In vitro effects of dexamethasone on bovine CD25+CD4+ and CD25−CD4+ cells

Research in Veterinary Science 93 (2012) 1367–1379

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In vitro effects of dexamethasone on bovine CD25+CD4+ and CD25 CD4+ cells Tomasz Mas´lanka ⇑, Jerzy Jan Jaroszewski Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowski Street 13, 10-718 Olsztyn, Poland

a r t i c l e

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Article history: Received 11 August 2011 Accepted 29 January 2012

Keywords: Dexamethasone CD4+ cells Foxp3 Cytokines Apoptosis Cattle

a b s t r a c t This paper investigates the in vitro effect of dexamethasone on bovine CD25highCD4+, CD25lowCD4+ and CD25 CD4+ T cells. Only a small percentage of bovine CD25highCD4+ (2–4%) and CD25lowCD4+ (1–2%) cells expressed Foxp3. Dexamethasone caused considerable loss of CD25 CD4+ cells, but it increased the relative and absolute numbers of CD25highCD4+ and CD25lowCD4+ lymphocytes, while at the same time reducing the percentage of Foxp3+ cells within the latter subpopulations. Considering all these, as well as the intrinsically poor Foxp3 expression in bovine CD25+CD4+, it can be concluded that the drug most probably increased the number of activated non-regulatory CD4+ lymphocytes. It has been found that changes in cell number were at least partly caused by proapoptotic effect of the drug on CD25 CD4+ cells and antiapoptotic effect on CD25highCD4+ and CD25lowCD4+ cells. The results obtained from this study indicate that the involvement of CD4+ lymphocytes in producing the anti-inflammatory and immunosuppressive effect of dexamethasone in cattle results from the fact that the drug had a depressive effect on the production of IFN-c by CD25 CD4+ cells. Secretion of TGF-b and IL-10 by CD4+ lymphocytes was not involved in producing these pharmacological effects, because the drug did not affect production of TGF-b and, paradoxically, it reduced the percentage of IL-10+CD4+ cells. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Regulatory cells (Tregs) comprise numerous sub-populations, including CD25+CD4+(Foxp3+) T cells, CD4 CD8 T cells, interleukin 10 (IL-10) producing Tr1 cells, transforming growth factor-b (TGF-b) producing T helper type 3 cells (Th3), natural killer (NK) T cells, and cdT cells (Tang and Bluestone, 2008). CD25+CD4+ Tregs were described for the first time in 1995 (Sakaguchi et al., 1995) as a population of naturally occurring cells that exhibited potent regulatory functions. Currently, CD25+CD4+ Tregs cells represent a major lymphocyte population engaged in the dominant control of self-reactive T responses and maintaining tolerance in several models of autoimmunity (Toda and Piccirillo, 2006). CD25+CD4+ Tregs can be divided into two principal subsets: naturally occuring Tregs (nTregs) and induced (also called inducible or adaptive) Tregs (iTregs). nTregs develop in the thymus (Fontenot et al., 2005) and constitute some 5–10% of peripheral CD4+ T cells in non-manipulated normal mice and humans (Jonuleit et al., 2001; Stephens et al., 2001), but in humans only the CD4+CD25high cells which constitute 2–3% of the CD4+ T cells are really regulatory (Baecher-Allan et al., 2001; Mottet and Golshayan, 2007). iTregs are derived de novo from a naive CD25 CD4+ precursor pool in peripheral lymphoid tissues following antigenic stimulation in

⇑ Corresponding author. Tel.: +48 89 523 3758; fax: +48 89 523 3440. E-mail address: [email protected] (T. Mas´lanka). 0034-5288/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2012.01.018

the presence of TGF-b and IL-2 (Chatila, 2009). However, CD25 is not specific marker for Tregs because is also expressed by activated T cells. To address this problem, scientists have been looking for marker molecules that might allow to discriminate activated and regulatory T cells within the population of CD25-expressing CD4+ cells. In 2003, three independent groups identified Forkhead Box P3 protein (Foxp3) as a unique marker for CD4+CD25+ T cells with suppressive/regulatory capacity in the mouse (Fontenot et al., 2003; Hori et al., 2003; Khattri et al., 2003). Foxp3 currently represents the most specific marker used to distinguish regulatory cells (Foxp3+CD25+CD4+) from activated effector cells (Foxp3 CD25+CD4+) within CD25+CD4+ cell subpopulation. The mechanisms by which Tregs cells mediate their suppressive effects can be broadly divided into those that target T cells (suppressor cytokines, IL-2 consumption, cytolysis) and those that primarily target antigen-presenting cells (decreased costimulation or decreased antigen presentation) (reviewed by Shevach, 2009). The concept of a soluble factor mediating Treg suppression is still controversial, considering the cell-to-cell contact dependence that was thought to be required to mediate suppression. However, there is a growing list of in vivo studies describing the importance of Treg-derived IL-10, TGF-b, and IL-35 in the suppression of various immune responses (Tang and Bluestone, 2008; Shevach, 2009; Workman et al., 2009). Since discovery CD25+CD4+ regulatory cells by Sakaguchi et al. (1995) an abundance of articles on this subject have been published, however, they concerned human beings and mice. On these

