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d -pinitol inhibits Th1 polarization via the suppression of dendritic cells
International Immunopharmacology 7 (2007) 791 – 804 www.elsevier.com/locate/intimp
D-pinitol
inhibits Th1 polarization via the suppression of dendritic cells
Jun Sik Lee a , In Duk Jung b , Young-Il Jeong c , Chang-Min Lee b , Yong Kyoo Shin d , Sang-Yull Lee e , Dong-Soo Suh f , Man-Soo Yoon f , Kyu-Sub Lee f , Yung Hyun Choi g , Hae Young Chung a , Yeong-Min Park b,⁎ b
a Department of Pharmacy, Pusan National University College of Pharmacy, Busan 609-735, South Korea Department of Microbiology and Immunology, and National Research Laboratory of Dendritic Cells Differentiation and Regulation, and Medical Research Institute, South Korea c Department of Microbiology, Pusan National University College of Nature Science, Busan 609-735, South Korea d Department of Pharmacology, Chungang University College of Medicine, Seoul 156-756, South Korea e Department of Biochemistry, Pusan National University College of Medicine, Busan 602-739, South Korea f Department of Obstetrics, Pusan National University College of Medicine, Busan 602-739, South Korea g Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052, South Korea
Received 21 December 2006; received in revised form 25 January 2007; accepted 25 January 2007
Abstract D-pinitol has been demonstrated to exert insulin-like and anti-inflammatory effects. However, the effects of the maturation and immunostimulatory functions of dendritic cells (DC) remain to be clearly elucidated. In this study, we have attempted to determine whether D-pinitol regulates surface molecule expression, cytokine production, endocytosis capacity, and underlying signaling pathways in murine bone marrow-derived DC. We also attempted to ascertain whether D-pinitol could influence Th1/Th2 immune response in vivo. The DC used in this study were derived from murine bone marrow cells, and were used as immature or LPSstimulated mature DC. The DC were then assessed with regard to surface molecule expression, dextran-FITC uptake, cytokine production, capacity to induce T-cell differentiation, and underlying signaling pathways. D-pinitol was shown to significantly inhibit CD80, CD86, MHC class I, and MHC class II expression in the LPS-stimulated mature DC. The DC also evidenced impaired IL-12 expression and IFN-γ production. The D-pinitol-treated DC were found to be highly efficient in regards to Ag capture via mannose receptor-mediated endocytosis. D-pinitol was also demonstrated to inhibit LPS-induced MAPKs activation and NF-κB nuclear translocation. Moreover, the D-pinitol-treated DC manifested impaired induction of Th1 responses, and normal cell-mediated immune responses. These novel findings provide new insight into the immunopharmacological role of D-pinitol in terms of its effects on DC. These findings also broaden current perspectives concerning our understanding of the immunopharmacological functions of D-pinitol, and have ramifications for the development of therapeutic adjuvants for the treatment of DC-related acute and chronic diseases. © 2007 Elsevier B.V. All rights reserved.
Keywords: D-pinitol; Dendritic cell; Th1 polarization; IFN-γ; NF-κB; IL-12
Abbreviations: DC, dendritic cells; MAPKs, mitogen-activated protein kinases; BM, bone marrow; APC, antigen-presenting cells; MHC, major histocompatibility complex; MLR, mixed lymphocyte reaction; TNCB, 2,4,6-trinitrochlorobenzene; CHS, contact hypersensitivity. ⁎ Corresponding author. Department of Microbiology and Immunology and National Research Laboratory of Dendritic Cell Differentiation & Regulation, Pusan National University College of Medicine, Ami-dong 1-10, Seo-gu, Busan 602-739, South Korea. Tel.: +82 51 240 7557; fax: +82 51 243 2259. E-mail address: [email protected] (Y.-M. Park). 1567-5769/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2007.01.018
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1. Introduction A substantial body of research is currently focused on the biology of dendritic cells (DC), specifically with regard to their possible clinical use as cellular adjuvants in the treatment of chronic infectious diseases and tumors. DC are professional antigen-presenting cells (APC) that have been determined to possess immune sentinel properties, initiating T-cell responses against microbial pathogens and tumors [1]. Immature DC capture and process exogenous agents within peripheral tissues, in which they begin to mature. Maturing DC migrate into the lymphoid organs, where they stimulate naive T cells via the signaling of both major antigen-peptide-presenting histocompatibility complex (MHC) molecules and costimulatory molecules [2,3]. Maturation and differentiation are generally understood to require the activation phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), p38 MAPK, and the transcription nuclear factor kappa B (NFκB) [4,5]. DC have also been demonstrated to be highly responsive to both inflammatory cytokines and bacterial products, such as TNF-α and lipopolysaccharides (LPS). When encountered in the peripheral organs, these products are known to induce a series of phenotypic and functional alterations in the DC. Similar changes, indicative of maturation, have also been reported after the infection of cells with mycoplasma, viruses, intracellular bacteria, and parasites [7]. DC located within the peripheral tissues generally tend to be both phenotypically and functionally immature. Immature DC do not induce primary immune responses, as they do not express the requisite costimulatory molecules. In addition, they don't express antigenic peptides as stable complexes with MHC molecules. While in an immature state, DC have been shown to effectively capture and process exogenous antigens (Ag) within the peripheral tissues, where the DC then begin to mature. DC maturation has previously been associated with decreased or absent Ag uptake, high levels of MHC class II and accessory molecule expression, as well as IL-12 generation upon stimulation [6]. The flavonoids comprise a family of common phenolic plant pigments, which have been identified as dietary anticarcinogens and antioxidants [7]. We reported previously that a variety of phytochemicals exhibit profound immunoregulatory activity, particularly in DC [8–11]. D-pinitol (3-O-methyl-chiroinositol), an active principle of the traditional antidiabetic plant, Bougainvillea spectabilis, reportedly exerts insulin-like effects [12]. Pinitol is also known to exert insulin-like
