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The Egyptian Journal of Histology:
doi: 10.1097/01.EHX.0000413854.99170.ea
Original articles

Effect of glucocorticoids on surface IL-10 receptors and intracellular IL-10 in mouse splenocytes, thymocytes, monocytes, and dendritic cells: immunofluorescence, confocal microscopy, and electrophoresis study

Abd Elrahman Ahmed, Mohamed

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Author Information

Department of Histology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Correspondence to Mohamed Abd Elrahman Ahmed, Department of Histology, Faculty of Medicine, Ain Shams University, Cairo, Egypt Tel: +20 111 437 7660; Fax: +20 2 26837673; e-mail: mekawy906@hotmail.com

Received November 4, 2011

Accepted January 12, 2012

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Abstract

Background: Glucocorticoids are capable of stimulating the secretion of interleukin-10 (IL-10) by leukocytes. The probable role of glucocorticoids in the susceptibility of immune cells to IL-10-mediated actions has not yet been studied. In this study, we examined the expression of IL-10 and IL-10 receptors (IL-10R) in mouse splenocytes, thymocytes, monocytes, and dendritic cells. In addition, we determined the effects of the glucocorticoid (methylprednisolone) on IL-10 secretion and IL-10R expression in the previous immune cells.

Methods: Forty-eight male mice with the C57BL/6 genetic background were used. The animals were 10–12 weeks of age and were divided equally into two groups. The animals in group I were provided with a balanced standard diet and water ad libitum. The animals in group II were provided with a balanced standard diet and methylprednisolone (0.5mg/kg/day) in the drinking water daily for 7 days. The mice in groups I and II were sacrificed after 7 days and the spleen and thymus were removed to isolate the splenocytes and thymocytes. The blood was also aspirated to isolate monocytes. Group III included mice with the mouse dendritic cell line (JAWS II). Cells of the mice in this group were divided into two subgroups. Group IIIa included the dendritic cells cultured in Iscove's modified Dulbecco's medium and group IIIb included the dendritic cells cultured in Iscove's modified Dulbecco's medium supplemented with methylprednisolone (2μg/ml) for 6 h. All the previous cells were processed for immunohistochemistry, western blotting, immunoassay analysis, and electrophoresis.

Results: All the cells examined showed an intense fluorescent surface expression of IL-10R as detected by the immunofluorescence technique. On using methylprednisolone, there were few and faint expressions of IL-10R. The intracellular IL-10 molecules were few in the dendritic cells but after treatment with methylprednisolone, the IL-10 molecules were numerous and intense. Western blotting and electrophoresis techniques showed intense precipitation of IL-10 at 35 kDa and faint precipitation of IL-10R at 110 kDa in all the cells examined after glucocorticoid treatment. There was also a highly significant (P < 0.001) increase in the level of IL-10 in all the cells examined after using methylprednisolone as detected by immunoassay.

Conclusion: Our findings could contribute toward understanding the immunomodulating mechanism of the action of glucocorticoids, which may result in greater therapeutic benefit in some diseases by enhancing the synthesis of the anti-inflammatory cytokine IL-10 and decreasing the expression of IL-10R in some immune cells.

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Introduction and aim of the work

Interleukin-10 (IL-10) is an anti-inflammatory and immunosuppressive cytokine produced as a result of immune activation of helper T cells [1], B cells [2], and macrophage/monocytes [3,4]. A previous study in mice showed that IL-10, a 35 kDa protein, could also be secreted by mast cells, counteracting the inflammatory effect that these cells have at the site of an allergic reaction [5].

IL-10 functions as an important regulator of the immune system. IL-10 has potent and broad-spectrum anti-inflammatory activity in infection, inflammation, and even in cancer [6]. It was found that IL-10 downregulates the T-helper response by attenuating the function of T cells, dendritic cells, monocytes, and macrophages and decreases the secretion of proinflammatory mediators such as tumor necrosis factor α, granulocyte–macrophage colony-stimulating factor, IL-4, and IL-5 [7]. The administration of IL-10 was found to suppress allergic inflammation in sensitized animals [8].

Some authors have reported that IL-10 inhibits macrophage cytokine production and the accessory functions of macrophages and enhances B-cell survival, proliferation, and antibody production [9]. In addition, IL-10 was found to suppress IFN-γ and monokine production [10–12].