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background, the current state of knowledge about bovine regulatory cells is very poor. Seo et al. (2007) showed that there are certain cells with regulatory properties within CD4+ cells in cattle. They demonstrated that staphylococcal enterotoxin type C1-stimulated CD4+ T cells suppressed the proliferation of naive peripheral blood mononuclear cells (PBMCs) in response to heat-killed-fixed Staphylococcus aureus. The suppression was partially mediated by IL-10 and TGF-b. They also detected an increase in the transcription of IL-10 and transforming growth factor b (TGF-b) genes in staphylococcal enterotoxin type C1 (SEC1)-stimulated cultures; it was attributed to the CD25+CD4+cell subpopulation. The expression of Foxp3 mRNA was also increased and was accompanied by the upregulation of CD152 and the downregulation of IL-2 transcription, suggesting that cells in this subpopulation are Tregs. A study by Hoek et al. (2009) found, to the contrary, that the regulatory effect in cattle to be that of WC1+, rather than of CD4+CD25high and CD4+CD25low lymphocytes. There have been only three reports in the available literature (Hoek et al., 2009; Seo et al., 2009; Gerner et al., 2010) which contain data on Foxp3 expression in bovine CD25+CD4+cells, although the results presented in them are highly inconclusive. It is noteworthy that, according to our knowledge, the data about Foxp3 expression, presented in the studies, are the first results obtained with the use of commercial anti-bovine Foxp3 antibody. Dexamethasone belongs to a group of steroidal anti-inflammatory (SAIDs) drugs known also as glucocorticosteroids, glucocorticoids or corticosteroids. It is known that glucocorticosteroids, commonly used to treat a variety of inflammatory and autoimmune diseases, exert a broad range of effects on the immune system, most of which are related to their capacity to inhibit cytokine production and other inflammatory mediators (Prado et al., 2011). Most glucocorticoid actions are mediated by its binding to the glucocorticoid receptors (GRs), additional nongenomic effects are discussed that may be exerted through nonrelated receptors or interactions with lipid membranes (Tuckermann et al., 2005; Song and Buttgereit, 2006; Wüst et al., 2008). There have been a number of papers published in recent years (Karagiannidis et al., 2004; Braitch et al., 2009; Provoost et al., 2009; Xie et al., 2009; Prado et al., 2011) on the effect of dexamethasone, or other SAIDs on the percentage of CD25+CD4+ cells and/or Foxp3 expression in them, as well as on production of some cytokines by them. The results presented in these papers have indicated or suggested that SAIDs could influence the regulatory cell population or even generate Tregs. However, the above mentioned papers deal only with human or mouse cells, whereas the knowledge of the effect of the drugs on the percentage of bovine CD25+CD4+ cells is actually based on one publication (Menge and Dean-Nystrom, 2008). Moreover, the effect of SAIDs on Foxp3 expression in CD4+ cells, or any other of this type, is not known in cattle. In view of the reports, published in recent years, on the influence of SAIDs on human and mouse CD25+CD4+ cells, the aim of this study has been to evaluate the effect of dexamethasone on bovine CD25highCD4+ and CD25lowCD4+, as well as CD25 CD4+ lymphocytes in peripheral blood with respect to: (a) percentage and absolute number of cells, (b) percentage of cells expressing Foxp3, (c) percentage of cells at early stage of apoptosis, (d) percentage of IL-10, TGF-b and IFN-c-producing cells. It is also noteworthy that the study provides a considerable contribution to the knowledge of bovine CD4+ lymphocytes.

2. Materials and methods Schematic diagram showing design of experiments is presented in Fig. 1.

2.1. Animals Studies were carried out on thirty six clinically healthy heifers (Polish Black and White breed), aged 12 months, kept indoors and originating from a dairy farm located in Bałdy (Poland). The animals were housed and treated in accordance with the rules approved by the Local Ethics Commission (Ethic Commission Opinion No 82/2010). One group of 18 heifers were used for experimental design A and a second group of 18 animals were used for experimental design B (Fig. 1). 2.2. Isolation of peripheral blood mononuclear cells and culture conditions Blood was aseptically drawn by venipuncture from the jugular vein into heparinized sterile vacutainer tubes (Becton Dickinson (BD) Biosciences, San Jose, CA, USA). PBMCs were isolated by Histopaque 1.077 (Sigma–Aldrich, Munich, Germany) density gradient centrifugation at 400g for 30 min at room temperature (RT). PBMCs were recovered from the interface, washed (300g for 10 min at 4 °C; these parameters were used for all cell-washing procedures) three times and resuspended in complete medium [CM; RPMI 1640, 10% FBS, 10 mM HEPES buffer, 10 mM nonessential amino acids, 10 mM sodium pyruvate and 10 U/ml penicillin/ streptomycin (all from Sigma–Aldrich)]. For phenotypic analysis and apoptosis detection PBMCs were adjusted to final concentration of 106 cells/mL in CM, seeded in 12-well plates in 2 mL aliquots and incubate/cultured for 12, 24, 48 and 168 h in the absence (control) or presence of dexamethasone (DEX). Apoptosis was evaluated after 12 and 24 h of incubation. For all experiments, cells were treated with drug in concentrations reflecting their plasma levels achieved in vivo at therapeutic doses (DEX 10 7 M) and at concentrations ten times lower (DEX 10 8 M). Dexamethasone (water soluble, cell culture tested) was purchased from Sigma– Aldrich; stock solutions (10 3 M) were prepared in ultra-pure water, frozen as 120 lL aliquots at 20 °C and thawed as required. To analyze the expression of intracellular IL-10, IFN-c and TGF-b PBMCs were adjusted to 5  106 cells/mL in CM and seeded in 24-well plates in 1 mL aliquots. Cells were pre-incubated for 1 h without (control) or with two concentrations of drug as described above followed by 6 (for evaluation of IL-10 and IFN-c production) or 12 h (for evaluation TGF-b production) stimulation with concanavalin A (Con A, Sigma–Aldrich) (5 lg/mL) in the presence of brefeldin A (Sigma–Aldrich) (10 lg/mL) during the last 5 h. Plates were incubated at 37 °C in an atmosphere of humidified incubator with 5% CO2 and 95% air. In order to ensure consistency in the absolute number of cells per test, each experiment included six wells of pooled PBMCs (obtained from six heifers) for each condition tested. All experiments were repeated independently three times, using six different animals for each experiment (overall n = 18). 2.3. Flow cytometry The sets of staining combinations are presented in Fig. 1. 2.3.1. The monoclonal antibodies (mAb) and other markers for flow cytometry The markers used in flow cytometry were as follows: FITC-conjugated mouse anti-bovine CD4 (CC8, IgG2a), PE-conjugated mouse anti-bovine CD25 (IL-A111, IgG1), AF647-conjugated human antibovine Foxp3 (7627, HuCAL Fab bivalent), AF647-conjugated hucal fab-dhlx-mh isotype (negative) control, AF647-conjugated mouse anti-bovine IFN-c (CC302, IgG1), AF647-conjugated mouse IgG1 isotype control, biotinylated mouse anti-bovine IL-10 (CC320, IgG1), biotinylated mouse IgG1 isotype control (all from Serotec,

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Fig. 1. Schematic diagram showing design of experiments and sets of staining combinations. Parallel sets of cell cultures were incubated/cultured for 12, 24, 48 and 168 h with and without dexamethasone (DEX 10 7 and DEX 10 8 M) to ascertain the effect of such treatment on the relative and absolute count of CD25highCD4+, CD25lowCD4+ and CD25 CD4+ cells, and their apoptosis (12 and 24 h) and Foxp3 expression (48 and 168 h) (A). To analyze the production of intracellular IL-10, IFN-c and TGF-b PBMCs were pre-incubated for 1 h without (control) or with the drug followed by 6 (for evaluation of IL-10 and IFN-c production) or 12 h (for evaluation TGF-b production) stimulation with concanavalin A (CON A) in the presence of brefeldin A during the last 5 h (B). Each experiment included six wells (using pooled PBMCs obtained from six heifers) for each condition tested. All experiments were repeated independently three times (overall n = 18).