effects, via the driving of creatine and other nutrients into muscle cells [12]. D-pinitol has been suggested to possess multifunctional properties, including anti-inflammatory properties [13]. In addition, it has also been implicated in the prevention of cardiovascular diseases [14]. Until now, the cellular targets of D-pinitol in the immune system have remained enigmatic, thereby leaving the issue of the global function of D-pinitol in relation to DC maturation and immuno-regulatory activities open to discussion in terms of future research. In this study, we have attempted to characterize the effects of a noncytotoxic concentration of D-pinitol on the maturational and functional properties of murine bone marrow (BM)-derived DC. Our findings demonstrated, for the first time, that D-pinitol treatment inhibited phenotypic and functional maturation in the DC. D-pinitol treatment also suppressed the LPSinduced activation of ERK1/2, JNK, and p38 MAPK, as well as NF-κB nuclear translocation, in the DC. The in vivo data indicate that, although the D-pinitol-treated DC were observed to migrate to the T-cell areas of secondary lymphoid tissue, they did not induce normal cell-mediated contact hypersensitivity (CHS). Moreover, this readily available drug may constitute a simple, inexpensive, and highly effective means for the manipulation of the immunostimulatory capacity of DC. Considering, then, the critical functions of these professional APC in the initiation and regulation of immune responses, coupled with the ready availability of D-pinitol, our findings appear to bear important implications with regard to the manipulation of the functions of DC in potential therapeutic applications. 2. Materials and methods 2.1. Animals and chemicals Male 8–12-week-old C57BL/6 (H-2Kb and I-Ab) and BALB/c (H-2Kd and I-Ad) mice were purchased from the Korean Institute of Chemistry Technology (Daejeon, Korea). They were housed in a specific pathogen-free environment within our animal facility for at least 1 week before use. D-pinitol was purchased from Sigma. 2.2. Reagents and Abs Recombinant mouse (rm)GM-CSF and rmIL-4 were purchased from R&D Systems. D-pinitol, dextran-FITC (molecular mass, 40,000), and LPS (from Escherichia coli 055:B5) were obtained from Sigma-Aldrich. An endotoxin filter (END-X) and an endotoxin removal resin (END-X B15) were acquired from Associates of Cape Cod. Cytokine ELISA kits for murine IL-12 p70, IL-4, and IFN-γ were purchased from BD Pharmingen. FITC-conjugated or PE-conjugated mAbs used to detect the
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expression of CD11c (HL3), CD80 (16-10A1), CD86 (GL1), CD54 (1C10), IAb β-chain (AF-120.1), H2Kb (AF6-88.5), CD3, and CD4 (L3T4), or the intracellular expression of IL-12 p40/ p70 (C15.6), and IL-10 (JESS-16E3) by flow cytometry, as well as isotype-matched control mAbs, biotinylated anti-CD11c (N418) mAb, Cytofix/Cytoperm kit and FITC-annexin V in combination with a propidium iodine kit were purchased from BD Pharmingen. To detect protein levels, anti-phospho-ERK, anti-ERK, anti-phospho-p38, anti-p38, anti-p-IκB, anti-β-actin anti-phospho-JNK, anti-JNK, and anti-p65 Ab from Santa Cruz. Bead-conjugated anti-CD11c mAb (Miltenyi Biotec) and paramagnetic columns (LS columns) were purchased from Miltenyi Biotec. 2.3. Generation and culture of DC DC was generated from murine BM cells, as described by Inaba et al. [15] with modifications. Briefly, BM was flushed from the tibiae and femurs of C57BL/6 and depleted of red cells with ammonium chloride. The cells were plated in sixwell culture plates (106 cells/ml; 3 ml/well) in OptiMEM (Invitrogen Life Technologies) supplemented with 10% heatinactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 × 10− 5 M 2-ME, 10 mM HEPES (pH 7.4), 20 ng/ml rmGM-CSF and rmIL-4 at 37 °C, 5% CO2. On day 3 of the culture, floating cells were gently removed and fresh medium was added. On day 6 or 7 of the culture, nonadherent cells and loosely adherent proliferating DC aggregates were harvested for analysis or stimulation. In some experiments, they were replated in 60-mm dishes (106 cells/ml; 5 ml/dish). On day 6, 80% or more of the nonadherent cells expressed CD11c. In certain experiments, in order to obtain highly purified populations for subsequent analyses, the DC were labeled with bead-conjugated antiCD11c mAb. This was followed by positive selection through paramagnetic columns (LS columns) according to the manufacturer's instructions. The purity of the selected cell fraction was N 95%.
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serum and 0.1% sodium azide in PBS). Cells were first blocked with 10% (v/v) normal goat serum for 15 min at 4 °C and stained with phycoerythrin (PE)-conjugated anti-H-2Kb [major histocompatibility complex (MHC) class I], anti-I-Ab (MHC class II), anti-CD80, and anti-CD86 with fluorescein isothiocyanate (FITC)-conjugated anti-CD11c for 30 min at 4 °C. The stained cells were analysed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). 2.6. Quantitation of Ag uptake Endocytosis was quantitated, as described by Lutz et al. [16,17]. In brief, 2 × 105 cells were equilibrated at 37 or 4 °C for 45 min and then pulsed with fluorescein-conjugated dextran at a concentration of 1 mg/ml. Cold staining buffer was added to stop the reaction. The cells were washed three times and stained with PE-conjugated anti-CD11c Abs, and then analyzed by FACSCalibur. Nonspecific binding of dextran to DC, determined by incubation of DC with FITC-conjugated dextran at 4 °C, was subtracted. The medium used in the culture, to stimulate DC with D-pinitol, was supplemented with GM-CSF, because the ability of DC to capture Ag is lost if DC are cultured without GM-CSF [18]. 2.7. Cytokines assay Cells were first blocked with 10% (v/v) normal goat serum for 15 min at 4 °C and then stained with FITC-conjugated CD11c+ antibody for 30 min at 4 °C. Cells stained with the appropriate isotype-matched Ig were used as negative controls. The cells were fixed and permeated with the Cytofix/ Cytoperm kit according to the manufacturer's instructions. Intracellular IL-12p40/p70, IL-10, and IFN-γ were stained with fluorescein R-phycoerythrin (PE)-conjugated antibodies in a permeation buffer. The cells were analyzed on a FACSCalibur flow cytometer using the CellQuest program. Furthermore, murine IL-12p70, IL-4, and IFN-γ from DC were measured using an ELISA kit according to the manufacturer's instructions.