IL-10 mediates its biological effects by interacting with the specific cell surface IL-10 receptor (IL-10R), which is a glycoprotein and consists of α and β chains [13]. IL-10R were detected on T cells, B cells, monocytes, macrophages, dendritic cells, NK cells, mast cell epithelial cells of the small and large intestines, epidermal cells, and keratinocytes [14,15].

The interaction of IL-10R with IL-10 seems to be highly complex and has been understood only partially. The murine IL-10R, 110 kDa, binds IL-10 to initiate the transduction of a signaling cascade, leading to a modification of biological responses [16]. A previous study has reported reduced expression of IL-10R on neonatal T lymphocytes [17]. This may play a crucial role in the deficient anti-inflammatory immune response in neonates. Endotoxin was found to increase the expression of IL-10R on fibroblasts [18]. Some authors have found that topical applications of glucocorticoids and vitamin D3 increase IL-10R expression in human epidermal cells [14,19]. Moreover, the expression of IL-10R was increased on monocytes after a Schistosoma mansoni infection [20]. Previous studies have shown that toll-like receptor ligands inhibit IL-10R signaling in lung dendritic cells, thus preventing the immunosuppressive activity of IL-10 [21].

Dendritic cells are part of the mammalian immune system. Paul Langerhans first described dendritic cells in the late 19th century. Dendritic cells process antigen and present it on its surface to other cells of the immune system. They are present in the skin (Langerhans cells), nose, lungs, stomach, and intestines. They are also found in an immature state in the blood, and once activated, they migrate to the lymph nodes, where they interact with T and B cells, initiating an immune response [22]. Freshly isolated Peyer's patch dendritic cells can produce IL-10 and induce the differentiation of Th2 cells [22].

Glucocorticoids are widely used to suppress inflammation in autoimmune and allergic diseases such as asthma and rheumatoid arthritis, but their mode of action remains unclear. It has been reported that the secretion of endogenous IL-10 might be augmented by glucocorticoids [23]. However, how glucocorticoids modulate the pharmacological action of chemotherapeutic agents remains to be clarified.

There are no sufficient data describing the effects of glucocorticoids on IL-10 and IL-10R in immune cells. In this study, we aimed to determine the effect of the glucocorticoid methylprednisolone on the secretion of IL-10 and the expression of IL-10R in mouse splenocytes, thymocytes, monocytes, and dendritic cells to examine the mechanism of action of corticosteroids, which result in greater therapeutic benefit.

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Materials and methods

Animals

Forty-eight male mice of the C57BL/6 genetic background and 10–12 weeks of age were used in this study. The animals were bred and maintained in a temperature-controlled (21±2°C) and light-controlled (lights on between 07:00 and 20:00h) room in the Department of Biological Sciences at Essex University, UK, and were provided with food and water ad libitum. The animals were divided equally into two groups.

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Grouping

Group I: This group included 24 animals that were provided with a balanced standard diet and water ad libitum.

Group II: This group included 24 animals that were provided with a balanced standard diet and received methylprednisolone at a dose of 0.5mg/kg/day in drinking water daily for 7 days. It was purchased from Imperial drug stores (North Promenade, Blackpool, UK).

Group III: This group included the JAWS II mouse dendritic cell line, which was divided into:

Group IIIa: This group included the JAWS II mouse dendritic cell line cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum (FCS) (v/v).

Group IIIb: This group included the JAWS II mouse dendritic cell line cultured in IMDM supplemented with 10% FCS (v/v) and methylprednisolone at a dose of 2μg/ml for 6h.

The mice in groups I and II were anesthetized by halothane inhalation. The spleen and the thymus gland were quickly removed and placed in separate Petri dishes. Each dish contained 5ml of PBS to isolate the splenocytes and thymocytes. The blood was also aspirated from groups I and II and placed in separate tubes. Each tube contained citrate to prepare a blood buffy coat to isolate monocytes.

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Dendritic cell line

The cell line JAWS II cell line was used in this study. This dendritic cell line was obtained from bone marrow cells of a p53-knockout C57BL/6 mouse [24]. It was purchased from the American Type Culture Collection (CRL-1194; ATCC, Manassas, Virginia, USA). Cells were grown in a CO2 incubator at 37°C and 5% CO2 in a complete culture medium consisting of IMDM with 10% FCS, 4mmol/l l-glutamine, 10U/ml penicillin and 100μg/ml streptomycin, 0.5mmol/l 2-mercaptoethanol, 1mmol/l sodium pyruvate, and 5ng/ml murine granulocyte-macrophage colony-stimulating factor. The medium was placed in the incubator for at least 15min to allow it to reach its normal pH (7.0–7.6) before cells were added. Cultures were maintained by transferring nonadherent cells to a centrifuge tube and treating attached cells with 0.25% trypsin-0.03% EDTA (Gibco, Renfirewshire, UK) at 37°C for 5min, followed by pooling the two populations of cells together, and dispensing them into new 90mm tissue culture dishes at a density of 6×105 per dish.

