTROL monocyte GROUPS E EXP

control DEX DEX CONTROL in CD14 EXPERR

control DEX CONTR control control DEX ONTRO L DEX O DEX DEX ONTRO L DEX total in CD8 in CD4 EXP ROUPS EX ROUPS EXP ROUPS lymphocyte

D

2 1,5 1 0,5 0

control control DEX CONTROL control DEX CONTR ODEX DEXCONTR O DEX DEX total EXP OUPS lymphocyte

E

70

% expression of TNF-α

% expression of CD25

80

60

C

DEX monocyte

B

70

0

151

control

F

DEX in CD4

152

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receptor expression and chemokine uptake through endocytosis. 3.3. Dexamethasone treatment of PBMC reduced cellular activation, and induced a down modulation of Th1 and Th2 cytokine profiles through an inhibition of the frequency of cytokine producing lymphocytes, as well as a decrease in cytokine production To determine the effects of DEX on lymphocytes and monocytes, the percentage of cell subpopulations was determined by flow cytometry. The only effect on the overall frequency of cell subpopulations was seen in a decrease of 17% of CD5 expression in CD19+ B cells (Fig. 4A) and an increase of 22% of CD14 + monocytes expression (Fig. 4B). The effects of DEX, on leukocyte activation, was detected in a decreased frequency of CD25 expressing lymphocytes. This decrease was seen both within the CD8+ T cells and CD4+ T cells (Fig. 4C). Moreover DEX induced a decrease in the frequency of CD69 expressing total lymphocytes, both within the CD8+ and CD4+ T cell subpopulations (Fig. 4D). The frequency of CD62L expression in lymphocytes did not differ from control culture (data not shown). The expression of cytokines by cells cultured in the presence of DEX was evaluated. A decrease in the frequency of total IFN-g producing lymphocytes was observed (Fig. 4E), which was reflected by a decrease in both IFN-g producing CD8+ and CD4+ T cells (Fig. 4E). DEX decreased by twice the frequency of CD4+ T cells expressing TNF-a as compared to control (Fig. 4F). Finally, DEX also decreased the frequency of IL-10 expression in CD14- monocytes by 17% (Fig. 4G); however, the frequency of IL-10 expression in total monocytes was increased by 23%. Again, DEX treated PBMC cultures displayed a significant decrease in the message for the Th1 cytokine, IL-2, and Table 5 Dexamethasone induces a decreased message expression for cytokines and for chemokines in activated PBMC

IL-2b IL-4b IL-5b IL-13b IL-8b MIP-1ab MCP-1b

Control (mean T S.D.)a

Dex (mean T S.D.)a

P-value

Fold change

32.87 T 20.16c 43.53 T 30.41c 33.78 T 7.07c 30.92 T 16.96c 33.85 T 18.71c 71.96 T 47.47c 29.40 T 27.2c

20.28 T 8.28c 30.49 T 13.42c 3.72 T 6.67c 2.20 T 5.38c 15.07 T 13.79c 24.17 T 16.01c 23.90 T 17.80c

0.02 0.01 <0.01 0.01 0.01 0.02 N.S.

0.61 0.70 0.11 0.07 0.44 0.33

N.S. — difference not significant ( P-value > 0.05). a Arbitrary units of mRNA as determined using semi-quantitative RTPCR as described in Materials and methods. b Cytokine/chemokine message expression was determined at 18 h following stimulation with anti-CD3/CD28 as described in Materials and methods. c The values represent the mean T S.D. for 9 people.

Table 6 Dexamethasone induces a decreased production of IFN-g and chemokines in activated PBMC

IFN-gb MIP-1ab MCP-1b MIGb RANTESb IP-10b Eotaxin-1b

Control (mean T S.D.)a

Dex (mean T S.D.)a

P-value

Fold change

5.89 T 4.40c 16.53 T 3.80c 6.66 T 0.63c 16.16 T 11.10c 3.68 T 0.81c 15.62 T 13.93c 0.05 T 0.02c

1.26 T 1.16c 9.01 T 5.09c 5.08 T 1.06c 11.38 T 6.51c 3.35 T 0.63c 20.13 T 17.80c 0.04 T 0.01c

0.01 <0.01 0.01 0.04 <0.01 0.01 N.S.

0.21 0.54 0.76 0.70 0.91 1.21

N.S. — difference not significant ( P-value > 0.05). a Nanogram per milliliter as determined using ELISA as described in Materials and methods. b Cytokine/chemokine production was determined at 18 hrs following stimulation with anti-CD3/CD28 as described in Materials and methods. c The values represent the mean T S.D. for 9 people.