Oxford, UK), APC-conjugated mouse anti-TGF-b (1D11, IgG1; this antibody reacts with human, mouse and bovine TGF-b1 and TGF-b2), APC-conjugated mouse IgG1 isotype control (both from R&D Systems, Minneapolis, MN, USA), APC-conjugated Annexin V, PerCP-conjugated streptavidin and 7-AAD (all from BD Biosciences).

2.3.2. Extracellular staining Cells were removed from the wells by pipetting and rinsing with FACS buffer [FB, 1 Dulbecco’s PBS (Sigma–Aldrich) devoid of Ca2+ and Mg2+ with 2% (v/v) heat-inactivated fetal bovine serum] and transferred into individual tubes and centrifuged. After

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additional washing in 2 mL FB, the cells were resuspended in FB and stained with FITC-conjugated anti-CD4 (1:20) and PE-conjugated anti-CD25 mAb (1:200). After 45 min incubation (on ice and in the dark), cells were washed in 2 mL FB. 2.3.3. Intracellular staining for Foxp3 Cells stained for surface markers (as described above) were fixed by adding 100 lL Leucoperm-Reagent A (Serotec) to each tube and then incubating for 15 min at RT in the dark. After this, the cells were washed with 3 mL FB and then permeabilized by adding 100 lL of Leucoperm-Reagent B (Serotec) and subsequently stained with AF647-conjugated anti-Foxp3 mAb (1:20) for 60 min at RT in the dark. After incubation cells were washed twice with 2 mL FB and analyzed by flow cytometry. Isotype control was performed as above except AF647-conjugated anti-Foxp3 mAb was replaced with hucal fab-dhlx-mh negative control-AF647. 2.3.4. Intracellular staining for IL-10 and IFN-c Cells stained for surface markers (as described above) were fixed with 200 lL 2% paraformaldehyde in Dulbecco’s PBS for 15 min on ice. After this, the samples were washed with 2 mL FB and then permeabilized by washing with 2 mL 0.2% (w/v) saponin (Sigma–Aldrich) in FB. Subsequently, cells were stained with biotinylated mouse anti-bovine IL-10 mAb (1:1000) for 45 min on ice in the dark, followed by washing with 2 mL 0.2% saponin in FB. Then, cells were stained with PerCP-conjugated streptavidin (1:400) and AF647-conjugated anti-IFN-c mAb (1:200) for 45 min on ice in the dark. Finally cells were washed twice with 2 mL FB and analyzed by flow cytometry. Isotype controls were performed as above except biotinylated mouse anti-bovine IL-10 and AF647conjugated anti-IFN-c mAb were replaced with biotinylated mouse IgG1 isotype control and AF647-conjugated mouse IgG1 isotype control, respectively. 2.3.5. Intracellular staining for TGF-b After extracellular staining (as described above) and fixing (200 lL 2% paraformaldehyde in Dulbecco’s PBS per sample for 15 min on ice) cells were permeabilized with 2 mL SAP buffer [0.1% (w/v) saponin, 0.05% (w/v) NaN3 in Hanks’ Balanced Salt

Solution (HBSS), (all from Sigma–Aldrich)] and stained with APCconjugated anti-TGF-b mAb (1:20) for 45 min at RT in the dark. After this step cells were washed twice with 2 mL SAP buffer and analyzed by flow cytometry. Isotype control was performed as above except APC-conjugated anti-TGF-b mAb was replaced with APC-conjugated mouse IgG1 isotype control. 2.3.6. Apoptosis Cells stained for surface markers were washed once in 1 mL of 1 Annexin V binding buffer (BD Biosciences). The supernatants were removed by centrifugation and the cells were suspended in 1 Annexin V binding buffer. A 5 lL of APC-conjugated Annexin V and 5 lL of 7-AAD were added to the cells. The cells were mixed gently and incubated for 15 min at RT in the dark, and then diluted with 400 ll of 1 Annexin V binding buffer and analyzed by flow cytometry within 1 h. 2.3.7. FACS acquisition and analysis Fig. 2 presents FACS analysis strategy. Flow cytometry analysis was performed using a FACSCanto II cytometer (BD Biosciences). Data were acquired by FACSDiva version 6.1.3 software (BD Biosciences) and analyzed by FlowJo software (Tree Star Inc., Stanford, CA, USA). Cytometry setup and tracking beads (CST, BD Biosciences) were used to initialize PMT settings. Unstained control cells as well as single stain control for every fluorochrome were prepared and used to set up flow cytometric compensation. For samples stained for cytokines a total of 200,000 events were acquired per sample. The absolute count represents the number of collected cells of each subset per sample taking into account that a fixed volume of sample (100 lL) was always analysed. The values obtained in this manner could reflect the changes in the absolute number of lymphocytes, because (a) for each time the same number of cells was seeded in each well, (b) after cell culture, the total well content was transferred to individual tubes, (c) the same volume of each sample (100 lL) was always analysed by flow cytometry (however, at least 120,000 events were acquired per sample), (d) all cytometry assays was performed using the same FSC threshold. Flowjo software directly converted a percentage count of a subpopulation of cells to an absolute count. Background fluorescence for intracel-

Fig. 2. FACS analysis strategy. Lymphocytes were gated based on forward and side scatter (FSC/SSC) parameters and then gated for expression of CD4 surface marker. Relative to the level of CD25 expression within CD4+ subpopulation, CD25highCD4+, CD25lowCD4+, and CD25 CD4+ cells were identified. Each of these cell populations was analyzed for cytokines (IFN-c, IL-10, TGF-b) and Foxp3 expression and for apoptosis as assessed by the surface exposures of plasma membrane phosphatidyl serine and loss of plasma membrane integrity using Annexin V/7-AAD staining. The dot plot of Annexin V versus 7-AAD was used for the assessment of apoptosis. Annexin V 7-AAD cells were considered viable; Annexin V+7-AAD cells were deemed early-apoptotic; and double positive cells (Annexin V+7-AAD+) were considered to be late apoptotic/necrotic.

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Fig. 3. The effect of dexamethasone on the relative and absolute count of CD25highCD4+ (A, A’), CD25lowCD4+ (B, B’), and CD25 CD4+ (C, C’) cells. PBMCs were cultured with medium alone or with dexamethasone (DEX 10 7 and DEX 10 8 M) for the indicated times. The relative count is expressed as a percentage of CD4+ cells expressing (high or low) or not expressing CD25. The absolute count represents the number of collected cells of each subset per sample. Results are the mean (±SEM) of three independent experiments with six samples per experiment (overall n = 18). ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 treated cells versus control cells. Typical cytograms demonstrating high and low expression of CD25 on CD4+ cells.