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Tissue culture

All manipulations were carried out using standard tissue culture techniques. All glass pipettes were sterilized at 180°C for 2h. In addition, disposable pipettes were used. IMDM, used for culturing dendritic cells, penicillin, streptomycin, 2-mercaptoethanol, and sodium pyruvate, was purchased from Sigma (St Louis, Missouri, USA). FCS and l-glutamine were obtained from Gibco. Murine granulocyte-macrophage colony-stimulating factor was purchased from R&D Systems (Minneapolis, Minnesota, USA). Tissue culture-grade plastics were (90, 50, and 35mm diameter Nunclon dishes) obtained from Nunc (Roskilde, Denmark).

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Chemicals

Paraformaldehyde (PFA) and saponin were purchased from Sigma (Hertfordshire, UK). 1,4-Diazobicyclo[2.2.2]octane (DABCO) was purchased from Fisher Scientific International Company (Loughborough, UK). All other reagents were from Fisons (Guildford, UK), or Anglia Chemicals (Norfolk, UK).

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Antibodies

The antibodies used for indirect immunofluorescence in this study were as follows:

1. IL-10 antibody (JES5-2A5) rat anti-mouse specific to detect mouse IL-10 was purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

2. IL-10R β (P-20) rat anti-mouse antibody was purchased from Santa Cruz Biotechnology Inc.

3. The secondary antibody, which was used in the indirect immunofluorescence technique, was mouse anti-rat fluorescein isothiocyanate (FITC) conjugated. The secondary antibody, which was used in enhanced chemiluminescence (ECL) and biotinylation techniques, was mouse anti-rat horseradish peroxidase (HRP). They were purchased from DAKO Ltd (Buckinghamshire, UK).

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Isolation of mouse splenocytes

Mice of groups I and II were anesthetized by Halothane inhalation. The spleen was then surgically removed by blunt dissection and placed in a Petri dish containing 5ml PBS prewarmed at 37°C [25]. A needle was used to disrupt the capsule of the spleen. Cells were extracted by pumping PBS through the spleen several times using a 1-ml syringe. The cell suspension was transferred gently by a Pasteur pipette to a 10ml centrifuge tube containing 3 ml of lymphoprep (Nycomed Pharma, Oslo, Norway) with a density of 1.077g/ml. The dish was washed with another 2ml of PBS to ensure that all cells were transferred to the tube so that lymphocytes could be separated from other cells by differential centrifugation on a density gradient [26]. The cells were then centrifuged at 2300 rpm for 20min at 22°C, after which the lymphocytes were visible as a white or a creamy ring located at the interface between the PBS and the lymphoprep. Other cells present had formed a pellet at the bottom of the centrifuge tube. The lymphocytes were removed using a Pasteur pipette and placed in a clean centrifuge tube, and then 5 ml PBS was added. Samples were spun at 1750 rpm at 22°C for 10min. The supernatant was aspirated off, leaving 0.25ml covering the pellet to prevent dehydration. The cell pellet was then washed twice with 5ml PBS and centrifuged as before. The cell pellet was then suspended in 5ml PBS and the cells were counted under a ×10 objective using a hemocytometer chamber.

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Isolation of mouse thymocytes

Thymocytes from mice were isolated by dissecting the thymus and disrupting the capsule by gentle flushing of 5ml of prewarmed PBS at 37°C into it a 5-ml syringe and a 25-G needle. The isolated cells were then collected and centrifuged at 1200rpm for 10min. The pellet was washed twice in PBS, suspended in warm PBS, and counted under a ×10 objective using a hemocytometer chamber.

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Isolation of mouse peripheral blood monocytes

Monocytes were isolated from a mouse-citrated blood buffy coat. The buffy coat was aspirated and diluted 1 : 2.2 with sterile saline and 6ml 6% dextran 500 (Sigma-Aldrich, Gillingham, Dorset, UK) was added and the tubes were placed at a 45° angle for 1h to allow red blood cells to sediment. The layer of leukocyte-rich plasma was removed and placed over Nycoprep 1.068 (Axis Shield, Kimbolton, UK). The mixture was centrifuged at 2000rpm for 15min. Following centrifugation, the top yellow layer was aspirated and discarded. The monocyte layer below was carefully removed and placed in a separate tube. The monocytes were washed twice with PBS and resuspended in a serum-free medium.