Th2 cytokines IL-4, IL-5 and IL-13 (Table 5). DEX also decreased the mRNA expression for MIP-1a/CCL-3 and IL8/CXCL-8 (Table 5). DEX reduced significantly the production of IFN-g by activated PBMC by almost 80% as compared to control (Table 6). DEX also diminished the chemokine producing activated PBMC for: MIP-1a/CCL-3; MCP-1/CCL-2, MIG/CXCL-9; and RANTES/CCL5 (Table 6). DEX, however, increased the PBMC production of IP-10/ CXCL-10. 3.4. Dexamethasone plus norepinephrine treatment of PBMC was immunosuppressive as demonstrated by the treatment of PBMC with just DEX In vivo, the most important hormones released during acute stress are both glucocorticoids and catecholamines (Moynihan, 2003). Then, PBMC were cultured simultaneously in the presence of NE and DEX to investigate their effects together on immune cells. The state of action of lymphocytes was detected through the analysis of CD25 and CD69 expression. A decrease in the total frequency of CD25 expressing lymphocytes, reflected through the commitment of CD8+ and CD4+ T cells to the expression of CD25 (Fig. 5A) was observed. Also, the frequency of CD69 expression by lymphocytes was decreased, reflected by a diminished frequency of CD69 expressing CD4+ T cells (Fig. 5B). The activities of NE in conjunction with DEX, related to the cytokines expression, in activated PBMC were detected by a decrease in the frequency of the IFN-g expressing lymphocytes that was reflected by a decrease in both IFN-g expressing CD8+ and CD4+ T cells (Fig. 5C). Also, decreased frequencies of total lymphocytes and within the CD4+ T cells expressing TNF-a were detected (Fig. 5D). In addition, NE plus DEX diminished the frequency of IL-10 producing lymphocytes (Fig. 5E).

K.C.L. Torres et al. / Journal of Neuroimmunology 166 (2005) 144 – 157

153

% expression of CD69

% expression of CD25

75 60 45 30 15 0

EX inROUPS CD8

EX ROUPS in CD4

20

control DEX control DEX CONTROLDEX + NE CONTROLDEX + NE + NE + NE total EXPE GROUPS R lymphocytes

EXPE R inGROUPS CD4

2

% expression of TNF-α

% expression of IFN-γ % expressionof IL-10

40

B

0,8 0,6 0,4 0,2 0 control DEX control DEX control DEX ONTR DEX NECONTRDEX O + NE + NE CONTRO DEX + NE + NE ++ NE total EX OUPS EXP EXP inOUPS CD8 inROUPS CD4 lymphocytes

C

60

0

control DEX control DEX control DEX CONTRO DEX L+ NE +NCONTRDEX O + NE + NE CONTRDEX O + NE +N total EXP GROUPS lymphocytes

A

80

1,5

1

0,5

0

D

control DEX control DEX CONTROLDEX + NE CONTROL + NE + NE total ROUPS EXPE lymphocytes

LinGROUPS R EXPE CD4

0,9

0,6

0,3 control CONTROL

E

DEX+ NE DEX + NE total lymphocytesGROUPS EXPERIMENTAL

Fig. 5. Norepinephrine plus dexamethasone induce an inhibition of the cell activation, and in Th1 and Th2 type cytokine expression and chemokine production. PBMC were stimulated with anti-CD3/CD28 in the presence of DEX 1 10 6 and NE 1 10 7 M. After 18 h in culture the cells were stained with antisurface markers and anti-cytokines mAbs and analyzed by flow cytometry. NE plus DEX decreased the expression of CD25 (A) and CD69 (B) in total lymphocytes, in CD4+ and in CD8+ T cells. NE plus DEX also decreased the frequency of IFN-g producing lymphocytes (C), and within CD4+ and CD8+ T cells, and TNF-a in total lymphocytes and in CD4+ T cells (D). The frequency of the IL-10 expression were decreased in lymphocytes (E) by treatment with NE plus DEX. Values, analyzed by paired T test when data fit a normal distribution or Wilcoxon test when results did not fit a normal distribution, are significantly different from control. P-values are less than 0.05 in all presented graphs. N = 11.

The message expression for Th1 cytokine IL-2, as of Th2 cytokines were decreased, for IL-4, IL-5 and IL-13 in PBMC treated with both NE plus DEX compared to control cultures (Table 7). Also, NE plus DEX decreased the chemokine production of PBMC for MIP-1a/CCL3,

MCP-1/CCL2, MIG/CXCL-9, RANTES/CCL5 and IP-10/ CXCL-10 compared to control (Table 8). These results show that the immunosuppressive effects of NE plus DEX are similar to the effects of DEX alone in PBMC cultures stimulated with anti-CD3/CD28.