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lular staining (controlled by labeled or biotinylated isotype controls) was very low or absent and never exceeded 2.0% of cells. The gates for the Foxp3 expression and IFN-c, IL-10 and TGF-b production within particular CD4+ cell subpopulations were set so that the positive signal for isotype control staining was less than 0.2%. 2.4. Statistical analysis All data are presented as the mean ± SEM. Student’s unpaired t test (GraphPad Prism3, GraphPad Software, San Diego, CA, USA) was used to compare the results between dexamethasone treated, at either concentration, and untreated cells. A P value less than 0.05 was considered to be statistically significant. All data were graphed with Sigmaplot software (version 12, Systat Software, Inc, Chicago, IL, USA).

(Fig. 3A) and CD25lowCD4+ cells [12 h: P < 0.001 (DEX 10 7), P < 0.05 (DEX 10 8); 24 h: P < 0.01; 48 and 168 h: P < 0.001] (Fig. 3B). It was also found that after 168 h of culturing with DEX 10 7 and DEX 10 8, a considerable (P < 0.001) increase in the absolute number of CD25highCD4+ (Fig. 3A’) and CD25lowCD4+ (Fig. 3B’) cells took place. Also, 2-day exposure to DEX 10 7 and DEX 10 8 increased (P < 0.001) the absolute number of CD25highCD4+ (Fig. 3A’), but not CD25lowCD4+. In cultures treated with DEX 10 7 and DEX 10 8 for 12, 24 and 48 h, a significant [12 and 24 h: P < 0.01 (DEX 10 7), P < 0.05 (DEX 10 8); 48 h: P < 0.001 (DEX 10 7), P < 0.01 (DEX 10 8)] decrease in the absolute number of CD25 CD4+ cells was observed (Fig. 3C’). The average loss in CD25 CD4+ lymphocytes in samples obtained from the cultures treated with the drug was 1816 ± 347 (12 h), 1846 ± 264 (24 h) and 1812 ± 210 (48 h) cells for DEX 10 7 and 1405 ± 394 (12 h), 1448 ± 303 (24 h) and 1435 ± 209 (48 h) cells for DEX 10 8.

3. Results high

+

3.1. Dexamethasone increases the percentage of CD25 CD4 and CD25lowCD4+ cells and reduces the absolute number of CD25 CD4+ cells The effect of dexamethasone on the percentage and number of CD25highCD4+, CD25lowCD4+ and CD25 CD4+ lymphocytes was evaluated after 12, 24, 48 and 168 h from PBMCs exposure to the drug. It was observed at each of the time points that DEX 10 7 and DEX 10 8 considerably increased the percentage of CD25highCD4+ (12 and 24 h: P < 0.05; 48 and 168 h: P < 0.001)

3.2. Dexamethasone reduces expression of Foxp3 in CD25highCD4+ and CD25lowCD4+cells The effect of dexamethasone on Foxp3 expression in CD25highCD4+, CD25lowCD4+ and CD25 CD4+ cells was evaluated after 48 and 168 h of PBMCs culture in the presence or absence of the drug. Surprisingly, it was found that after 48 h of culture in the presence of DEX 10 7 and DEX 10 8, considerable [CD25highCD4+: P < 0.01 (DEX 10 7), P < 0.05 (DEX 10 8); CD25lowCD4+: P < 0.05] reduction of percentage of Foxp3+ cells among CD25highCD4+ and CD25lowCD4+ lymphocytes took place (Fig. 4A). After 168 h exposure

Fig. 4. The effect of dexamethasone on the proportion of Foxp3+CD25highCD4+ and Foxp3+CD25lowCD4+ cells. PBMCs were cultured with medium alone or with dexamethasone (DEX 10 7 and DEX 10 8 M) for 48 (A) and 168 h (B). The results are expressed as a percentage of CD25highCD4+ and CD25lowCD4+ cells expressing Foxp3. Data reported are the mean (±SEM) of three independent experiments with six samples per experiment (overall n = 18). ⁄P < 0.05, ⁄⁄P < 0.01, treated cells versus control cells. Typical cytograms demonstrating Foxp3 expression within the CD25highCD4+ and CD25lowCD4+ cells.

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Fig. 5. The effect of dexamethasone on apoptosis of CD25highCD4+, CD25lowCD4+ and CD25 CD4+ cells. PBMCs were cultured with medium alone or with dexamethasone (DEX 10 7 and DEX 10 8 M) for 12 (A) and 24 h (B). The results are expressed as a percentage of CD25highCD4+, CD25lowCD4+ and CD25 CD4+ cells in early apoptosis (Annexin V+/7AAD cells). The data represent the mean (±SEM) of three experiments with six samples per experiment (overall n = 18). ⁄P < 0.001, treated cells versus control cells. Typical cytograms showing flow cytometry analysis assessing apoptosis in particular lymphocyte subpopulations.

to the drug, a significant [P < 0.05 (DEX 10 7), P < 0.01 (DEX 10 8)] reduction in the relative number of Foxp+CD25highCD4+ cells was still observed; however, the percentage of CD25lowCD4+ cells expressing Foxp3 did not differ significantly from the control values (Fig. 4B). No expression of Foxp3 in CD25 CD4+ cells was observed. 3.3. Dexamethasone increases apoptosis of CD25 CD4+ cells and it reduces the number of early apoptotic cells within the CD25highCD4+ and CD25lowCD4+subpopulations Evaluation of the effect of the drug on apoptosis of CD25highCD4+, CD25lowCD4+ and CD25 CD4+ cells was performed

after 12 and 24 h from incubation of PBMCs in the absence or presence of the drug. It was found that 12 h after exposure to DEX 10 7 and DEX 10 8 the percentage of early apoptotic cells (Annexin V+7-AAD ) in the subpopulation of CD25 CD4+ cells increased significantly (P < 0.001), whereas no effect of the drug was observed on apoptosis of CD25highCD4+ and CD25lowCD4+ cells (Fig. 5A). Furthermore, the percentage of early apoptotic cells within CD25highCD4+ and CD25lowCD4+ subpopulations in samples obtained from cells incubated for 24 h in the presence of DEX 10 7 and DEX 10 8 was found to decrease significantly (P < 0.001), but no effect of the drug on apoptosis of CD25 CD4+ lymphocytes was observed (Fig. 5B).