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Immunofluorescence staining of interleukin-10 receptors on the surface of splenocytes, thymocytes, dendritic cells, and monocytes

A suspension of at least 5×105 cells were placed in a small microcentrifuge tube and centrifuged for 1.5 min to pellet the cells. The supernatant was discarded and the pellet was washed twice using 300μl of PBS and resuspended by gentle pipetting several times. The tube was then centrifuged to pellet the cells and the supernatant was discarded. The cells were fixed by adding 400μl of 4% PFA to the pellet, resuspended by gentle pipetting, and incubated for 15min at room temperature (RT). The tube was centrifuged and 500μl of a blocking solution (PBS/0.02% FCS/0.02% NaN3) was then added to the cell pellet, resuspended, and incubated for 30min at RT. The tube was centrifuged and 50 μl of IL-10R antibody was added at a dilution of 1 : 20 to the cell pellet and resuspension was carried out by gentle pipetting. The cells were incubated overnight at 4°C. The tube was then centrifuged and the pellet was washed thrice with 500μl of the same previous blocking solution. The pellet of cells was resuspended and incubated with 50μl of diluted (1:20) FITC-conjugated secondary antibody for 2h in the dark at RT. After incubation with the secondary antibody, the cells were spun down using the bench-top microcentrifuge. The supernatant was carefully removed without touching the pellet and 500μl of the previous blocking solution was then added to the pellet to resuspend it. The last step was repeated thrice to ensure the complete removal of any unbound antibody, thus reducing the background fluorescence. Forty microliters of PBS/0.02% FCS/0.02% NaN3 was added to the pellet to resuspend it. A drop of DABCO was placed on a microscope slide along with a drop from the cell suspension. DABCO prevents fading of the fluorochrome with time. The cells were then covered with a clean cover slip and sealed by a colorless nail varnish. The slides were then examined under a ×60 oil immersion objective of an inverted immunofluorescence microscope (IMT2; Olympus, Tokyo, Japan). This microscope was equipped with a krypton/argon laser scanning confocal imaging system (MRC600; Biorad, Cambridge, Massachusetts, USA). All the images were taken using the 586-nm laser line, Kalman filtering (five scans), and natural density filter 1. Aperture, gain, and black levels were adjusted such that the nonspecific fluorescence detected in negative controls was removed.

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Immunofluorescence staining of intracellular interleukin-10 receptor in mouse dendritic cells

Cells cultured on cover slips in 35mm dishes were washed twice by PBS for 30 s each time. The cells were then fixed by 4% PFA for 15min at RT. The cells were permeabilized using 0.01% saponin in a special blocking solution (0.02% FCS/0.02% NaN3/1×PBS/0.01% saponin) for 20min at RT. The dishes were then washed twice with a blocking solution 0.02% FCS/0.02% NaN3/1×PBS/0.01% saponin. The cells on the cover slips were then incubated with 50μl of diluted primary antibody for 1h at RT. This was done by inversion of the cover slip over a small drop of the primary antibody on a square of parafilm in a moist chamber. The cover slip was then removed with the cell side up and placed in a small dish for washing. The cover slip was then washed thrice using the previous blocking solution. The cells were then incubated with 50μl of diluted (1 : 20) FITC-conjugated secondary antibody for 2h in the dark. This was also done by inversion of the cover slip over a small drop of secondary antibody on a square of parafilm in a moist chamber. The cover slip was then removed with fine tweezers and placed cell side up in a small dish and washed thrice by PBS for 5min each time. It was then mounted on a slide containing a drop of DABCO to prevent fading of the fluorochrome with time. Each cover slip was then sealed by a colorless nail varnish. The slides were then examined under a ×60 oil immersion objective of the confocal microscope using both the phase contrast and the blue filter.

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Cell lysis and membrane solubilization

Splenocytes or thymocytes or monocytes or dendritic cells in suspension were counted using the hemocytometer and washed twice by PBS. The cell suspension was divided into small mcc tubes; each tube contained about five million cells. The tubes were then subjected to centrifugation at 1200rpm for 5min at RT. A known volume (0.5ml) of SDS sample buffer [10% (v/v) glycerol, 50mmol/l DTT, 5% (w/v) SDS, 0.06mol/l Tris base, pH 6.8, and 0.02% (w/v) bromophenol blue] was added to the cell pellet and resuspended and kept for 5min. The tubes containing the cell lysates were then kept at −20°C until used for loading into the gel, followed by western blotting and ECL detection.