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4. Discussion Stress situations activate the HPA axis and the SNS resulting in elevated plasma glucocorticoid and catecholamine levels and inducing profound effects on various immune functions. The majority of studies performed related to the effects of stress on the immune response have used clinical studies and in vivo animal models. Development of an in vitro system working with human cells and defined glucocorticoids and catecholamines allows for a detailed investigation of the cellular mechanisms that stress-related alterations have on the activation and functional state of the T cell subpopulations. Through these types of studies we can determine which cytokines and chemokines are modulated, and which cell subpopulations are most affected by the given neuroendocrine molecules. Therefore, the current study was undertaken to investigate the role of glucocorticoid (DEX) and catecholamines (NE and DA), and the combination of both DEX and NE on the modulation of immune activity using PBMC of healthy human donors. The findings determined that the well known immune suppressing activities of DEX are due to a reduction of both cell activation, and cytokine and chemokine production. Moreover, similar results were seen from cultures with both DEX and NE together. On the other hand, these studies demonstrated that catecholamines induce an increased lymphocyte activation accompanied by augmented Th1 and Th2 type cytokine production. These parameters were evaluated by the determination of both the frequency of cytokines producing T cells, as well as message and secreted protein. The literature contains contradictory data concerning the effects of norepinephrine on immune cells (Moynihan et al., 2004). This is because in vitro and in vivo models are used with different concentrations of NE and under different experimental conditions. In this study, the use of 1 10 7 M of NE, a concentration between plasma levels (1 10 9 M / 1 10 8 M) and the ones liberated in lymphoid tissues (1 10 6 M / 1 10 5 M), was used to try and replicate

Table 7 Dexamethasone plus norepinephrine induces a decreased message expression for cytokines in activated PBMC

IL-2b IL-4b IL-5b IL-13b

Control (mean T S.D.)a

Dex + NE (mean T S.D.)a

P-value

6.51 T 6.31c 19.5 T 17.79c 8.55 T 9.26c 16.43 T 11.77c

3.24 T 6.49c 9.97 T 14.47c 0.78 T 2.60c 0.49 T 1.18c

0.07 0.05 0.01 <0.01

Fold change 0.51 0.09 0.03

N.S. — difference not significant ( P-value > 0.05). a Arbitrary units of mRNA as determined using semi-quantitative RTPCR as described in Materials and methods. b Cytokine message expression was determined at 18 h following stimulation with anti-CD3/CD28 as described in Materials and methods. c The values represent the mean T S.D. for 11 people.

Table 8 Dexamethasone plus norepinephrine induces a decreased chemokine production in activated PBMC

IFN-gb MIP-1ab MCP-1b MIGb RANTESb IP-10b Eotaxin-1b

Control (mean T S.D.)a

Dex + NE (mean T S.D.)a

P-value

0.06 T 0.04c 2.49 T 1.40c 7.29 T 1.03c 9.10 T 7.44c 5.00 T 1.99c 1.32 T 0.08c 0.05 T 0.02c

0.05 T 0.03c 0.85 T 0.84c 5.16 T 0.73c 2.60 T 2.94c 4.01 T 2.08c 0.83 T 0.07c 0.04 T 0.02c

N.S. <0.01 <0.01 <0.01 0.01 <0.01 0.07

Fold change 3 0.3 3.5 0.2 0.4

N.S. — difference not significant ( P-value > 0.05). a Nanogram per milliliter as determined using ELISA as described in Materials and methods. b Cytokine/chemokine production was determined at 18 h following stimulation with anti-CD3/CD28 as described in Materials and methods. c The values represent the mean T S.D. for 11 people.