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Fig. 6. The effect of dexamethasone on IFN-c (A), IL-10 (B) and TGF-b (C) production. Expression of intracellular IFN-c, IL-10 and TGF-b was investigated in PBMCs preincubated for 1 h with medium alone or with dexamethasone (DEX 10 7 and DEX 10 8 M) followed by 6 h (for IL-10 and IFN-c) or 12 h (for TGF-b) stimulation with Con A in the presence of brefeldin A during the last 5 h. The results are expressed as a percentage of CD25highCD4+, CD25lowCD4+ and CD25 CD4+ cells producing IFN-c or IL-10 or TGFb. Results are the mean (±SEM) of three independent experiments with six samples per experiment (overall n = 18). ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001, treated cells versus control cells. Typical cytograms illustrating cytokine expression in particular lymphocyte subpopulations.

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3.4. Dexamethasone reduces the percentage of CD25 CD4+ cells producing IFN-c as well as the percentage of IL-10+ cells within the CD25highCD4+, CD25lowCD4+ and CD25 CD4+subpopulations The effect of dexamethasone on the production of selected cytokines was evaluated in cells incubated with or without the drug, with accompanying Con A stimulation in the presence of brefeldin A. DEX 10 7 and DEX 10 8 were found to significantly (P < 0.001) reduce the percentage of IFN-c-producing CD25 CD4+ cells (Fig. 6A), but it did not affect synthesis of IFN-c in CD25highCD4+ or CD25lowCD4+ cells. It has also been shown that DEX 10 7 and DEX 10 8 significantly reduced the percentage of IL-10 producing cells within CD25highCD4+ (P < 0.05), CD25lowCD4+ (P < 0.01) and CD25 CD4+ (P < 0.05) subpopulations (Fig. 6B). No effect of the drug was observed on the percentage of TGF-b+ cells in any of the evaluated lymphocyte subpopulations (Fig. 6C).

4. Discussion As has been said before, there have been few reports in the available literature (Hoek et al., 2009; Seo et al., 2007, 2009) on bovine CD25+CD4+ lymphocytes, and their findings are frequently contradictory (reviewed in Mas´lanka, 2011). The obtained results have shown that bovine CD25highCD4+ and CD25lowCD4+ lymphocytes express Foxp3, but this applies only to a few percent of the cells. Assuming that Foxp3 determines regulatory potential of CD25+CD4+ cells, the findings concerning Foxp3 expression remain in line with those of a study by Hoek et al. (2009), who have shown bovine CD25highCD4+ and CD25lowCD4+ cells not to have any regulatory properties. Therefore, the results show that in terms of Foxp3 expression and suppressor properties (Hoek et al., 2009), bovine CD25highCD4+ and CD25lowCD4+ cells are not adequate to human or mouse cells with such phenotype. Obviously, one cannot claim that there are no regulatory cells among bovine CD4+ lymphocytes, because there are regulatory cells Tr1 (Roncarolo et al., 2006) and Th3 (Faria and Weiner, 2006), known to occur in humans and in mice, which are also included in CD4+ lymphocyte subpopulation, but there are no data about their occurrence in ruminants. Moreover, the results of a study conducted by Seo et al. (2007) showed that there are certain cells with regulatory properties among bovine CD4+ cells. Expression of Foxp3 in CD25+CD4+ cells has also been determined in other studies (Hoek et al., 2009; Seo et al., 2009), but due to very vague information about it, it is impossible to perform an exact comparison of those findings with those of our study. Hoek et al. (2009) found expression of Foxp3 to occur only in CD25highCD4+ lymphocytes, whereas this factor was not detected in CD25lowCD4+ cells; however, they contained its mRNA. Seo et al. (2009) evaluated expression of Foxp3 with respect to total population of CD25+CD4+ and found some naive lymphocytes in the population to be Foxp3+. However, neither of the publications provides the percentage of cells which expressed this factor. Histograms shown in Seo et al. (2009) suggest (no actual figures are provided) that it took place only in a small percentage of CD25+CD4+ and the percentage of Foxp3+CD25+CD4+ increased after stimulation with SEC1. The most precise and comparable data are contained in a recently published paper by Gerner et al. (2010), whose findings indicate that the percentage of Foxp3+ CD25highCD4+ and CD25lowCD4+ cells was equal to 34.8% and 4.5%, respectively. It is difficult to find an explanation for such a great discrepancy between the findings presented in Gerner et al. (2010) and those of this study in terms of Foxp3 expression in CD25highCD4+ cells; however, it should be pointed out that only in our study was commercial anti-bovine Foxp3 mAb used. Hoek et al. (2009) and Gerner et al. (2010) used nonspecies-specific antibodies, although they showed cross reactivity

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with bovine Foxp3. Only Seo et al. (2009) used an anti-bovine Foxp3 mAb, although that was an antibody produced for their own needs. Regardless of the differences between the findings of experiments conducted by different research teams with respect to Foxp3 expression in bovine CD25+CD4+ cells, it is beyond doubt that the cells contain little Foxp3 as compared to human or mouse CD25+CD4+ cells. It was found in the study of You et al. (2007) that about 98% of mouse CD25highCD4+ expressed Foxp3, whereas there were 83.5% (BALB/c mice) or 91.3% (C57BL/6 mice) Foxp3+ cells in the CD25lowCD4+ subpopulation. It has been shown by Meier et al. (2009) that the percentage of Foxp3+ cells among the CD25highCD4+ cells in human PBMCs was about 83%; however, its value was much lower for CD25lowCD4+ PBMCs – it was about 13%. There have been other studies (Chen et al., 2008), whose findings also concerned human PBMCs; it has been found that the percentage of Foxp3+ cells was equal to 95.34% in the CD25highCD4+ and 38.01% in the CD25lowCD4+ subpopulation. Considering all this, it is beyond doubt that bovine lymphocytes of the CD25highCD4+ and CD25lowCD4+ subpopulations are not equivalent to human or mouse regulatory cells of this phenotype. With the current knowledge, it is difficult to say whether those small subpopulations of bovine Foxp+CD25highCD4+ and Foxp+CD25lowCD4+ lymphocytes play any role in inducing immune tolerance. No presence of Foxp3 in CD25 CD4+ cells has been detected in this study, which is in line with the findings of Seo et al. (2009) and Hoek et al. (2009), although Gerner et al. (2010) observed Foxp3 expression in about 0.5% of CD25 CD4+ cells. It is possible that in cattle, as in humans, transient expression of Foxp3 may take place in CD25 CD4+ cells (Wang et al., 2007). There have been some reports in the available literature on the percentage of CD25+ cells in bovine CD4+ PBMCs, but only the publication of Hoek et al. (2009) provide some data on the distinction between high and low CD25 expression. It has been found in the present study that 2.18% and 7.68% of CD4+ PBMCs on average showed high and low expression of CD25 molecule, respectively, therefore, a total of 9.86% of the cells were CD25+ (those are control values, obtained 12 h after incubation, but the values obtained for 24, 48 and 168 h were very close). Furthermore, it was found by Hoek et al. (2009) that an average of 1.9% and 23.1% CD4+ PBMCs were CD25high and CD25low, respectively. Therefore, the results shown here differ only slightly from the data on the percentage of CD25highCD4+ cells, but it has been shown in this study that there are three times fewer CD4+ cells with low CD25 expression as compared to what Hoek et al. (2009) noted. The reason for the discrepancy probably lies in the difference between the ages of animals used for the experiments, as numerous studies have shown that the percentage of CD25+CD4+ PBMCs in cattle increases with age. In this study, PBMCs were obtained from 12-month-old animals, whereas Hoek et al. (2009) conducted their experiments on cows at the age of over 2. It has been shown in other studies (Isaacson et al., 1998) conducted on cattle at the age of over 4.5 that about 31–33% of freshly isolated CD4+ PBMCs, incubated for 20 h, showed expression of CD25. Furthermore, Seo et al., (2007) detected the presence of CD25 on 13.5% of CD4+ PBMCs from steers aged 10–18 months. It was observed in the study of Kampen et al. (2006), conducted on calves at the age of 6 months or younger that only 6.6% of CD4+ PBMCs showed expression of CD25 molecule. The findings of the study of Quade and Roth (1999), conducted on calves at the age of 3–6 months, show that the presence of the CD25 antigen was present on about 10% of CD4+ PBMCs. Therefore, considering the significant effect of age on the percentage of CD25+CD4+ PBMCs, it may be claimed that the obtained results with respect to total (high + low) expression of CD25 on CD4+ PBMCs are relative consistent with the above mentioned results. Moreover, confrontation of the results of this study with the findings of Hoek et al. (2009) may produce a hypothesis that the