2.4. Stimulation of DC by D-pinitol 2.8. Mixed lymphocyte reaction D-pinitol
was dissolved in DMSO, or DMSO alone (0.01% v/v) was added to cultures of isolated DC in six-well plates (106 cells/ml; 3 ml/well). DMSO (0.1%) alone was used as a control due to the lack of no cytotoxicity in DC. For the analysis of apoptosis, DC were stimulated with LPS or left without any stimuli. Additionally, apoptosis was analyzed over time by staining of phosphatidylserine translocation with FITC-annexin V in combination with a propidium iodine kit according to the manufacturer's instructions. 2.5. Flow cytometric analysis On day 6, BM-DC were harvested, washed with phosphate buffered saline (PBS) and resuspended in fluorescence activated cell sorter (FACS) washing buffer (2% fetal bovine
Responder T cells, which participate in allogeneic T-cell reactions, were isolated from spleen of BALB/c mice with a MACS column. Staining with fluorescein isothiocyanate (FITC)-conjugated anti-CD3 antibodies revealed that they consisted mainly of CD3+ cells (N 95%). The lymphocyte population (95% of CD3+ cells) was then washed twice in PBS and labeled with CFSE, as previously described [19]. The cells were resuspended in 5 μM CFSE in phosphate-buffered saline (PBS). After being shaken for 8 min at room temperature, the cells were washed once in pure fetal bovine serum (FBS) and twice in PBS with 10% FBS. DC (1 × 104) or DC exposed to D-pinitol (80 μM/ml) or LPS (100 ng/ml) for 24 h were cocultured with 1 × 105 allogenic CFSE-labeled T lymphocytes in 96-well, U-bottom plates (Nunc). A negative control (CD3+
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BCA assay kit (Pierce, Rockford, IL). Equivalent amounts of proteins were separated by SDS-10% PAGE and analyzed by Western blotting using an antiphospho-ERK1/2 (p-ERK), antiphospho-JNK (p-JNK), or anti-phospho-p38 (p-p38) MAPK mAb for 2 h, as described by the manufacturer of the antibodies. Following washing three times with TBST, membranes were incubated with secondary HRP-conjugated anti-mouse IgG for 1 h. After washing, the blots were developed using the ECL system (Amersham) by following the manufacturer's instructions. DC nuclear extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL), according to the manufacturer's instructions. NF-κB p65 subunits in the nuclear extracts were determined by Western blot analysis with anti-NF-κB p65 subunit Ab. 2.10. Generation of DC from spleen and culture
Fig. 1. D-pinitol has no cytotoxicity to BM-DC up to 80 μM. BMderived DC were generated as was described in the Materials and methods section, and analyzed via flow cytometry on day 7. The DC were stained for CD11c and annexin-V and PI. The percentage within each positive cell represents the incidence of annexin-V+ PI+. DC were generated as described in the Materials and methods section of this article.
lymphocytes alone) and a positive control (CD3+ lymphocytes in 5 μg of Concanavalin A) were created for each experiment. After 4 days, the cells were harvested and washed in PBS. CFSE dilution optically gated lymphocytes were assessed by flow cytometry. 2.9. Nuclear and cytoplasmic extracts and Western blot The cells were exposed to LPS (200 ng/ml) in the absence or presence of 80 μM in a D-pinitol pretreatment. Following 15 or 30 min of incubation at 37 °C, cells were washed twice with cold PBS and lased with modified RIPA buffer (1.0% NP-40, 1.0% sodium deoxycholate, 150 nM NaCl, 10 mM Tris–HCl [pH 7.5], 5.0 mM sodium pyrophosphate, 1.0 mM NaVO4, 5.0 mM NaF, 10 mM/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride) for 15 min at 4 °C. Lysates were cleared by centrifuging at 14,000 ×g for 20 min at 4 °C. The protein content of cell lysates was determined using the Micro
Mice were injected intraperitoneally with D-pinitol (10 mg/kg) 3 times (1 time/day) for 3 days before the administration of 1 mg/kg of LPS in a lateral vein of the tail. Twenty-four hours after the LPS challenge, they were scarified, their spleens were disrupted and the cells were centrifuged at 400 ×g for 5 min, resuspended in OptiMem supplemented with 10% heatinactivated FBS, L-glutamine, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, HEPES and 2-ME for 2 h. Finally, the non-adherent cells were washed out. The residual adherent cells were maintained in the culture medium and incubated overnight at 37 °C in a 5% CO2 atmosphere. After incubation, DC (which exhibit adherence capacity in the first hours of culture) became non-adherent and floated in the medium. The cells were gated on CD11c+ for DC. 2.11. Isolation of CD4+ T cell from spleen and culture Mice were injected intraperitoneally with D-pinitol (10 mg/kg) every 3 days before the administration of 1 mg/kg of LPS in a lateral vein of the tail. Twenty-four hours after the LPS challenge, they were killed, their spleens were disrupted and the cells were centrifuged at 400 ×g for 5 min, resuspended in OptiMem supplemented with 10% heat-inactivated FBS, Lglutamine, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, HEPES and 2-ME for 4 h. The residual adherent cells were maintained in the culture medium and incubated overnight at 37 °C in a 5% CO2 atmosphere. The cells were fixed and permeated with the Cytofix/Cytoperm kit according to manufacturer's instructions. Intracellular IFN-γ was stained with fluorescein R-phycoerythrin (PE)-conjugated antibodies in a permeation buffer. The cells were gated on CD4+ T cell for T cell. 2.12. 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced CHS In the sensitization phase, bead-sorted CD11c+ DC were pulsed with 0.1% (w/v) TNBS (Sigma Aldrich) in PBS for
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Fig. 2. D-pinitol suppresses the expression of CD80, CD86, MHC class I, and MHC class II in a dose-dependent manner during DC maturation. DC were generated as described in the Materials and methods section of this article. On day 6, the cells were harvested and analyzed using two-color flow cytometry. The cells were gated on CD11c+. D-pinitol was added for 24 h to the DC at concentrations of 20, 40, and 80 μM. Surface molecule expression was then analyzed. A, The DC were left untreated (control) or stimulated for 24 h with 200 ng/ml LPS, in the absence or presence (gray line, control isotype; thin line, D-pinitol with LPS; thick line, LPS) of 80 μM D-pinitol on day 6. B, Overlay of histogram A in one box for clarification, respectively. UN represents the chemically untreated control group. The histogram is from one out of four representative experiments.
15 min at 37 °C. After three washes in PBS, the cells were counted, and their viability was assessed by trypan blue exclusion. One million cells were injected s.c. into the abdomen of animals shaved and painted with 7% w/v 2,4,6trinitrochlorobenzene (TNCB; Sigma-Aldrich) diluted in acetone-olive oil, 4/1 (v/v) (vehicle). Negative controls included animals injected with unpulsed DC (without hapten) and animals treated with the vehicle alone. Seven days after
sensitization, the mice were painted on the dorsal and ventral side of the left ear with 10 μl of 1% TNCB in vehicle. The thickness of the left (challenged) and the right (control) ear were measured after 48 h by using an engineer's spring-loaded micrometer (Mitutoyo). The percentage increase in ear thicknesses was calculated using the following formula: 100 × ((thickness of challenged ear-thickness of unchallenged ear) / thickness of unchallenged ear).
