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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Acknowledgment
  9. References

Glucosamine (GlcN), like N-acetylglucosamine (GlcNAc), is salvaged into the hexosamine pathway and is converted to UDP-GlcNAc. Golgi N-glycan branching enzymes produce N-glycans, using UDP-GlcNAc as a substrate, which attach to the T cell receptor (TCR) and cytotoxic T-lymphocyte antigen-4 (CTLA-4). These findings suggest that GlcN exerts the immunoregulation through TCR signalling, which could be involved not only in cytokine production but also activated T cell apoptosis. In fact, a preliminary study showed that GlcN reduced the number of CD3+ T cells of NC/Nga mice with AD-like skin lesions. Therefore, whether apoptosis of T cells would be one of the potential molecular mechanisms of GlcN-induced immunosuppression was investigated. Cultured human primary along with Jurkat T cells and purified T cells from NC/Nga mice with or without Df-induced AD-like skin lesion were used for the study. Glucosamine treatment increased the number of T cells expressing β1,6GlcNAc-branched N-glycans, with reduced ZAP-70 phosphorylation and enhanced CTLA-4 expression. Glucosamine treatment reduced the number of activated T cells from both the human primary and Jurkat cells and the dermatitis-induced mice. The expression of FasL and activated caspases, particularly caspase-3, was increased, whereas the phosphorylation of PI3K, Akt and NF-κB was decreased by GlcN treatment. Therefore, in addition to down-regulating TCR signalling and promoting CTLA-4 expression, GlcN may also suppress T cell function by enhancing apoptosis of activated T cells, through both extrinsic and intrinsic apoptotic signalling pathways, which were regulated by the inhibition of PI3K/Akt and NF-κB phosphorylation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Acknowledgment
  9. References

Glucosamine (GlcN), a natural amino monosaccharide (hexosamine), is widely taken as a dietary supplement to treat osteoarthritis or other inflammatory conditions [1-5]. It has been also suggested that GlcN has the potential immunoregulatory capacity, although details have been different; GlcN suppresses the unprimed T cell response [6] and the Th1 response in Th1 cytokine-producing experimental autoimmune encephalitis [7]. We also showed that GlcN suppressed the Th2 immune response in peripheral blood leucocytes of patients with atopic dermatitis (AD) as well as Th2-dominant NC/Nga mice [8]; however, the mechanism for the suppression is unclear. In the absence of antigenic stimuli, naïve T cells require basal growth signalling for long-term survival, whereas activation of antigen-specific T cells by antigenic stimuli is supposed to pass through proliferation, growth arrest, differentiation either Th1 or Th2 and finally apoptosis. These basal and activation processes are coordinated by protein N-glycosylation and metabolism to control homoeostasis and self-tolerance in T cells [9, 10]. Glucosamine, like N-acetylglucosamine (GlcNAc), enters the hexosamine pathway after 6-phosphorylation and is converted to UDP-GlcNAc, a substrate for the Golgi N-glycan branching enzymes [10]. The Golgi pathway is sensitive to hexosamine flux, with production of tri- and tetra-antennary N-glycans which bind multivalent galectins, forming a molecular lattice. The number of N-glycans, which attach to the surface receptors of the cell, including T cell receptor (TCR) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) [10], regulate surface glycoprotein levels. Increase in TCR signalling enhances T cell proliferation, whereas CTLA-4, an inhibitory receptor, induced to the cell surface about 4–5 days following T cell activation, promotes growth arrest [11]. The interaction between β1,6GlcNAc-branched complex-type N-glycans on TCR, which increases after T cell activation, and galectins reduces TCR signalling by opposing agonist-induced TCR clustering at the immune synapse [12]. In addition, β1,6GlcNAc-branched complex-type N-glycans enhance CTLA-4 through suppression of its endocytosis [10].