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percentage of CD25+CD4+ PBMCs which increases with age is rather that of CD25lowCD4+ subpopulation than CD25highCD4+ cells. Taking into account the intrinsically poor Foxp3 expression in bovine CD25+CD4+, and the fact that other researchers have shown that in bovine co-culture suppression assays these cells were neither anergic nor suppressive (Hoek et al., 2009), it becomes rather clear that bovine CD25highCD4+ and CD25lowCD4+ cells are mostly activated effector lymphocytes. This is because the CD25 molecule is a lymphocyte activation indicator. Therefore, despite the fact that the mouse CD4+CD25+ cells are regarded as equivalent to Tregs, the cells in fact share the phenotype with activated CD4+ lymphocytes. Unlike activated CD4+ cells with only a transient presence of the CD25 molecule, their expression in mouse regulatory lymphocytes is constant, hence they are called ‘‘regulatory T lymphocytes with constitutive expression of CD25’’, although it does not make it possible to identify proper regulatory cells within the CD4+CD25+ subpopulation (Malek, 2008). Regulatory cells in mice occur both in the CD25highCD4+ and in the CD25lowCD4+ subpopulation, with Foxp3 being the marker which identifies them. Human CD25lowCD4+ lymphocytes do not have regulatory properties and are regarded as activated effector lymphocytes (Condomines et al., 2006), although a considerable percentage of them show the presence of Foxp3+. ‘‘True’’ regulatory cells are found only in CD25highCD4+ subpopulation (Baecher-Allan et al., 2001), although the subpopulation also contains activated effector cells. This study has shown that dexamethasone increases the percentage, and the absolute number after prolonged exposure of CD25highCD4+ and CD25lowCD4+ cells, whereas it reduces the number of CD25 CD4+ lymphocytes. The obtained results indicate that changes in the number of the cells are the consequence of the effect of the drug on apoptosis of the cells from those subpopulations (although the effect of the drug on expression of CD25 cannot be ruled out). The reduction of the absolute number of CD25 CD4+ cells was undoubtedly caused by proapoptotic effect of the drug on those cells, because the percentage of early apoptotic cells in CD25 CD4+ lymphocytes was found to increase significantly after 12 h of incubation in the presence of the drug. Such an effect was not observed after 24 h of exposure of the cells to the drug, which is in line with the results for reduction of the total number of CD25 CD4+ lymphocytes. The reduction rate of the number of those cells after 12 h did not increase; it remained relatively constant after 24 and 48 h, whereas no significant differences were observed after 7 days in terms of the absolute number of CD25 CD4+ lymphocytes between control samples and those cultured in the presence of the drug. This leads to the conclusion that the first 12 h revealed the proapoptotic effect of dexamethasone on CD25 CD4+ cells, but this effect was disappeared after 24 h of incubation. The transient nature of proapoptotic effect of the drug’s effect may imply that only certain pool of CD25 CD4+ lymphocytes may be sensitive to its action. It was found at each of the time points that dexamethasone increased the percentage of CD25highCD4+ and CD25lowCD4+ cells. Initially, the increase must have been relative because it was the consequence of actual reduction of the number of CD25 CD4+ cells as a result of intensified apoptosis. Prolonged exposure to the drug resulted in an increase in the absolute number of CD25highCD4+ and CD25lowCD4+ cells. This was at least partly caused by the antiapoptotic effect of the drug on CD25highCD4+ and CD25lowCD4+ cells, because dexamethasone was found to significantly reduce the number of early apoptotic cells in those subpopulations. Chen et al. (2004) have shown that murine CD25+CD4+ cells are more resistant to dexamethasone-mediated cell death than are CD25 CD4+ cells. A comparison of those findings with this study shows that the tendency is consistent; however, it must be emphasized that this study has revealed that dexamethasone has a proapoptotic effect on CD25 CD4+ lymphocytes in cattle and antiapoptotic effect on CD25+CD4+ ones,