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Table 1 D-pinitol markedly inhibits the expression of costimulatory molecules (CD80, CD86 and CD54), MHC class I and II on LPS-stimulated CD11c + DC % of positive cell a (MFI) b Surface Ag
Medium
D-pinitol
LPS
D-pinitol + LPS
CD80 CD86 MHC I MHC II CD54
71 ± 2 (263 ± 32) 72 ± 1 (167 ± 21) 74⁎ ± 3 (168 ± 17) 76 ± 4 (395 ± 43) 74⁎ ± 4 (315 ± 24)
72⁎ ± 3 (270 ± 17) 73 ± 4 (154 ± 21)⁎ 72⁎ ± 3 (151 ± 17)⁎ 74 ± 2 (263 ± 42) 73 ± 1 (310 ± 25)⁎
86⁎ ± 2 (596 ± 42) 87 ± 1 (582 ± 47) 88⁎ ± 2(286 ± 12) 84⁎ ± 2 (721 ± 52) 89 ± 1 (893 ± 95)
77 ± 4 (420 ± 23)⁎⁎ 79 ± 2 (379 ± 42)⁎⁎ 82 ± 2 (215 ± 7)⁎⁎ 81 ± 3 (559 ± 36)⁎⁎ 83 ± 1 (712 ± 71)
⁎, ⁎⁎, The statistical significance between samples with and without D-pinitol is indicated (⁎, P b 0.05 vs unstimulated DC (medium); ⁎⁎, P b 0.01 vs LPS-stimulated DC). a BM-derived DC were cultured in the absence or presence of D-pinitol (80 μM) following the LPS (200 ng/ml) stimulation for 24 h. The expression of surface molecules was analyzed by FACSCalibur. A two-color flow cytometry was used to determine the level of surface molecules expression on CD11c + DC. b MFI, (mean fluorescence intensity). The results are from one experiment of three performed.
2.13. Statistics All results were expressed as the means ± SD of the indicated number of experiments. Statistical significance was estimated using a Student's t-test for unpaired observations. Afterwards the differences were compared with regard to statistical significance by one-way ANOVA, followed by the Bonferroni's post hoc test. The categorical data from the fertility test were subjected to statistical analysis via a Chisquare test. A P of b 0.05 was considered significant. 3. Results 3.1. D-pinitol inhibits the maturation of BM-derived murine DC In the initial series of experiments, we attempted to determine whether D-pinitol influences DC maturation. BMDC were cultured for 6 days in OptiMEM supplemented with 20 ng/ml of granulocyte-macrophage-colony stimulating factor (GM-CSF) and 20 ng/ml of IL-4. Different concentrations of D-pinitol were added to the cultures on day 6, with or without 200 ng/ml of LPS. D-pinitol was determined to be noncytotoxic to BM-DC at concentrations of 80 μM. There were no marked differences in the percentage of dead cells, as evidenced by CD11c+ cell and annexin-V/propidium iodide (PI) staining (Fig. 1); for this reason, it was adjusted to concentrations below 80 μM. We then investigated the effects of a range of D-pinitol concentrations on DC maturation. BMDC were cultured for 24 h in the presence of 0 to 80 μM D-pinitol, as is described in the Materials and methods section of this article. As is shown in Fig. 2A and Table 1, D-pinitol 80 μM significantly attenuated CD80, CD86, CD54, and MHC class I and II expression on the surface of the CD11c+, in contrast to LPS. These inhibitory effects occurred in a dosedependent manner, and were most pronounced with regard to the expression of CD80, CD86, CD54, MHC class I and MHC class II molecules. These molecules were also upregulated within 24 h of LPS exposure (Fig. 2B, thick lines). Exposure to 80 μM D-pinitol in the presence of LPS was accompanied by an impaired expression of MHC class I and MHC class II molecules.
Interestingly, a significant downregulation of the costimulatory molecules, CD80 and CD86, was also observed under these conditions (Fig. 2B, thin lines). 3.2. D-pinitol impairs IL-12 secretion, and does not influence IL-10 production during LPS-induced DC maturation It has been previously hypothesized that DC, as well as macrophages and monocytes, function as sources of proinflammatory molecules [18,20]. We assessed the ability of the BM-DC to generate pro-inflammatory cytokines. IL-12 expression was identified previously as a specific marker of DC activity. It is also known to be an important marker for DC maturation, and can be used to select Th1-dominant adjuvant. The secretion of bioactive IL-12 p70 requires the coordinated expression of two of its subunits, namely p35 and p40, which are encoded by two separate genes, and regulated independently [16]. We analyzed the production of both intracellular IL-12p40/p70 and bioactive IL-12p70 in D-pinitol-treated DC. As is shown in Fig. 3A, the intracellular staining of FITClabeled anti-CD11c+ DC with PE-labeled anti-IL-12 p40/p70 or anti-IL-10 mAbs revealed that DC stimulated with 80 μM D-pinitol expressed small amounts of IL-12 p40/p70, in contrast to unstimulated DC (D-pinitol-untreated, i.e., LPStreated DC), whereas IL-10 was merely detected. When the supernatants were analyzed via ELISA, IL-10 was also barely detectable 24 h after stimulation with 200 ng/ml of LPS. As is shown in Fig. 3B, ELISA analysis revealed high levels of IL12 p70 upon the stimulation of DC with LPS (95.4 ± 12.2 ng/ ml) for 24 h. D-pinitol (54.2 ± 7.9 ng/ml) was shown to alleviate the effects of LPS. These results indicated that exposure to D-pinitol impairs the ability of DC to generate large amounts of IL-12 p70 and pro-inflammatory cytokines. The findings also indicate that D-pinitol suppresses the functional maturation of LPS-stimulated DC. 3.3. D-pinitol suppresses endocytic activity of DC The expression of surface molecules on DC and the observed changes in IL-12 production show that D-pinitol exposure leads
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Fig. 3. D-pinitol impairs IL-12 secretion and has no influence on IL-10 in LPS-induced DC maturation. A, BM-DC was stimulated by 80 μM D-pinitol for 24 h with or without LPS. This figure also represents the analysis of IL-12 p40/p70 and IL-10 expression in CD11c+ DC by intracellular cytokine staining after 24 h of LPS stimulation. The numbers indicate the percentages of CD11c+ cells expressing IL-12 p40/p70 or IL-10. The results are representative of four separate experiments. B, Analysis of IL-12 p70 production by magnetic bead-purified DC (1 × 106 cells) over time (12–48 h) after LPS stimulation (200 ng/ml) using ELISA. The data represent the means (± SD) of four separate experiments. (⁎⁎, P b.01 vs DC-stimulated with D-pinitol + LPS).