Because activated T cells are supposed to be eliminated quickly after cessation of immune challenge, enhanced β1,6GlcNAc-branching by GlcN may also regulate apoptosis of activated T cells. We also examined that number of activated T cells purified from NC/Nga mice with AD-like skin lesions was reduced after treatment with GlcN (data not shown). Activated T cell apoptosis occurs through multiple pathways, some of which involve Fas [13]. Fas, an apoptosis-inducing molecule, can be activated by FasL, which leads to caspase-8 activation and cell extrinsic apoptosis [14, 15]. Caspase-8 cleaves Bid, generating a truncated form, tBid [16, 17], which in turn induces the release of caspase-activating factors including cytochrome c from mitochondria, leading to intrinsic apoptosis [18]. Fas ligation can activate other major signalling pathways, such as mitogen-activated protein kinase (MAPK) pathways [19, 20], nuclear factor kappaB (NF-κB) transcription factor [21, 22], and phosphoinositide-3-kinase (PI3K) signalling [14, 23]. PI3K is an upstream mediator of apoptosis signal-regulating kinase (Akt), and PI3K/Akt pathway has been described as a major signalling pathway in the regulation of cell proliferation and survival. PI3K/Akt stimulates phosphorylation of pro-apoptotic factors, such as Bcl-2-associated death (BAD) protein and caspase-9 [24], which inhibits the mitochondrial death cascade, thereby promoting cell survival. Additionally, PI3K/Akt activates NF-κB-dependent expression of the anti-apoptotic c-Myc gene, which also promotes cell survival [14, 25].

In the present study, we investigated whether apoptosis of T cells would be one of the potential molecular mechanisms of GlcN-induced immunosuppression, using cultured primary normal human and Jurkat T cells and purified cells from NC/Nga mice with or without Df-induced AD-like skin lesions. Glucosamine enhanced surface expression of β1,6GlcNAc-branched N-glycans on the activated T cells with reduced phosphorylation of Zeta-associated protein (ZAP)-70, which is recruited by TCR signalling [26], and increased expression of CTLA-4, while stimulating the activated T cell apoptosis via the inhibition of PI3K and NF-κB pathways.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Acknowledgment
  9. References
Reagents

3-(4,5-Dimethyl-thiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT), foetal bovine serum (FBS) and antibiotics (penicillin and streptomycin) were obtained from GIBCO (Carlsbad, CA, USA). RPMI-1640 media was purchased from Welgene Inc. (Daegu, Korea). Antibody for β-actin, sodium dodecyl sulphate and dimethylsulphoxide (DMSO) and Phaseolus vulgaris leucoagglutinating lectin (PHA-L) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for Bad, c-Myc, FasL and CTLA-4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for Bid, phospho-Bad, caspase-3, caspase-8, caspase-9, NF-κB p65, phospho-NF-κB p65, Cytochrome c, PI3K, phospho-PI3K, Akt, phospho-Akt, ZAP-70 and phospho-ZAP-70 were purchased from Cell Signaling Technology (Danvers, MA, USA). Nitrocellulose (NC) membrane, phenylmethylsulfonyl fluoride, sodium orthovanadate, leupeptin, aprotinin, β-mercaptoethanol, HEPES and dithiothreitol (DTT) were purchased from Amresco (Solon, OH, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit, rabbit anti-goat and rabbit anti-mouse IgG and Western blotting detection reagents were purchased from Thermo (Rockford, IL, USA). Annexin-V-FITC and propidium iodide (PI) were purchased from Beckman Coulter (Brea, CA, USA). Ionomycin and PI3K inhibitors and LY294002 were purchased from Calbiochem (La Jolla, CA, USA). The anti-CD3 antibody and PE mouse anti-human CTLA-4 were purchased from BD Biosciences (San Diego, CA, USA). The Ficoll was purchased from GE Healthcare (Buckinghamshire, UK) The CD90.2 and CD3 microbead and LS columns were purchased from Miltenyi Biotec (Auburn, CA, USA). Dermatophagoides farina (Df) body ointment was obtained from Biostir Inc. (Kobe, Japan). Glucosamine was purchased from Sigma-Aldrich and was prepared in phosphate-buffered saline (PBS) and stored at −20 °C.

Induction of AD and GlcN treatment in NC/Nga mice

Six-week-old male NC/Nga mice, purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan), were maintained under uncontrolled conventional air conditions in the Laboratory Animal Facility at Dongguk University Ilsan Hospital. The animal care and use committee of the research institute at the Hospital approved all studies.