physiological relevant concentrations of catecholamines (Sanders and Straub, 2002). NE could increase the activation of the lymphocytes seen through the increased frequency of CD4+ T cells expressing CD69. It was demonstrated by Gan et al. (2002) that NE inhibits the expression of CD69 in IL-2 activated NK (Natural Killer) cells, but NK cells express more h2-adrenergic receptors than CD4+ T cells (Wahle et al., 2001). Thus, our findings indicate that NE has different effects on T and NK cells, and this may be due to differences in their expression of h2-adrenergic receptors. Previous studies indicated that NE can influence the trafficking of lymphocytes and their distribution within the body (Dhabhar, 2002). Our in vitro results showing that NE induced a decrease in the frequency of CD8+ T cells expressing CD62L may reflect the findings from Sanders and Straub (2002) showing a redistribution of CD8+ T cells and provide a possible mechanism by which NE could lead to this altered homing. NE induced an increased production of several cytokines characteristic of both Th1 and Th2 type response at the level of either transcription or expressed protein, as well as determined by the frequency of cytokine producing cell subpopulations. Interestingly, previous in vivo and in vitro findings in humans suggest that catecholamine exposure and h2-adrenoreceptor (h2AR) stimulation may skew the effector cell balance toward a Th2 cell cytokine secretion profile (Sanders and Straub, 2002), through an inhibition of T cell synthesis of Th1 related cytokines IFNg, TNF-a and IL-2 (Kalinichenko et al., 1999). However, our results showed an exacerbated Th1 and Th2 cytokine production from PBMC stimulated in the presence of NE and also DA, demonstrating the potential of both catecholamines to stimulate both pro- and anti-inflammatory cytokines. In the current work we did not investigate the presence of AR (adrenoreceptors) in the cell subpopulations. However, it is known that the most common adrenergic receptor expressed on T and B cells is the

K.C.L. Torres et al. / Journal of Neuroimmunology 166 (2005) 144 – 157

h2-adrenergic receptor (Ramer-Quinn et al., 1997). The h2-adrenergic receptor is not expressed on resting or activated Th2 cells (Ramer-Quinn et al., 1997; Kohm and Sanders, 2000). Therefore, the potential of NE and DA in increasing Th2 type cytokines, showed by our results, might reflect indirect effects of other cells present in the culture. Some results have shown the capability of h2AR engagement to induce a decrease in Th1 type cytokines (Kalinichenko et al., 1999; Sanders and Straub, 2002). However, the Th1 cytokine profile induced by catecholamines from PBMC might reflect h2AR activation in conjunction with other stimuli from other cells such as cytokines and co-stimulatory molecules. The balance of activated intracellular pathways by catecholamines, cytokines, chemokines and co-stimulatory molecules resulted in an increased Th1 type cytokine profile in our system. However, the determination of exactly what intracellular pathway networks led to this profile remains to be investigated. Several studies have noted opposing effects with respect to cytokine modulation on the engagement of aAR and hAR in monocytes (Hasko and Szabo, 1998). While our studies did not show any significant effects of NE on the monocyte population, further studies to determine which AR pathway is dominant in monocytes would help to clarify differences in the literature concerning effects of NE on immune cytokine profiles. Our studies demonstrated that NE induced a decreased production of the chemokines, MIG and eotaxin-1, as determined by ELISA using supernatants from activated PBMC. However, mRNA for IL-8 was increased by NE, which could suggest that MIG and eotaxin-1 are decreased due to consumption in culture rather than decreased production, or a post-transcriptional process resulting in less protein synthesis. It is important to remember that the levels of chemokines as determined by ELISA could be affected by consumption as seen by Ewen and Baca-Estrada (2001) for IL-4. Interestingly, Hasko et al. (1998) demonstrated that catecholamines inhibited MIP-1a through a hAR mediated mechanism. Therefore, the mechanisms that control chemokine and cytokine expression on immune cells under NE effects may be differently regulated. The results obtained from PBMC cultures performed in the presence of DA were very similar to that seen for NE effects on activated immune cells. The concentration used in the PBMC cultures (5  10 7 M) were a slightly higher than plasma levels from chronic stressed individuals that varies from 6.5  10 9 to 3  10 8 M (Ghosh et al., 2003). Even so, we confirmed that this dose was not inducing cell death or apoptosis. Higher activation was seen through an increase in CD4+CD69+ T cells, as well as an increase in the frequency of cytokine producing cells following activation in the presence of DA. In our studies, cells cultured in the presence of DA had no difference in CD25 expression as compared to control cultures (data not shown). Carr et al. (2003) observed that L-dopa treatment, in vivo, induced lymphocyte proliferation through a