to a profound inhibition of the phenotypic and functional maturation of DC in vitro. However, these results did not permit us to exclude the possibility that D-pinitol may induce a general inhibition of the physiological functions of DC. Thus, we attempted to determine whether the stimulation of DC with D-pinitol alters the ability of the cells to capture antigens. We exposed the DC to D-pinitol, with or without LPS, adding dextran-FITC to the culture media. The percentage of doublepositive cells (CD11c+ + dextran-FITC) in the D-pinitol-treated and non-treated DC was identical. The percentage of LPSstimulated DC was lower than the percentage of untreated DC. The D-pinitol-treated DC exhibited a higher capacity for endocytosis of dextran-FITC than did the LPS-stimulated DC (Fig. 4A, B). Again, this shows that the D-pinitol-treated DC were both phenotypically and functionally immature. A set of identical experiments were also performed at 4 °C, and the results of these indicated that the uptake of dextran-FITC by DC
was inhibited at low temperatures. This data indicates that D-pinitol induces immaturity in the DC. 3.4. D-pinitol impairs the allostimulatory capacity of DC The fluorescein-based dye, CFSE, has biochemical properties which render it particularly appropriate for this application. Specifically, CFSE dye is loaded into cells in vitro and the CFSE in a given cell is monitored over time. Upon division, the CFSE segregates equally between the daughter cells in such a way that the intensity of cellular fluorescence decreases twofold with each successive generation. This property of CFSE allows for the accurate tracking of the number of divisions that a given cell has undergone, either in vitro or following transfer in vivo [19]. In order to determine whether D-pinitol exerts detectable effects on allogeneic T-cell stimulation, we subjected the DC to 24 h of stimulation with
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Fig. 4. D-pinitol-treated DC exhibit enhanced endocytic capacity. DC (1 × 105 cells) were treated with 80 μM D-pinitol, with or without LPS (200 ng/ml), for 24 h. The endocytic activities of the DC were assessed via flow cytometry after FITC-dextran treatment. The cells were then washed twice in cold HBSS and stained with a PE-conjugated anti-CD11c+ antibody. The endocytic activity of the controls was determined after exposure to FITC-dextran at 4 °C. A, The numbers represent the percentages of FITC-dextran-/CD11c+-PE double positive cells. B, The profile of endocytic capacity was also demonstrated as a histogram by mean fluorescence intensity (MFI). Medium designates the chemically untreated control group. The results represent the means (± SD) of three separate experiments, all of which yielded similar results. D-pinitol. As is shown in Fig. 4, the LPS-treated DC exhibited a far more profound rate of proliferation than did the controls, whereas D-pinitol appeared to impair proliferation responses in the allogeneic T cells elicited by the LPS-stimulated DC. Importantly, the maturation induced via LPS stimulation (24 h at 200 ng/ml) profoundly promoted the allostimulatory capacity of the untreated DC, whereas D-pinitol exposure significantly impaired their allostimulatory capacity (Fig. 5B). We also attempted to determine the potency of DC in terms of their ability to adhere to T cells, and their ability to form
clusters. The size of the DC/T-cell clusters decreased with exposure to D-pinitol in contrast to the LPS-treated group. The cluster number of LPS-treated DC in the presence of D-pinitol was markedly inhibited when compared with LPS-stimulated DC in the absence of D-pinitol (Fig. 5A). Considering the inhibitory effects of D-pinitol on the IL-12 (a Th1-inducing cytokine) production in DC, we attempted to characterize the quality of the primary T-cell response in DC that had matured in the presence of D-pinitol. Naive allogenic T cells primed with mature DC were observed to differentiate into Th1
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Fig. 5. D-pinitol treated DC impair cluster formation and proliferation of allogeneic T-cells, and inhibit Th1 responses in vitro. The DC were incubated for 24 h in medium alone, in 80 μM D-pinitol, 200 ng/ml LPS, or in D-pinitol coupled with LPS. The DC were washed and cocultured with T cells. A, Cluster formation was assessed after 72 h. One representative experiment of three is also included in the figure, showing a representative field in a culture well photographed using an inverted phase contrast microscope. B, DC were cultured in medium with or without D-pinitol for 24 h. The treated DC were harvested and thoroughly washed to remove the D-pinitol. A mixed lymphocyte reaction was allowed to proceed for 3 days, as described in the Materials and methods section of this article. T-cell proliferation was analyzed by flow cytometry and presented as a percentage of dividing cells. C, Cells were then examined for cytokine release after 48 h. IFN-γ and IL-4 were measured by ELISA in culture supernatants. Data are expressed as ng/ml/106 cells ± SD of triplicate cultures (⁎⁎, P b.01 vs T-cells primed with LPS-treated DC). Medium represents the chemically untreated control group. Similar results were obtained and expressed as the means (±SD) from four separate experiments.
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immature DC to D-pinitol, prior to LPS stimulation. Pretreatment with 80 μM D-pinitol resulted in a marked inhibition of the LPS-induced upregulation of p-p38, p-ERK1/2, and pJNK. The total ERK1/2 proteins were constitutively expressed (Fig. 6A). Furthermore, other researchers have reported that LPS signal transduction activates a variety of signal pathways, including the NF-κB pathway [18]. These data indicate that Dpinitol inhibits MAPKs expression, which is clearly relevant to the regulation of LPS-induced DC maturation. To characterize the role of NF-κB translocation more precisely, we stimulated immature DC with LPS prior to exposing them to D-pinitol. Then, in order to determine whether D-pinitol affects the blockade of LPS-induced NF-κB translocation, we prepared nuclear extracts from DC that had been treated with both LPS and D-pinitol. Nuclear translocation of the NF-κB p65 subunit was then evaluated via Western blotting. LPS was determined to enhance the nuclear translocation of the NF-κB p65 subunit within 45 min of exposure. Conversely, pretreatment with 80 μM D-pinitol resulted in suppression of the LPS-enhanced NF-κB p65 nuclear translocation (Fig. 6B). 3.6. Intraperitoneal administration of LPS-induced DC maturation
D-pinitol
inhibits
In order to determine whether the apparent inhibitory effects of D-pinitol on splenic DC maturation in vivo was mediated solely by drug toxicity or by interference with DC Fig. 6. D-pinitol suppresses MAPKs and NF-κB activation in LPSstimulated DC. The DC were pretreated with 80 μM D-pinitol for 15, 30 and 45 min prior to LPS stimulation (200 ng/ml). The cell lysates were prepared and blotted with anti-phospho-ERK1/2 (p-p44/42), antiERK1/2 (p44/42), anti-phospho-JNK1/2, anti-JNK1/2, anti-phosphop38 (p-p38), and anti-p38 (p38) Abs (A). LPS-induced NF-κB nuclear translocation was inhibited by D-pinitol (B). The DC were pretreated for 30 min with D-pinitol, and stimulated with 200 ng/ml LPS for the indicated lengths of time. The nuclear extracts were blotted with antip65 Ab. The bound Abs were visualized using biotinylated goat antirabbit IgG. The results shown represent three independent experiments. (−), Represents the chemically untreated control group.