Atopic dermatitis was induced using Df body ointment as described previously [27]. The hair on the backs of the NC/Nga mice was removed. The barrier was disrupted with a 4% sodium dodecyl sulphate (SDS) treatment on the dorsal skin and both surfaces of each ear 3 h before the Df body ointment (100 mg/mouse) was applied. These procedures were repeated once every 3 days for 21 days.

Glucosamine (10 mg/mouse), which has been identified as an optimal dose in mouse [7], was administered intraperitoneally once a day for 14 days, starting after 7 days followed by Df application. Phosphate-buffered saline was administered instead of GlcN in the control group. Each group consisted of six mice.

Purification of T cells

Spleens were removed from NC/Nga mice, and erythrocyte-depleted single-cell suspensions were prepared in RPMI-1640 medium. Splenic T cells were purified using anti-CD90.2 magnetic microbeads and the columns, according to the manufacturer's instructions.

Human primary CD3+ T cells were isolated from peripheral blood of the three normal healthy donors by a Ficoll gradient centrifugation at 400 g, which was followed by a positive selection using anti-CD3 magnetic beads. The purity of each T cells was >95%.

Cell culture

The human primary CD3+ T cell and lymphoblast-like Jurkat T cell line (no. 40152; KCLB, Seoul, South Korea) and mouse T cells were cultured in RPMI-1640 medium containing 2.0 g/l of glucose with 100 units/ml penicillin, 100 units/ml streptomycin and 10% (v/v) dialysed heat-inactivated FBS at 37 °C in a humidified atmosphere of 95% air and 5% CO2.

Cell stimulation and treatment with GlcN

The human CD3+ T cells (1 × 10cells/well) and Jurkat T cells (5 × 104/well) cultured in 96-well plates were treated with anti-CD3 (5 μg/ml) plus 1 μm ionomycin and the indicated concentrations of GlcN for 24, 48 or 72 h. Mouse T cells were cultured in 96-well plates (5 × 104/well) in 200 μl of RPMI 1640 medium supplemented with 10% FBS for 48 h.

T cell viability assays

Cell viability was evaluated by the MTT reduction method. The cells were stained with MTT for 4 h. The precipitated formazan was dissolved in DMSO and the optical density was measured at 570 nm with background subtraction at 630 nm using a spectrophotometer. Effects of GlcN on inhibition of cell growth were calculated from the ratio of the cell viability with GlcN relative to that with PBS.

FACS analysis

Apoptosis was detected by the annexin-V-FITC binding assay. The cells were resuspended in binding buffer and then treated with 5 μl of Annexin-V-FITC staining solution for 10 min and with 10 μl of PI for another 5 min in the dark.

For cell cycle analysis, T cells were fixed with ice-cold 70% (v/v) ethanol for 24 h. The cells were resuspended in PBS containing PI (50 μg/ml), Triton X-100 (0.1%, v/v), 0.1% sodium citrate and DNase-free RNase (10 μg/ml), and then left at room temperature for 30 min. DNA content was determined by fluorescence-activated cell sorter (FACS) analysis. The percentage of viable and dead cells and the percentage of cells in the G0/G1, S and G2-M phases of the cell cycle were determined for 20,000 events per sample.

For analysis of CTLA-4 or β-1,6GlcNAc-branched N-glycan level, T cells were stained with CTLA-4 PE orPHA-L FITC for 30 min in the dark. Fluorescence-activated cell sorter analysis used Cytomics™ FC500 Flow Cytometry (Beckman Coulter).

Western blot analysis

T cells were resuspended in lysis buffer (10 mm Tris–HCl containing 50 mm NaCl, 50 mm NaF, 10 mm EDTA, 1 mm DTT, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 1 mm phenylmethylsulfonyl fluoride (PMSF), 5 mm leupeptin, and 10 μg/ml aprotinin, pH 7.5) for 30 min on ice. After centrifugation, the supernatant was added to one volume of 4 × Laemmli buffer containing 20% β-mercaptoethanol, boiled for 5 min and then frozen on dry ice. The samples were stored at −80 °C.