155

mechanism independent of IL-2 and IL-2R. Moreover, IL-2 message expression from cells cultured in the presence of DA did not differ from control cultures. These data suggest an early activation event in the CD4+ T cell population treated with DA that was independent on an early production of IL-2. Like NE, DA induced an increased expression of both pro- and anti-inflammatory cytokines. The DA receptors D2, D3, D4 and D5 are weakly expressed on human T cells (McKenna et al., 2002), but DA can interact with cells through expression of oligo-heterodimers of D2 and D3, or through hAR. Ghosh et al. (2003) detected that DA inhibited the release of Th1 and Th2 cytokines from isolated T cells through engagement of D2 and D3 receptors. However, the current work shows that DA increased the frequency of CD4+ and CD8+T cells expressing Th1 and Th2 cytokines at the level of message transcription and cytokine expression. While we did not investigate which dopamine receptors were involved, it is possible that our results reflect indirect effects of DA upon other leukocytes, or activity through h-adrenoreceptors (Hasko et al., 2002). In contrast to the increase in cytokines, DA induced a decrease in MIG, IP-10 and eotaxin production. These findings suggest that the mechanisms underlying control of chemokine production differ from those mechanisms for cytokine production and, thus are, differentially modulated by catecholamines. The concentration of DEX used in PBMC cultures was 1 10 6 M, which did not reduce the cell viability. This concentration is close to the higher physiological concentrations of glucocorticoids released during physical or psychological stress circumstances that vary in the range of 3.5  10 7 to 9.5  10 7 M (Webster et al., 2002). An inhibition of cell activation, by DEX, was detected not only in total lymphocytes, but also as a decreased frequency of CD4+ and CD8+ T cells expressing CD25, the a chain of IL-2 receptor (IL-2R). Similar results were seen by Batuman et al., (1994) where DEX (1 10 6 M) could inhibit the a and h chain of IL-2R in PHA activated T cells. Moreover the present study showed that DEX inhibited activation of both CD4+ and CD8+ T cells as measured by the expression of CD69, another recent activation marker. DEX induced a decreased expression of Th1 and Th2 cytokines at the level of message transcription and secreted protein, as well as a decreased frequency of cytokine producing CD4+ and CD8+ T cells. Glucocorticoids have been shown to induce a shift in type1/type2 cytokine to a Th2 type cytokine profile in physiologic stress concentrations. It may be possible that the inhibition of both proand anti-inflammatory cytokines seen by us was due to the concentration of DEX used or due to the fact that DEX can bind with greater affinity to GR (glucocorticoid receptor) than natural glucocorticoid hormones (Webster et al., 2002). The mechanisms of down regulation by glucocorticoids could then be exacerbated in these cells, through the high engagement of GR and the subsequent prevention of the

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transcription and expression of Th1 and Th2 type cytokine genes. DEX increased the frequency of monocytes expressing CD14 following activation. Interestingly, this increase was accompanied by a decrease in the frequency of IL-10 expressing CD14 cells. Thus, the inhibitory effects of DEX likely inhibited the transition of activated CD14+ monocytes to the CD14 negative phenotype. These findings agree with findings by Haller et al. (2002) showing that activated CD14 negative monocytes produced IL-10 which down regulated pro-inflammatory cytokines. Cultures of activated PBMC in the presence of both NE plus DEX were performed in vitro, to evaluate the role of both glucocorticoids and catecholamines acting together, in attempts to simulate an environment that would occur during stress situations. Interestingly, the data collected were very similar to the results obtained by activated PBMC cultured in the presence of DEX alone. Decreased Th1 and Th2 cytokine profiles at the level of message transcription, protein secretion, and the frequencies of CD4+ and CD8+ T cells expressing both pro- and anti-inflammatory cytokines, were all detected. Moreover, decreased activation was also seen. The HPA axis is believed to play a pivotal role in the stress response (Carrasco and Van de Kar, 2003). Our, in vitro, results suggest that the DEX mediated effects are dominant over those of NE. Therefore, the engagement of GR and AR together with other secondary effects leads to immunosuppression as determined by a number of criteria. Interestingly the production of MIG/CXCL-9 was decreased by DEX treatment in 30% (Table 6) and by NE by approximately 25% (Table 2); and by more than 70% when PBMC were treated with NE plus DEX, suggesting an additive effect of both molecules in the MIG/CXCL-9 production. In conclusion, our findings show that DEX inhibits human leukocyte activation and production of pro- and antiinflammatory cytokines, as well as a decrease in the frequencies of cytokine producing cells, similar to effects of DEX plus NE. In contrast, the catecholamines, NE and DA, believed to be immunosuppressive for Th1 type responses, exerted opposite effects to DEX and increased cell activation and production of pro- and anti-inflammatory cytokines from several subpopulations. Thus, the description of the multifaceted activities of neurotransmitters and hormones on human leukocyte subpopulations will aid in our understanding of immunoregulation via these important stress molecules.

Acknowledgements Financial support was provided by CAPES (Brazilian PhD student fellowships), CNPq (Brazilian National Government Science Foundation) and FINEP, CT-Infra. KJG, MMT, and WOD are CNPq Research Fellows.

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