lymphocytes when they generated high levels of INF-γ and low levels of IL-4 (Fig. 5C). By way of contrast, T cells primed with DC that had matured in the presence of D-pinitol inhibited INF-γ production. These results show that the majority of D-pinitol's effects on the T-cell-differentiating properties of DC are a consequence of the inhibition of IL-12 production. 3.5. D-pinitol suppresses LPS-induced activation of MAPKs and nuclear translocation of the NF-κB in DC LPS stimulation has been shown to affect MAPK and NFκB signal pathway activation in DC. MAPK and NF-κB activation are important events in DC maturation [21]. LPS activates p-p38 MAPK, p-ERK1/2, and p-JNK (Fig. 6A). In order to characterize the effects of D-pinitol on the expression of p-p38 MAPK, p-ERK1/2, and p-JNK in DC, we exposed
Fig. 7. In vivo D-pinitol administration suppresses the phenotypic maturation of LPS-challenged splenic DC. Mice were intraperitoneally injected with 10 mg/kg D-pinitol 3 times (1 time/day) for 3 days. One hour after the final injection, the mice were injected in a lateral tail vein with 1 mg/kg LPS. At 24 h, the mice were killed and the splenic DC were generated, as was described in the Materials and methods section of this article. The cells were harvested and we analyzed the expression of CD80, CD86, MHC I, and MHC II via two-color flow cytometry. The cells were firstly gated for CD11c+, after which we analyzed the expression of CD80, CD86 and MHC class II by flow cytometry as histogram with mean fluorescence intensity (MFI) (A) and distribution of positive populations (B). The histogram shown is from one of three representative experiments. All data points represent the average (±SD) of three separate experiments, which were conducted with five mice per group. (⁎⁎, P b 0.01 vs LPS-treated mice).
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Fig. 8. D-pinitol impaired INF-γ production in the CD4+ T-cells of LPS-treated mice. Mice were injected i.p. with 10 mg/kg of D-pinitol 3 times (1 time/day) for 3 days. One hour after the final injection, the mice were injected with 1 mg/kg of LPS in a lateral tail vein (this trial was conducted with five mice per group). The mice were killed at 24 h, and T-cells were generated as was described in the Materials and methods section of this article. The CD4+ T-cells were subsequently analyzed using an intracellular cytokine staining technique. Data are presented as a dotted plot (% of positive cells) and as a histogram mean fluorescence intensity (MFI) (A) and also presented as a histogram with MFI (B). The histogram (B) is from one representative experiment out of 3 (⁎⁎, P b 0.01 vs LPS-treated mice).
production, we assessed the effects of D-pinitol on phenotypic characteristics in LPS-stimulated mice. We isolated spleenderived DC from all experimental groups (5 mice per group), and confirmed the phenotypic characteristics of the mice using flow cytometry. We determined that 95% of the evaluated DC expressed CD11c+ molecules (Fig. 7A, B). Representative FACS
histograms showed that only the LPS-exposed splenic DC had expressed detectable levels of costimulatory and MHC molecules. However, in cells receiving 3 days of D-pinitol pretreatment, CD80, CD86, and MHC class I and II molecules were found to have been markedly downregulated 24 h after LPS challenge. These in vivo results showed that D-pinitol
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challenged with a hapten. By way of contrast, D-pinitol-treated DC, regardless of tested D-pinitol concentration, failed to elicit significant immune responses under identical conditions (Fig. 9). Additionally, the responses of animals that had been sensitized with TNBS-pulsed, D-pinitol-treated DC were shown to be comparable with those evidenced by the unsensitized animals. The results for the control group, which received injections with unpulsed DC (negative control), and for the group sensitized via epicutaneous hapten applications (positive control), confirmed that the observed immune responses were antigen-specific.
4. Discussion Fig. 9. D-pinitol-treated DC impairs cell-mediated immune response. A culture consisting of approximately 106 purified DC in the presence or absence of 80 μM. D-pinitol were pulsed with 0.1% (w/v) TNBS and injected with SC on day 0, as described in the Materials and methods section of this article. In the control groups, DC were either not TNBSpulsed ([−] control) and injected s.c., or the animals were shaved and the skin of their abdomen was painted with 7% w/v TNCB ([+] control). After 7 days, ear thickness was measured in the experimental mice. The results are expressed as the mean ± SD percentage increase in ear swelling for the 5 treatment group animals and the 5 control group animals. Treatment with vehicle alone induced no detectable swelling. (⁎⁎, P b 0.01 vs D-pinitol-treated DC or [−] control).