For preparation of nuclear protein, the cells (1 × 106) were resuspended in 10 μl of hypotonic solution (10 mm HEPES (pH 7.9), 10 mm KCl, 2 mm MgCl2, 1 mm DTT, 0.1 mm EDTA, 0.1 mm PMSF, 1 mm sodium orthovanadate, 10 mm NaF, 5 mm leupeptin and 10 μg/ml aprotinin) for 10 min at 4 °C; 3 μl of 10% Nonidet P-40 was then added. After centrifugation, the pellet was resuspended in 40 μl of extraction buffer (50 mm HEPES, 50 mm KCl, 300 mm NaCl, 0.1 mm EDTA, 1 mm DTT, 0.1 mm PMSF, 10% glycerol, 5 mm leupeptin and 10 μg/ml aprotinin, pH 7.9) for 30 min on ice. After centrifugation at 10,000 g for 10 min, the supernatant was recovered and frozen on dry ice. Samples were stored at −80 °C.

Equal amounts of proteins (20–60 μg) were separated by 8% or 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a NC membrane. After blocking, the membrane was incubated with 1:1000 diluted antibodies against c-Myc, FasL, Bad, phospho-Bad, NF-κB p65, phospho-NF-κB p65, PI3K, phospho-PI3K, Akt, phospho-Akt, JNK, caspase-8, caspase-9, caspase-3, Bid, cytochrome c or CTLA-4. After incubation with appropriate HRP-conjugated secondary antibodies (diluted 1:3000), the blots were visualized with ECL Plus™ Western Blotting Detection Reagents (Amersham, Piscataway, NJ, USA), and the signals were captured on an Image Reader (LAS-3000; Fuji Photo Film, Tokyo, Japan).

Statistical analysis

All data are expressed as mean ± SD of three or five independent experiments. Statistical analysis was performed using the Student's t-test or anova with post-test Bonferroni correction to evaluate the significance of differences.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Acknowledgment
  9. References

GlcN decreases number of activated human T cells

Effect of GlcN on cell viability was examined using primary CD3+ T cells obtained from healthy controls and human lymphoblast-like Jurkat T cells, which were stimulated with 5 μg/ml anti-CD3 plus 1 μm ionomycin. MTT assay suggested that the number of viable primary and Jurkat T cells was significantly (< 0.05) reduced in a dose-related manner at the indicated concentrations and duration up to 72 h (Fig. 1A). Flow cytometric analysis revealed that the percentage of Annexin-V/PI double-positive cells increased gradually to keep pace with the GlcN concentration, showing statistical significance at 10 and 20 mm compared to untreated control (< 0.05) (Fig. 1B).

image

Figure 1. Effect of glucosamine on survival of activated human T cells. The cells were incubated with 5 μg/ml anti-CD3 plus 1 μm ionomycin and 0, 2, 5, 10 or 20 mm glucosamine for 24, 48 and 72 h. (A) The MTT assay results. (B) The result of flow cytometry analysis with annexin-V and PI double staining in the cells incubated for 48 h. The graph represents the mean ± SD. from three independent experiments. Statistical analysis was performed using Student's t-test to evaluate the significance of differences.

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GlcN enhances β1,6GlcNAc-branched N-glycans on the activated Jurkat T cells

Effect of GlcN on N-glycan expression was examined using stimulated Jurkat T cells. Fluorescence-activated cell sorter analysis showed that the number of T cells expressing β1,6GlcNAc-branched N-glycans on their surface was significantly (< 0.05) increased at 20 mm GlcN concentration (Fig. 2).

image

Figure 2. Effect of glucosamine on N-glycan expression of Jurkat T cells. Jurkat T cells were stimulated with 5 μg/ml anti-CD3 plus 1 μm ionomycin and treated with 0, 5 or 20 mm glucosamine for 72 h. For N-glycan expression analysis, flow cytometry was used. The graph is presented as the mean ± SD. from three independent experiments. Y axis shows mean fluorescence intensity (MFI). Statistical analysis was performed using Student's t-test to evaluate the significance of differences.