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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting

Cell lysates were analyzed on 10% acrylamide gels by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto a nitrocellulose filter (Schleicher-Schuell, Dassel, Germany) for 1h at 220 mA in the presence of transfer buffer (20mmol/l Tris-acetate, 0.1% SDS, 20% isopropanol, pH 8.3). After transfer, the membrane was blocked for 1h in blocking solution (5% low-fat dried milk dissolved in PBS-T) and washed with PBS-Tween (PBS-T), two rinses, 15-min wash. Membranes were probed with the appropriate dilution of the primary antibody (1:1000) against IL-10 or IL-10R for 1h at 22°C, followed by diluted (1:2000) HRP secondary antibody for 2h at RT, followed by washing with PBS-T. The antigen was visualized on the blotted membrane using the ECL procedure (Amersham Pharmacia, Hertfordshire, UK) according to the manufacturer's instructions.

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Enhanced chemiluminescence technique

The ECL technique is a highly sensitive technique. Protein detection by ECL may be achieved in less than 1min. The IL-10 and IL-10R proteins were detected in the immunoblotted membrane using ECL reagents. The detection reagents of ECL (Amersham Pharmacia) consist of detection solution 1 and detection solution 2. Equal volumes of detection solution 1 (1ml) were mixed with detection solution 2 (1ml) to cover the membrane. The membrane was placed on a piece of Saran Wrap present in an x-ray film cassette. The protein side of the membrane was up. The detection reagent mixture was added to the protein side of the membrane, so that the reagents were held by surface tension on the surface of the membrane, which was not allowed to become uncovered. The membrane was incubated for precisely 1 min with the mixture at RT without agitation. The excess of detection reagent was then drained off, the membrane was wrapped in Saran Wrap, and any air pockets were gently smoothed out. The membrane was placed protein side up in the film cassette and a sheet of autoradiography Hyperfilm-ECL (Amersham Pharmacia) was placed on top of the membrane after switching the light off in the dark room. The cassette was then closed to expose the film. Exposure times varied between 30 s and 15min. The cassette was then carefully opened and the film was removed immediately and developed in an automatic film processor (Geva Matic 60; Agfa Gevaert, Bonn Germany) according to the manufacturer's instructions.

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Interleukin-10 assay

Cells from all groups were placed in serum-free medium for 5h. The supernatants were collected and frozen until the cytokine assay was performed. The Becton Dickinson (Oxford, UK) Th1/Th2 cytokine bead array system was used to determine the level of IL-10 according to the manufacturer's instructions. The cytokine bead array Th1/Th2 kit uses bead population conjugated with the antibody specific for IL-10. The bead has a discrete fluorescence intensity that can be determined by flow cytometry.

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Morphometric and statistical study

The mean values and SDs were calculated and the levels of IL-10 were measured in the different groups that were subjected or not to methylprednisolone glucocorticoid. The number of readings were 15 for each cell type. Student's t-test was used for comparison and the P value was calculated using the SPSS program version 15, (Barcelona, Spain). Statistical significance was determined at a level of P value less than 0.05. Thus, P value greater than 0.05 was considered nonsignificant and P value less than 0.001 was considered highly significant.

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Results

Immunofluorescence detection of interleukin-10 receptor on the surface of splenocytes and thymocytes

After isolating the splenocytes and thymocytes from the spleen and the thymus of group I animals, the cells were centrifuged. The pellet of cells was subjected to the primary monoclonal antibody against IL-10R, followed by the secondary FITC-conjugated secondary antibody. The splenocytes appeared under the confocal microscope as small and rounded with intense multiple yellow fluorescent granules of IL-10R that were expressed on its surface (Fig. 1). The thymocytes also appeared as small and rounded cells with multiple deep yellow fluorescent granules of IL-10R on its surface (Fig. 2). Animals in group II were administered oral glucocorticoid. The splenocytes isolated from the animals of this group showed faint and a few yellow fluorescent granules of IL-10R on the surface (Fig. 3). Moreover, the thymocytes isolated from the animals in group II also showed a few and faint yellow fluorescent granules of IL-10R on the surface (Fig. 4). For the negative control, no primary antibody was added but the splenocytes or thymocytes were immunostained only with the FITC-conjugated secondary antibody. The cells showed negative staining and no granules were detected on the surfaces (Fig. 5).