whereas Chen et al. (2004) observed the proapoptotic effect of the drug on cells in both of the subpopulations, with CD25+CD4+ lymphocytes being less susceptible to the effect than CD25 CD4+ cells. There has been only one report about the effect of SAIDs on the percentage of CD25+CD4+ cells in cattle (Menge and Dean-Nystrom, 2008), whereas there have been none on their effect on Foxp3 expression in the species. Menge and Dean-Nystrom (2008) observed an increase in the percentage of CD25+CD4+ cells in blood of calves 48 h after parenteral administration of dexamethasone. They regarded these cells as activated effector lymphocytes, although they considered a possibility that they may have been Tregs. Other researchers (Anderson et al., 1999) determined the effect of dexamethasone on the percentage of CD25+ cells in cattle, but that applied only to PBMCs. It was shown in that study that the administration of dexamethasone to animals resulted in an increase in the percentage of CD25+ PBMCs, which was also interpreted as an increase in the number of activated cells. This interpretation is supported by results obtained by Chung et al. (2004). These authors cultured human CD4+CD25 and CD4+CD25+ cells in the presence of dexamethasone and/or IL-7 for 4 days. Although two thirds of CD4+CD25 cells became CD4+CD25+ cells, they had no suppressive activity. In contrast, the original CD4+CD25+ cells maintained suppressive activity after dexamethasone /IL-7 treatment, however, there was not a significant expansion in their cell number. Thus, dexamethasone and IL-7 did not induce additional Tregs cells, but additively induced the expression of the activation marker CD25 by CD4+CD25 T cells (Chung et al., 2004). Moreover, it has been shown that glucocorticosteroids enhance the expression of high-affinity IL-2 receptors (CD25) in different mouse T cell lines, by specifically inducing transcription of the IL-2Ra gene (Lamas et al., 1993). However, later, and especially in recent years, opinions about the nature of human and mouse CD4+CD25+ generated with the use of glucocorticosteroids have changed, because the findings of a number of studies have shown or implied (Karagiannidis et al., 2004; Braitch et al., 2009; Provoost et al., 2009; Xie et al., 2009) that those are regulatory cells rather than activated lymphocytes. Braitch et al. (2009) reported that methylprednisolone increased the percentage of CD25highCD4+ cells, plasma IL-10 and Foxp3/CD3 ratio in patients with multiple sclerosis. Findings of others (Provoost et al., 2009) suggest that treatment with inhaled glucocorticosteroids in asthmatics may increase Foxp3 expression within the CD25highCD4+ cells. Also, Karagiannidis et al. (2004) demonstrated that glucocorticosteroids upregulated Foxp3 and CD25+CD4+ cells in asthmatic patients. Furthermore, dexamethasone induced IL-10 and Foxp3 expression in short-term and long-term cultures. Other results (Xie et al., 2009) indicate that co-stimulation with dexamethasone and IL-2 selectively expand the functional Foxp3+CD25+CD4+ cells in vivo. Moreover, it was found that in mice treated with dexamethasone the proportion of CD25+CD4+ cells and the ratio of CD25+CD4+ cells to CD25 CD4+ cells was enhanced in the lymphoid organs (Chen et al., 2004). An increase in the percentage of CD25+CD4+ cells in all of those studies was interpreted as an increase in the number of regulatory cells, which was sometimes supported by results of additional studies. In general, the results of the studies have shown that an important, if not the leading, role in immunosuppressive and anti-inflammatory properties of glucocorticosteroids is played by generating regulatory cells with the Foxp3+CD25+CD4+ phenotype. However, the results of the latest studies (Prado et al., 2011) have contradicted the theory and they seem to go back to the original concept, according to which CD25+CD4+ generated with the use of glucocorticosteroids are not regulatory cells. Prado et al. (2011) have shown that dexamethasone increased expression of Foxp3 in human CD25highCD4+ cells, which were generated in vitro from CD25 CD4+ lymphocytes after appropriate stimulation. However,

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the cells did not show any significant suppressive activity in functional assays. Foxp3 expression has been shown to increase in CD25+CD4+ cells generated by means of TGF-b (TGF-b Tregs) in the presence of dexamethasone. The cells showed suppressive activity, but it was not greater than TGF-b Tregs generated without the drug. Moreover, in this study, it was demonstrated that CD4+CD25high cells from systemic lupus erythematosus (SLE) patients under glucocorticoid therapy did not exhibit a more enhanced suppressive function than those from SLE patients not receiving this treatment (Prado et al., 2011). Therefore, the results indicate that SAIDs may generate in human cells with Treg phenotype, but the cells do not have regulatory properties. Considering the fact that human effector lymphocytes may temporarily show the presence of Foxp3 (Wang et al., 2007) and that glucocorticosteroids increase expression of CD25, it is justified to suppose that CD25+CD4+ cells generated by means of those drugs could be just a fraction of activated effector lymphocytes. Hopefully, the nearest future will bring a clear answer about the real nature of Foxp3+CD25+CD4+ generated with the use of glucocorticosteroids. It has been shown in this study that dexamethasone increased the number of CD25+CD4+ by modifying apoptosis of CD4+ lymphocytes; however, its effect on expression of CD25 in CD25 CD4+ cells has not been examined; therefore, it would not be justified to claim that the drug generated CD25+CD4+ lymphocytes from CD25 CD4+, cells, although it cannot be ruled out that such a process may have taken place and contributed to an increase in the number of lymphocytes in the CD25highCD4+ and CD25lowCD4+ subpopulations. Summarizing the results concerning the effect of dexamethasone on the number of CD25+CD4+ lymphocytes, it can be said that the drug increased the percentage, and, after prolonged exposure, also the absolute number of Foxp3 CD25+CD4+ cells, which were most probably activated non-regulatory lymphocytes. Knowing the immunosuppressive properties of dexamethasone, this effect may seem paradoxical, but apparently it exists, and the evaluation of its importance requires more research. The immunosuppressive effect of dexamethasone is beyond doubt; however, this does not mean that the drug: (a) may not manifest any effect which could be associated with activating an element contributing to such a complex reaction as immunological or inflammatory, (b) must inhibit both of those reactions on all levels. Taking into account the literature reports on the effect of SAIDs on expressions of Foxp3, surprisingly, we found that dexamethasone reduced the percentage of Foxp3+ cells in the CD25+CD4+ subpopulation, however, it was compensated (data not provided) by increasing the absolute number of lymphocytes in this subpopulation. The reducing effect of dexamethasone on the percentage of Foxp3+ cells is in contradiction with the results quoted above (Braitch et al., 2009; Karagiannidis et al., 2004; Provoost et al., 2009; Xie et al., 2009). Most probably, the drug effect is species specific, although, interestingly, a report has been published recently (Xiang and Marshall, 2011), which claims that human PBMCs treated with dexamethasone showed reduced expression of mRNA for Foxp3 as compared to control samples. Our study has shown that dexamethasone reduced the percentage of IL-10-producing cells, both in the subpopulations of CD25highCD4+ and CD25lowCD4+ lymphocytes, and within CD25 CD4+ cells. We have not found the drug to affect production of TGF-b by cells in the subpopulations mentioned above. Therefore, the results of our study indicate that production of the two most important anti-inflammatory cytokines, i.e. IL-10 and TGFb, by bovine CD4+ is not involved in triggering anti-inflammatory or immunosuppressive effect of dexamethasone. The findings indicate that the participation of bovine CD4+ lymphocytes in the two pharmacological effects of dexamethasone is rather associated with the depressive effect of the drug on the production of the key inflammatory cytokine IFN-c. We have shown dexamethasone