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GlcN inhibits ZAP-70 phosphorylation and enhances CTLA-4 surface expression on the activated Jurkat T cells

Effect of GlcN on TCR and CTLA-4 surface expression was examined using stimulated Jurkat T cells. Because TCR signalling leads to tyrosine phosphorylation of the CD3 immunoreceptor tyrosine-based activation motifs [28-30] and, in turn, recruitment of ZAP-70 [26], TCR signalling was examined by the levels of ZAP-70 phosphorylation on activated Jurkat T cells. Western blot and FACS analysis suggested that ZAP-70 phosphorylation was reduced (Fig. 3A), whereas CTLA-4 surface expression was increased, along with the GlcN concentration compared to untreated control (< 0.05) (Fig. 3B, C).

image

Figure 3. Effect of glucosamine on the ZAP-70 phosphorylation and CTLA-4 surface expression of Jurkat T cells. Jurkat T cells were stimulated with 5 μg/ml anti-CD3 plus 1 μm ionomycin and treated with 0, 5 or 20 mm glucosamine for 20 min or 72 h. Western blot analysis of (A) phosphorylation of ZAP-70 and (C) expression of CTLA-4. The graphs are presented as the mean ± SD. from three independent experiments. Y axis shows relative intensity of phosphorylation of ZAP-70 or CTLA-4 protein normalized to the total form or β-actin. (B) Flow cytometry for surface expression CTLA-4 analysis. The graph represents the mean ± SD. from three independent experiments. Y axis shows surface expression of CTLA-4 (%). Statistical analysis was performed using Student's t-test to evaluate the significance of differences.

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GlcN induces activated human T cell apoptosis through intrinsic and extrinsic apoptosis pathways

The effect of GlcN, on the viability of activated T cells, was similar for both the primary cells and the Jurkat T cell line (Fig. 1A, B). To examine the mechanism of GlcN effect on the cell viability, the need for a large number of cells should be considered. Therefore, the mechanism was examined in stimulated Jurkat T cells. A cell cycle analysis suggested that the relative percentage of G1 arrest increased significantly (< 0.05) in a dose-dependent manner from 5 mm, following the GlcN treatment (Fig. 4A). A Western blot assay for apoptotic protein expression showed that GlcN increased the expression of FasL, cleaved Bid, cleaved caspases-8, -9 and -3, and cytochrome c in a dose-dependent manner (Fig. 4B).

image

Figure 4. Effect of glucosamine on apoptosis of Jurkat T cells. Jurkat T cells were stimulated with 5 μg/ml anti-CD3 plus 1 μm ionomycin and treated with 0, 5, 10 or 20 mm glucosamine for 48 h. (A) Flow cytometry for cell cycle analysis. The data are presented as the mean ± SD from five independent experiments. (B) Western blot analysis of FasL, caspase-8, Bid, c, caspase-9 and caspase-3 protein expression in total cell lysates. β-actin was used as the internal standard. The graph represents the mean ± SD from three independent experiments. Y axis shows relative intensity of each protein or cleaved protein normalized to β-actin. The cleaved protein form is indicated by black arrow. Statistical analysis was performed using Student's t-test to evaluate the significance of differences.

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GlcN inhibits PI3K/Akt phosphorylation with NF-κB nuclear translocation of activated human T cells

PI3K/Akt plays a critical role regulating cell survival through BAD phosphorylation [24] and NF-κB-dependent c-Myc expression [14, 25]. Therefore, the effect of GlcN on this pathway was examined in the presence or absence of the specific PI3K inhibitor, LY294002. Glucosamine inhibited phosphorylation of PI3K, Akt and BAD, as well as the expression of c-Myc gene dose dependently (Fig. 5A). The inhibition at 20 mm GlcN was comparable to that of LY294002 (Fig. 5A). Phosphorylation and nuclear translocation of classical NF-κB (p65/p50) were significantly (< 0.05) reduced in a dose-dependent manner from 5 mm GlcN (Fig. 5B).

image

Figure 5. Effect of glucosamine on the expression of PI3K/Akt-related proteins. Jurkat T cells were treated with 5 μg/ml anti-CD3 plus 1 μm ionomycin and indicated concentrations of glucosamine for 2 h. (A) Western blot analysis of PI3K, p-PI3K, Akt, p-Akt, Bad, p-Bad and c-Myc protein expression in total cell lysates in the presence or absence of LY294002. The graph represents the mean ± SD from three independent experiments. Y axis shows the relative intensity of each protein or phosphorylated protein normalized to β-actin or the total form. (B) Western blot analysis of NF-κB and p-NF-κB p65 expression in total cell and nuclear lysates. The graph represents the mean ± SD from three independent experiments. Y axis shows the relative intensity of each phosphorylated protein normalized to β-actin or the total form. Statistical analysis was performed using Student's t-test to evaluate the significance of differences.