pretreatment inhibits the phenotypic maturation of LPSexposed DC. 3.7. D-pinitol impairs the production of INF-γ by CD4+ T cells in LPS-treated mice Naive allogenic T cells that had been primed with mature DC were shown to differentiate into Th1 lymphocytes when they produced high levels of INF-γ. In addition, high levels of IFN-γ in CD4+ T cells induce the production of IL-12 in DC. In an effort to characterize the effects of D-pinitol on INF-γ production in the CD4+ T cells of LPS-exposed mice, we isolated spleen-derived CD4+ T cells from all of the experimental mice and determined INF-γ production via flow cytometry. We determined that 95% of the evaluated T cells expressed CD4+ molecules. Representative FACS histograms showed that only the LPS-exposed splenic CD4+ T cells expressed detectable INF-γ levels. However, in cells pretreated for 3 days with D-pinitol, INF-γ production was markedly downregulated 24 h after LPS challenge (Fig. 8A, B). These in vivo data indicated that pretreatment with D-pinitol impaired INF-γ production in LPS-stimulated splenic CD4+ T cells. These results show that the majority of the effects of D-pinitol on the T-cell-differentiating properties of DC occur via INF-γ inhibition. 3.8. D-pinitol-treated DC fail to induce normal cell-mediated immune responses A single s.c. injection of 106 TNBS-pulsed purified DC was demonstrated to induce a significant CHS response, which was visualized 7 days after injection when the animals were
This is, to the best of our knowledge, the first report concerning the effects of D-pinitol on the phenotypic and functional maturation of murine BM-derived DC. This is also the first study in which D-pinitol-exposed DC have been analyzed with regard to their capacity to sensitize recipients for cell-mediated immune responses. Many researchers have previously shown that D-pinitol exerts a variety of biological effects, including anti-diabetic [12] and anti-inflammatory properties [13]. Nevertheless, the exact modulation pathways of D-pinitol remain unclear. Also, the effects of D-pinitol on the maturation and immunological responses of DC remain largely unknown. DC are also considered to play important roles in the establishment of hypersensitivity and transplantation tolerance [22]. We conducted a series of functional assays in order to ascertain the phenotype and function of D-pinitol-treated DC, and also conducted in vivo assessments of their capacity for hypersensitivity. Our findings indicate that the effects of D-pinitol principally involved the suppression of MAPKs and NF-κB activation. To ensure that the observed effects of D-pinitol could be attributed to DC and not to contaminating cells persisting in the BM-derived cell cultures, the DC were purified (N 95%) prior to analysis in each of the conducted assays. Our findings conclusively indicated that D-pinitol is a potent inhibitor of LPS-induced DC maturation. These findings provide new insights into the immunopharmacology of D-pinitol. D-pinitol inhibited DC maturation in a dose-dependent manner. The profound suppressive effects of D-pinitol on DC maturation can be safely attributed to a nonspecific inhibitory effect. Therefore, we also assessed the ability of D-pinitol-treated DC to internalize FITC-dextran via mannose receptor-mediated endocytosis. This mechanism is a defining characteristic of mature DC, as opposed to immature DC [17]. The endocytic capacities of the D-pinitol-treated DC were found to be profoundly elevated, thereby suggesting that D-pinitol inhibited both the phenotypic and functional maturation of DC. We conducted an evaluation of MAPKs and NF-κB
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signal pathways, in order to characterize the mechanism underlying the preventive effects of D-pinitol on LPS-induced DC maturation. The MAPKs and NF-κB signaling pathways appear to be extremely relevant to the process of DC maturation [21]. Within the MAPKs and NF-κB signaling pathways, several molecules, including ERK, JNK, p38 and IκB, have been shown to be crucial. In this study, D-pinitol exerted a detectable suppressive effect on the phenotypic and functional maturation of murine BM-DC, via ERK1/2 and JNK inhibition. This mechanism may also be relevant to the observed protective effects of D-pinitol against certain autoimmune diseases, including arthritis, allergy, and diabetes. We also found that the NF-κB signaling pathway may be closely involved in the DC maturation-inhibitory effects of D-pinitol. Recently collected evidence suggests that DC cytokine production is dependent on either the particular subset of DC, or on the stimuli received by the DC [23]. IL-12, in particular, exerts multiple immunoregulatory functions, inducing the activation of the Th1 subset, which performs a pivotal role in the induction of inflammation. A growing body of evidence suggests that development toward Th1 cells is primarily regulated by DC-derived cytokines, including IL-12 [24] and IFN-γ [25]. The results obtained in this report indicate that D-pinitol caused CD11c+ DC to generate IL-12 in the presence of LPS, and also confirm that D-pinitol exerts inhibitory effects on the expression of intracellular IL-12 p40/p70 and IL-12 p70. Additionally, this study demonstrates that LPS-stimulated CD11c+ DC skews naive T cells toward development into IFN-γ-producing T cells. D-pinitol was determined to significantly impair the capacity of these cells to proliferate and initiate Th1 responses, under both in vitro and in vivo conditions. Naive T cells stimulated with D-pinitol-treated DC generated lower IFN-γ levels, but did not exhibit significantly altered abilities to generate IL-4. These results indicated that D-pinitol is a potent DC maturation inhibitor. Because Th1 cells exert functionally immunogenic or protective effects against invading pathogens, the inhibition of DC-mediated Th1 polarization might constitute a D-pinitol-associated immunosuppressive mechanism. However, the inhibition of Th1 development exerts negative regulatory roles on a wide variety of immune cells [24]. Consequently, D-pinitol-mediated inhibition of IL-12 generation in LPS-stimulated CD11c+ DC may also contribute to the induction of an immunosuppressive state. Based on observations in our in vitro and in vivo experiments, we hypothesized that D-pinitol-treated DC
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should exhibit deficiencies in their ability to stimulate naive T cells in vivo, in addition, they should initiate cellmediated immune responses. It was demonstrated recently that as few as 105 TNBS pulsed murine BM-derived DC (TNBS-DC) induced a profound CHS response [26]. The ability of the DC to induce T-cell-mediated immune responses was evaluated using TNBS-DC to sensitize for CHS in naive syngeneic recipients. CHS sensitization was verified in recipients of s.c. injections containing 105 TNBS-DC, but was not observed in recipients of D-pinitol-pretreated TNBS-DC. This shows that the decreased T-cell-stimulatory capacity of the D-pinitoltreated, BM-derived DC cannot be readily reversed after the removal of D-pinitol is sustained in vivo [27]. In conclusion, we have characterized a variety of effects exerted by D-pinitol on BM-derived DC. D-pinitol was demonstrated to inhibit phenotypic maturation and modulate cytokine production in these DC, resulting in a significant inhibition of Th1 development. Moreover, our experimental data showed that D-pinitol suppressed LPSinduced costimulatory molecules/MHC class types and IFN-γ, under in vivo experimental conditions. These findings illustrate the immunopharmarcological functions of D-pinitol. Moreover, exposure to this readily available drug may constitute a nontoxic and highly effective means for the modulation of DC immunoregulatory capacity. Acknowledgement This work was supported by the Korea Science and Engineering Foundation through National Research Laboratory Program Grant M1050000000806J00 0000810. References [1] Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991;9:271–96. [2] Rock KL, Clark K. Analysis of the role of MHC class II presentation in the stimulation of cytotoxic T lymphocytes by antigens targeted into the exogenous antigen-MHC class I presentation pathway. J Immunol 1996;156(10):3721–6. [3] Austyn JM. Dendritic cells. Curr Opin Hematol 1998;5(1):3–15. [4] Cella MR, Del Prete A, Vecchi A, Corada M, Martin-Padura I, Motoike T, et al. Increased DC trafficking to lymph nodes and contact hypersensitivity in junctional adhesion molecule-Adeficient mice. J Clin Invest 2004;114(5):729–38. [5] Salio M, Cerundolo V, Lanzavecchia A. Dendritic cell maturation is induced by mycoplasma infection but not by necrotic cells. Eur J Immunol 2000;30(2):705–8. [6] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392(6673):245–52. [7] Chen JW, Zhu ZQ, Hu TX, Zhu DY. Structure-activity relationship of natural flavonoids in hydroxyl radical-scavenging effects. Acta Pharmacol Sin 2002;23(7):667–72.