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Immunofluorescence detection of interleukin-10 receptor on the surface of dendritic cells

The JAWS II dendritic cell line of group IIIa was cultured and collected in a suspension. After centrifugation, the primary monoclonal antibody, P-20, was used against IL-10R. It was added to the cell pellet, followed by the FITC-conjugated secondary antibody. The cells appeared under the confocal microscope as large and rounded. The cell surfaces of some cells were covered with multiple intense yellow fluorescent granules of IL-10R (Fig. 6). However, the dendritic cells of group IIIb, which were exposed to glucocorticoid for 6h in the culture, showed few and faint yellow fluorescent granules of IL-10R on the surface (Fig. 7). For the negative control, no primary antibody was added but the cells were immunostained only with the FITC-conjugated secondary antibody. The dendritic cells showed negative staining, with no granules detected on the surface (Fig. 8).

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Immunofluorescence detection of interleukin-10 receptor on the surface of monocytes

Peripheral blood monocytes of groups I and II appeared under the confocal microscope as large and rounded. The cell surface was covered with multiple intense yellow fluorescent granules of IL-10R (Fig. 9). However, the monocytes of group II, which were exposed to methylprednisolone, showed few faint and yellow fluorescent granules of IL-10R on the surface (Fig. 10). For the negative control, no primary antibody was added but the cells were immunostained only with the FITC-conjugated secondary antibody. The monocytes showed negative staining, with no granules detected on the surface (Fig. 11).

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Immunofluorescence detection of intracellular interleukin-10 in dendritic cells

The intracellular expression of IL-10 was examined on the adherent dendritic cell line of group IIIa. These cells were grown in culture and subcultured on coverslips. The cells were fixed and examined by the indirect immunofluorescence technique using the JES5-2A5 antibody specific against mouse IL-10. Immunofluorescence was detected and recorded using confocal laser microscopy. The cells appeared branched with multiple processes. The cytoplasm was filled with a few yellow granules of IL-10 (Fig. 12). However, the dendritic cells of group IIIb, which were exposed to glucocorticoid for 6 h in the culture, showed many intense yellow fluorescent granules of IL-10 in their cytoplasm (Fig. 13). For the negative control, no primary antibody was added but the cells were immunostained only with the FITC-conjugated secondary antibody. The dendritic cells showed negative staining, with no granules detected in their cytoplasm (Fig. 14).

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Immunoblotting and enhanced chemiluminescence detection of the molecular weight of interleukin-10 receptor in splenocytes, thymocytes, monocytes, and dendritic cells

The SDS cell lysates of splenocytes, thymocytes, and monocytes of group I and the dendritic cell lysate of group IIIa were separated by SDS-PAGE through a 12% gel. The P-20 primary antibody was used against IL-10R, followed by the HRP secondary antibody and detected by ECL reagents. Western blotting and the ECL profile from cell lysates showed dark bands for the molecular weight of mouse IL-10R at approximately 110 kDa (Fig. 15). However, the SDS cell lysates of splenocytes, thymocytes, and monocytes of group II and the dendritic cell lysate of group IIIb, which were exposed to methylprednisolone, showed faint bands for the molecular weight of mouse IL-10R also at approximately 110 kDa (Fig. 16).

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Immunoblotting and enhanced chemiluminescence detection of the molecular weight of interleukin-10 in splenocytes, thymocytes, monocytes, and dendritic cells

The SDS cell lysates of splenocytes, thymocytes, and monocytes of group I and the dendritic cell lysate of group IIIa were separated by SDS-PAGE through a 12% gel. The JES5-2A5 primary antibody was used against IL-10. This was followed by the HRP secondary antibody and detected by ECL reagents. Western blotting and the ECL profile from cell lysates showed faint bands for the molecular weight of mouse IL-10 at −35 kDa (Fig. 17). However, the SDS cell lysates of splenocytes, thymocytes, and monocytes of group II and the dendritic cell lysate of group IIIb, which were exposed to methylprednisolone, showed dark bands for the molecular weight of mouse IL-10 also at −35 kDa (Fig. 18).