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GlcN reduces viable T cell number in NC/Nga mice with Df-induced atopic dermatitis-like skin lesion but not in healthy control mice

The results of in vitro studies suggested that GlcN may stimulate apoptosis of the activated T cells, although the effect of GlcN on resting T cells was not examined. To confirm the GlcN effect in vitro, the parameters were compared, using NC/Nga mice with or without Df-induced AD-like skin lesions in the presence or absence of an optimal dose, 10 mg/mouse [7], of GlcN administration. The clinical skin scores were measured by the sum of individual scores based on the symptoms of erythema/haemorrhage, scarring/dryness, oedema and excoriation/erosion. The skin scores were significantly (< 0.05) reduced in GlcN-treated group compared to the untreated group of Df-induced AD model. However, the reduction of such scores by GlcN administration was not seen in the healthy control mice (Fig. 6A).

image

Figure 6. Effect of glucosamine on T cell viability in NC/Nga mice with or without dermatitis induction. NC/Nga mice with (Df+) or without Df-induced atopic dermatitis-like skin lesions (Df−) were treated with (GlcN+) or without glucosamine (GlcN−). (A) Representative clinical features of Df-induced and healthy control mice, which were treated with or without glucosamine. The clinical skin score from six mice, which was measured by the sum of individual scores based on the symptoms of erythema/haemorrhage, scarring/dryness, oedema and excoriation/erosion. (B) The MTT assay results. The graph represents the mean ± SD from six mice. Statistical analysis was performed using anova with Bonferroni post-test to evaluate the significance of differences.

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MTT assay suggested that the viable number of purified splenic T cells was significantly (< 0.05) lower in GlcN-treated compared to untreated group of Df-induced AD model mice (Fig. 6B). In contrast, no significant decrease in viable T cells was observed in healthy control mice (Fig. 6B).

GlcN-induced apoptosis of activated T cells with reducing ZAP-70 phosphorylation and enhancing CTLA expression in the Df-induced NC/Nga atopic dermatitis model

As in in vitro results (Fig. 3A, B), Western blot analysis showed that ZAP-70 phosphorylation was significantly (< 0.05) reduced, whereas CTLA-4 expression was enhanced in the GlcN-treated group compared to the untreated one (Fig. 7A).

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Figure 7. Mechanism of glucosamine-induced decreased T cell number in NC/Nga mice with or without dermatitis induction. NC/Nga mice with (Df+) or without Df-induced atopic dermatitis-like skin lesions (Df−) were treated with (GlcN+) or without glucosamine (GlcN−). (A) Western blotting of p-ZAP70, ZAP70 and CTLA-4 (B) FasL and cleaved caspase-3, (C) PI3K, p-PI3K, AKT, p-AKT, NF-κB and p-NF-κB expression on purified splenic T cells. The graph represents the mean ± SD from three or six mice. Y axis shows the relative intensity of each protein or phosphorylated protein normalized to β-actin or the total form. The cleaved protein form is indicated by black arrow. Statistical analysis was performed using anova with Bonferroni post-test to evaluate the significance of differences.

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For apoptosis signalling molecules, treatment with GlcN significantly (< 0.05) upregulated FasL expression with caspase-3 cleavage compared to those of the untreated group in Df-induced AD model. However, this result was not observed in healthy control mice (Fig. 7B). In addition, administration of GlcN significantly (< 0.05) reduced PI3K/Akt and NF-κB phosphorylation in Df-induced AD model, but not in healthy control mice (Fig. 7C).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Acknowledgment
  9. References