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to have a significant reducing effect on the percentage of IFN-cproducing cells in the CD25 CD4+ subpopulation. Considering additionally the fact that the drug caused temporarily serious reduction of the absolute number of CD25 CD4+ lymphocytes, it becomes clear that the total reduction of IFN-c+CD25 CD4+ was much greater than is shown by the percentage data. The decrease of IFN-c production could be due to an influence of dexamethasone on nuclear factor kappa B (NF-kB). This assumption is based on the following two reasons: (a) NF-kB plays central role in induction of a large number of important immunoregulatory genes, including this encoding IFN-c (Baeuerle, 1993; Grilli et al., 1993), (b) it has been demonstrated that glucocorticoids are potent inhibitors of NF-kB activation in mice and cultured cells (Auphan et al., 1995). Analysis of the available literature shows that it is not possible to precisely compare the results of this study with those of other studies because there are no data available on evaluation of the effect of any anti-inflammatory drugs on the percentage of CD4+ cells producing IFN-c, or any other cytokine in cattle. There are also no data on the effect of SAIDs on the percentage of IL-10+, TGF-b+ and IFN-c+ cells among CD4+ lymphocytes in other animals or humans. However, there are results of studies which examined the effect of dexamethasone on secretion of IL-10 and IFN-c by sorted CD4+ lymphocytes. Ramirez (1998) found dexamethasone to reduce secretion of IFN-c by rat’s CD4+ lymphocytes. In line with those findings are the observations made by Choy et al. (1999) who found that dexamethasone caused dose-dependent suppression of IFN-c mRNA expression in CD4+ cells. Thus, the data quoted above are in agreement with the results of this study. It is different in terms of the effect of dexamethasone on production of IL-10. The drug has been found to increase the secretion of interleukin by human CD4+ cells (Richards et al., 2000), or not affect this process (Bartels et al., 2007), whereas this study has shown considerable reduction of the percentage of IL-10 producing CD4+ (both CD25+ and CD25 ) induced by dexamethasone. Such an effect of the drug may seem paradoxical because knowledge of the anti-inflammatory and immunosuppressive properties makes one expect the opposite effect, or no effect of the drug on production of IL-10. The experiment results and the current knowledge do not provide a basis for elucidation of the phenomenon. However, it does seem possible that reduction of the number of IL-10+CD4+ lymphocytes is not a direct result of the effect of dexamethasone, but it is an attempt to compensate suppression of the Th1 type response, resulting from the drug-induced heavy impairment of IFN-c production by CD25 CD4+ lymphocytes. Considering the obtained results in view of the current knowledge on the effect of dexamethasone on production of IL-10 and IFN-c one must not fail to mention the results of a study of the effect of the drug on cytokine production by PBMCs. Menge and Dean-Nystrom (2008) showed that the amount of mRNA for IFN-c in properly stimulated PBMCs obtained from cattle which received dexamethasone was significantly reduced as compared to the control values. However, other studies (Anderson et al., 1999), in which the level of IFN-c in the medium obtained from culture of PBMCs of cattle which the received dexamethasone was determined, did not show the drug to affect production of this cytokine. This finding remains in contrast with the results of similar studies on human PBMCs (Franchimont et al., 1998; Agarwal and Marshall, 2001; Brandl et al., 2007; Salicrú et al., 2007). Franchimont et al. (1998) and Brandl et al. (2007) have shown dexamethasone to reduce both production of IFN-c and IL-10 by human PBMCs. On the other hand, Salicrú et al. (2007) also observed an inhibiting effect of the drug on IFN-c synthesis by human PBMCs, but they obtained different results in terms of its effect on production of IL-10, as they found that dexamethasone significantly increased production of this interleukin. The same results were achieved by Agarwal and Marshall (2001). Those researchers have postulated that dexamethasone promotes

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type 2 cytokine production primarily through inhibition of type 1 cytokines. It is clear that the data from above mentioned studies are not exactly comparable with respect to the findings presented here because our study has evaluated the effect of dexamethasone on production of IL-10 and IFN-c only by CD4+ lymphocytes, whereas the drug could affect the production of those cytokines by other subpopulations of T cells (e.g. WC1+), or other cell types (NK) which comprise PBMCs. Nevertheless, it is noteworthy that nearly all the results of the studies quoted above have shown dexamethasone to reduce IFN-c production, which is correlated with the findings of this study. In addition, we have only contradictory data on the effect of the drug on IL-10 synthesis, which nevertheless show that observations made in this study on inhibiting effect of dexamethasone on production of IL-10 are not isolated. Summarizing all the findings of this study, it should be first raised that bovine CD25highCD4+ and CD25lowCD4+ lymphocytes are not equivalent to human or mouse regulatory cells with this phenotype in terms of Foxp3 expression, because only a small percentage of the cells shows the presence of this factor. Dexamethasone has been found to cause a considerable reduction of CD25 CD4+ cells, but it increased the relative and the absolute numbers of CD25highCD4+ and CD25lowCD4+ lymphocytes, while at the same time reducing the percentage of Foxp3+ cells in those subpopulations. Considering this and the intrinsically poor Foxp3 expression in bovine CD25+CD4+, it can be concluded that the drug the most probably increased the number of activated non-regulatory lymphocytes. The increase in the absolute number of CD25highCD4+ and CD25lowCD4+ cells compensated the reduced percentage of Foxp3+ lymphocytes, therefore a decrease in the absolute number of Foxp+CD25highCD4+ and Foxp+CD25lowCD4+ cells did not take place. It has been found that changes in cell number were at least partly caused by proapoptotic effect of the drug on CD25 CD4+ cells and antiapoptotic effect on CD25highCD4+ and CD25lowCD4+ cells. The proapoptotic effect of dexamethasone had a transient character, which may imply that only a certain pool of CD25 CD4+ was sensitive to it. The obtained results indicate that the involvement of CD4+ lymphocytes in forming the anti-inflammatory and immunosuppressive effect of dexamethasone resulted from the fact that the drug has a depressive effect on production of key proinflammatory cytokine – IFN-c – by CD25 CD4+ cells. Secretion of TGF-b and IL-10 by CD4+ lymphocytes was not involved in forming these pharmacological effects because the drug did not affect production of TGF-b and, paradoxically, it reduced the percentage of IL-10+CD4+ cells.

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