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Level of interleukin-10 in response to glucocorticoid

Splenocytes, thymocytes, and monocytes of group I and dendritic cells of group IIIa were placed in a serum-free medium for 5h. The supernatants were collected and a cytokine bead array system was used to determine the IL-10 level. The amount of IL-10 secreted was detected at a low level. In contrast, splenocytes, thymocytes, and monocytes of group II and dendritic cells of group IIIb, which were exposed to glucocorticoids, showed considerably high levels of IL-10 in the supernatants of the cultured cells. The cells respond to the presence of glucocorticoid by secreting a high amount of IL-10. The mean and SD are presented. There was a highly significant (P < 0.001) increase in the IL-10 level in all the cell types examined of all groups, which were exposed to methylprednisolone glucocorticoid, as compared with the cells that were not exposed to methylprednisolone glucocorticoid (Table 1 and Histogram 1).

Table 1
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Histogram 1
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Discussion

In the current paper, we investigated the expression of IL-10 and IL-10R in the mouse splenocytes, thymocytes, monocytes, and dendritic cells. We also studied the expression of these molecules when the previous cells were exposed to glucocorticoid for a short period of time. The present work showed that splenocytes, thymocytes, monocytes, and dendritic cells expressed IL-10R intensely on their surfaces. SDS-PAGE and western blotting techniques showed intense precipitation of IL-10R at 110 kDa. In contrast, the administration of methylprednisolone glucocorticoid caused a decrease in the IL-10R expression on the surface of all the cells studied and faint precipitation of IL-10R at 110 kDa. Therefore, downregulation of IL-10R by glucocorticoids is not restricted to only one type of cells but also comprises many types of cells including the antigen-presenting cells. Consequently, this indicates the existence of a general pattern of responsiveness to IL-10-mediated signals.

Splenocytes and thymocytes in the current study expressed high levels of IL-10R. This finding was in agreement with a previous study that detected high levels of IL-10R on peripheral blood T cell subsets [27]. Our current data suggest that splenocytes and thymocytes are susceptible to IL-10-mediated signals. In the present study, high levels of IL-10R were also expressed on the peripheral blood monocytes. This was in agreement with a previous study that detected high levels of IL-10R on airway macrophages of asthmatic patients [28]. Moreover, some investigators detected IL-10R on peripheral blood mononuclear cells or on natural killer cells and neutrophils [29,30]. Monocytes have been considered to have the ability to transform into tissue macrophages and IL-10 treatment has been reported to accelerate this process [31]. Dendritic cells are the most potent antigen-presenting cells, and it is becoming increasingly clear that they play an essential instructive role during both T cell differentiation and T cell activation [32]. This study also detected an intense expression of IL-10R on the surface of the antigen-presenting dendritic cells. This was in accordance with the finding of a previous study that identified IL-10R on the surface of human dendritic cells [19]. Consequently, dendritic cells are also able to interact with IL-10 through its surface IL-10R. The IL-10/IL-10R interaction in immune cells results in the transcriptional activation of several hundred genes and activates the tyrosine kinases [33]. The receptor engagement and tyrosine phosphorylation activate the cytoplasmic inactive transcription factors, resulting in translocation and gene activation [34].

The administration of methylprednisolone in our study resulted in a decrease in the expression of IL-10R on splenocytes and thymocytes. Our finding was in agreement with a previous study that detected low levels of IL-10R on peripheral blood T cell subsets after glucocorticoid treatment [27]. It could be hypothesized that IL-10R downregulation by glucocorticoids might be associated with a decrease in the frequency of T cell subsets with high levels of IL-10R. In addition, glucocorticoids reduced the expression of IL-R on the surface of dendritic cells in this work. It is clear that the decrease in IL-10R expression observed in this study was caused by the direct action of glucocorticoids exerted on surface IL-10R molecules. This was found in a previous study that showed the occurrence of programmed cell death in the rat tracheal mucosa dendritic cells after treatment with glucocorticoids [35]. Peripheral blood monocytes in our current work also showed a decrease in the expression of IL-10R after glucocorticoid treatment. Therefore, the possibility that glucocorticoids induced programmed cell death in cells expressing IL-10R cannot be excluded in the current paper and this should be studied further. The decrease in IL-10R expression on the cells examined is particularly interesting in light of the widely described glucocorticoid-induced improvement in IL-10 production [23,36]. We assume that such reverse actions of glucocorticoids in increasing IL-10 and decreasing IL-10R could represent mechanisms of common oppose-regulation of IL-10-mediated effects. Besides the mechanism, the clinical implications need to be assessed in more detailed studies. Many of the therapeutic effects of corticosteroids can be attributed to their ability to modulate immune responses. T lymphocytes in particular have been used to establish some of the key models by which corticosteroids inhibit cell activation and gene expression. There is now substantial evidence that corticosteroids potently suppress pulmonary immune responses driven by T-helper cells.