Immunosuppressive effects of GlcN have been presented. We also have previously reported that GlcN suppresses the Th2 immune response in NC/Nga mice with AD-like skin lesions, which may involve the GATA3 reducing the Th2 differentiation from naïve splenic T cells [8]. T cell receptor is a key receptor in T cell homoeostasis, and GlcN reduced TCR signalling, which was identified by reduced ZAP-70 phosphorylation, and enhanced CTLA-4 expression (Figs. 3A,B and 7A). Because ZAP-70 regulates TCR-medicated activation of the nuclear factor of the activated T cell (NFAT), an essential transcription factor of Th1 or Th2 cytokines [31, 32], the ZAP-70 reduction may play a role in the suppression of Th2 immune response by GlcN treatment. In addition, increased N-glycan expression on activated T cell surface by GlcN treatment (Fig. 2) suggested that GlcN-induced N-glycans could be involved in the reduced TCR signalling (Fig. 3A, B). Although it has been reported that the immunosuppressive effects of GlcN have not been attributed to cell death [2], GlcN reduced the number of cultured human T cells, activated ex vivo (Fig. 1A) and purified splenic T cells from dermatitis-induced (Fig. 6B). Although the effect of GlcN was not examined in cultured human T cells without stimulation, no numerical change of the T cells in the healthy control NC/Nga mice (Fig. 6B) suggests that GlcN induces cell death of the activated T cells, but not the constitutive T cells. In addition, the GlcN effect was identified by the treatment with an optimal mouse dose of GlcN, 10 mg/mouse [7] (Fig. 6B), although, 20 mm of GlcN concentration, which was used in in vitro study, was too high to reach in nature. The clinical effect of GlcN in Df-induced mice (Fig. 6A) also supported the relationship between reduced activated T cell number and therapeutic effect.

The reduction of viable cell number suggests that cell death exceeds cell proliferation. There are two different mechanisms of cell death, apoptosis and necrosis. The results of FACS analysis, using Annexin-V and PI (Fig. 1B), and the increased proportion of G1 phase cells (Fig. 4A) [33] in cultured human T cells supported the apoptotic processes in the T cells. Moreover, the increased caspase-3 cleavage in Jurkat T cells was activated ex vivo (Fig. 4B), and Df-induced NC/Nga mice (Fig. 7B) indicates that apoptotic processes are involved in the reduced viable T cell number. Glucosamine administration increased FasL expression in the activated Jurkat T cells (Fig. 4B) and Df-induced NC/Nga mice, but not in the healthy control mice (Fig. 7B). The increases in FasL expression indicate the involvement of the extrinsic apoptosis signalling pathway [14, 15]. Although changes in tBid, cytochrome c and cleaved caspase-9 were not examined in NC/Nga mice, these parameters were increased in the activated Jurkat T cells (Fig. 4B). Because the extrinsic pathway is connected to the intrinsic pathway through tBid, the increase in tBid, cytochrome c and cleaved caspase-9 indicates that the intrinsic pathway is also involved in the process.

Because the PI3K/Akt pathway is a major signalling pathway in the regulation of cell proliferation and survival, reduced phosphorylation of PI3K and Akt may explain the cell death. Glucosamine reduced phosphorylation of PI3K and Akt in activated T cells from Jurkat cell line (Fig. 5A) and the mice (Fig. 7C). With relation to cell survival, PI3K/Akt has been shown to stimulate BAD phosphorylation [24] and c-Myc expression [14, 25]. In fact, the treatment with a specific inhibitor, LY294002, reduced both BAD phosphorylation and c-Myc expression, either did GlcN treatment (Fig. 5A). Stimulation of c-Myc expression by NF-κB (p65/p50) [25] may explain the reduced phosphorylation of NF-κB in response to GlcN (Figs. 5B and 7C). In addition, reduced expression of NF-κB in the nuclear fraction of activated Jurkat T cells, which occurred with as low as 5 mm of GlcN (Fig. 5B), indicates reduced activity of NF-κB [34]. Because PI3K/Akt activates NF-κB [14], the reduced NF-κB phosphorylation could indicate that PI3K/Akt is involved in the GlcN-induced apoptosis.

In summary, GlcN reduced the number of viable T cells, which resulted from apoptosis of the activated, but not the constitutive T cells. The apoptosis underwent extrinsic and intrinsic pathways, and phosphorylation of PI3K and NF-κB was involved in regulating apoptosis. In addition to Reduced TCR and enhanced CTLA-4 expression, GlcN-induced increase surface N-glycan expression levels may play a role in apoptosis.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Acknowledgment
  9. References

This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No.: A103017).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Acknowledgment
  9. References