The present study showed that IL-10 was secreted by splenocytes, thymocytes, monocytes, and dendritic cells of mice under normal conditions. Our results are in agreement with previous studies that detected IL-10 production by some subtypes of T cells [37]. In addition, a previous study showed the production of large quantities of IL-10 by monocytes [38]. The production of IL-10 by dendritic cells in our study was in agreement with a previous study that showed that pulmonary dendritic cells produce a significant amount of IL-10 [39]. Western blotting analysis of the molecular weight of the IL-10 in all the cells examined in this study detected it at 35 kDa. This was in agreement with a previous study that determined the molecular weight of mouse IL-10 at 35 kDa and this cytokine contained intrachain disulfide bonds that are essential to the biological function of IL-10 [40].

IL-10 is a potent anti-inflammatory and immunosuppressive cytokine. It interacts with its receptor (IL-10R), which mediates its major immunosuppressive function by inhibiting antigen-presenting cell function and cytokine production by macrophages, monocytes, and dendritic cells, leading to the inhibition of T cell-mediated immunity [33,41]. Previous studies considered IL-10 as an important anti-inflammatory cytokine in asthma [42]. Other studies proved that IL-10 produced by dendritic cells induces T-cell hyporesponsiveness and apoptosis [43].

In the present study, treatment with glucocorticoids increased the secretion of IL-10 in all the cells examined and the dendritic cells were filled with IL-10 intense yellow fluorescent granules. We also detected intense precipitation of IL-10 molecules at 35 kDa in all the cells, which were subjected to glucocorticoid treatment. Our finding was in agreement with a previous study that reported that a normal daily increase in the glucocorticoid levels resulted in an enhanced production of IL-10 [44]. Previous studies have reported that the secretion of endogenous IL-10 might be augmented by the intake of glucocorticoids [45]. However, if these increases in glucocorticoids levels become too frequent, the cells might develop resistance to the action of glucocorticoid [46]. The increased production of IL-10 by monocytes after glucocorticoid treatment in our study was in agreement with a previous study that detected increased production of IL-10 by monocytes following pretreatment with dexamethasone for at least 12h [47].

In peripheral tissues, immature dendritic cells capture antigens efficiently. Upon exposure to danger signals, such as bacterial products, dendritic cells undergo a maturation process characterized by an increased formation of IL-10 [48]. Dendritic cells are a key component of the innate immune system, and slight differences in the maturation of dendritic cells can qualitatively alter T-cell activation and the subsequent immune response. Increased plasma levels of IL-10 have been detected in patients under glucocorticoid therapy, indicating that steroids may exert their suppressive effect by increasing IL-10 production [49]. Several studies have also reported that treatment with corticosteroids resulted in the differentiation of T cells that secrete high levels of the immunoregulatory cytokine IL-10 [50]. Upregulation of IL-10 secretion after treatment with glucocorticoids may occur at both the protein and the mRNA level, probably indicating that glucocorticoids enhance gene transcription for IL-10.

The anti-inflammatory actions of glucocorticoids at the molecular level have been reported previously by some authors. Glucocorticoids bind to glucocorticoid receptors in the cytoplasm, which then dimerize and translocate to the nucleus, where they bind to glucocorticoid response elements on glucocorticoid-responsive genes, resulting in increased transcription. Glucocorticoids may increase the transcription of genes coding for anti-inflammatory proteins, including IL-10, but this is unlikely to account for all of the widespread anti-inflammatory actions of glucocorticoids. The most striking effect of glucocorticoids is to inhibit the expression of multiple inflammatory genes (cytokines, enzymes, receptors, and adhesion molecules). This is more likely to be because of a direct inhibitory interaction between activated glucocorticoid receptors and activated transcription factors, such as nuclear factor-kB and activator protein-1, which regulate the inflammatory gene expression [51].

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Conclusion

The present study showed that the expression of IL-10R was decreased and IL-10 secretion was increased in some immune cells after glucocorticoid treatment. The therapeutic benefit of glucocorticoids results in an increase in the synthesis of IL-10 and a decrease in the expression of IL-10R. Future studies are needed to understand the mechanisms of action of corticosteroids.

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Acknowledgements
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Conflicts of interest

There are no conflicts of interest.

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Keywords:

dendritic cells; glucocorticoids; IL-10; IL-10R; splenocytes; thymocytes

© 2012 The Egyptian Journal of Histology

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