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[8] Kim GY, Kim KH, Lee SH, Yoon MS, Lee HJ, Moon DO, et al. Curcumin inhibits immunostimulatory function of dendritic cells: MAPKs and translocation of NF-kappa B as potential targets. J Immunol 2005;174(12):8116–24. [9] Kim SH, Park HJ, Lee CM, Choi IW, Moon DO, Roh HJ, et al. Epigallocatechin-3-gallate protects toluene diisocyanate-induced airway inflammation in a murine model of asthma. FEBS Lett 2006;580(7):1883–90. [10] Lee JS, Kim SG, Kim HK, Lee TH, Jeong YI, Lee CM, et al. Silibinin polarizes Th1/Th2 immune responses through the inhibition of immunostimulatory function of dendritic cells. J Cell Physiol 2006;210(2):385–97. [11] Kim GY, Lee MY, Lee HJ, Moon DO, Lee CM, Jin CY, et al. Effect of water-soluble proteoglycan isolated from Agaricus blazei on the maturation of murine bone marrow-derived dendritic cells. Int Immunopharmacol 2005;5(10):1523–32. [12] Davis A, Christiansen M, Horowitz JF, Klein S, Hellerstein MK, Ostlund Jr RE. Effect of pinitol treatment on insulin action in subjects with insulin resistance. Diabetes Care 2000;23(7): 1000–5. [13] Singh RK, Pandey BL, Tripathi M, Pandey VB. Anti-inflammatory effect of (+)-pinitol. Fitoterapia 2001;72(2): 168–70. [14] Kim JI, Kim JC, Kang MJ, Lee MS, Kim JJ, Cha IJ. Effects of pinitol isolated from soybeans on glycaemic control and cardiovascular risk factors in Korean patients with type II diabetes mellitus: a randomized controlled study. Eur J Clin Nutr 2005;59(3):456–8. [15] Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/ macrophage colony-stimulating factor. J Exp Med 1992;176 (6): 1693–702. [16] Lutz MB, Assmann CU, Girolomoni G, Ricciardi-Castagnoli P. Different cytokines regulate antigen uptake and presentation of a precursor dendritic cell line. Eur J Immunol 1996;26(3):586–94. [17] Sallusto F, Cella M, Danieli C, Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J Exp Med 1995;182(2):389–400.
[18] Rescigno M, Martino M, Sutherland CL, Gold MR, RicciardiCastagnoli P. Dendritic cell survival and maturation are regulated by different signaling pathways. J Exp Med 1998;188(11): 2175–80. [19] Lyons AB. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. J Immunol Methods 2000;243(1–2):147–54. [20] Mosca PJ, Hobeika AC, Clay TM, Nair SK, Thomas EK, Morse MA, et al. A subset of human monocyte-derived dendritic cells expresses high levels of interleukin-12 in response to combined CD40 ligand and interferon-gamma treatment. Blood 2000;96(10):3499–504. [21] An H, Yu Y, Zhang M, Xu H, Qi R, Yan X, et al. Involvement of ERK, p38 and NF-kappaB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells. Immunology 2002;106(1):38–45. [22] Shlomchik WD, Couzens MS, Tang CB, McNiff J, Robert ME, Liu J, et al. Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 1999;285(5426):412–5. [23] Brinkmann V, Geiger T, Alkan S, Heusser CH. Interferon alpha increases the frequency of interferon gamma-producing human CD4+ T cells. J Exp Med 1993;178(5):1655–63. [24] Kadowaki N, Antonenko S, Ho S, Rissoan MC, Soumelis V, Porcelli SA, et al. Distinct cytokine profiles of neonatal natural killer T cells after expansion with subsets of dendritic cells. J Exp Med 2001;193(10):1221–6. [25] Rissoan MC, Soumelis V, Kadowaki N, Grouard G, Briere F, de Waal Malefyt R, et al. Reciprocal control of T helper cell and dendritic cell differentiation. Science 1999;283(5405):1183–6. [26] Lappin MB, Weiss JM, Delattre V, Mai B, Dittmar H, Maier C, et al. Analysis of mouse dendritic cell migration in vivo upon subcutaneous and intravenous injection. Immunology 1999;98(2): 181–8. [27] Yoon MS, Lee JS, Choi BM, Jeong YI, Lee CM, Park JH, et al. Apigenin inhibits immunostimulatory function of dendritic cells: implication of immunotherapeutic adjuvant. Mol Pharmacol 2006;70(3):1033–44.
D-pinitol
inhibits Th1 polarization via the suppression of dendritic cells
Jun Sik Lee a , In Duk Jung b , Young-Il Jeong c , Chang-Min Lee b , Yong Kyoo Shin d , Sang-Yull Lee e , Dong-Soo Suh f , Man-Soo Yoon f , Kyu-Sub Lee f , Yung Hyun Choi g , Hae Young Chung a , Yeong-Min Park b,⁎ b
a Department of Pharmacy, Pusan National University College of Pharmacy, Busan 609-735, South Korea Department of Microbiology and Immunology, and National Research Laboratory of Dendritic Cells Differentiation and Regulation, and Medical Research Institute, South Korea c Department of Microbiology, Pusan National University College of Nature Science, Busan 609-735, South Korea d Department of Pharmacology, Chungang University College of Medicine, Seoul 156-756, South Korea e Department of Biochemistry, Pusan National University College of Medicine, Busan 602-739, South Korea f Department of Obstetrics, Pusan National University College of Medicine, Busan 602-739, South Korea g Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052, South Korea
Received 21 December 2006; received in revised form 25 January 2007; accepted 25 January 2007
Abstract D-pinitol has been demonstrated to exert insulin-like and anti-inflammatory effects. However, the effects of the maturation and immunostimulatory functions of dendritic cells (DC) remain to be clearly elucidated. In this study, we have attempted to determine whether D-pinitol regulates surface molecule expression, cytokine production, endocytosis capacity, and underlying signaling pathways in murine bone marrow-derived DC. We also attempted to ascertain whether D-pinitol could influence Th1/Th2 immune response in vivo. The DC used in this study were derived from murine bone marrow cells, and were used as immature or LPSstimulated mature DC. The DC were then assessed with regard to surface molecule expression, dextran-FITC uptake, cytokine production, capacity to induce T-cell differentiation, and underlying signaling pathways. D-pinitol was shown to significantly inhibit CD80, CD86, MHC class I, and MHC class II expression in the LPS-stimulated mature DC. The DC also evidenced impaired IL-12 expression and IFN-γ production. The D-pinitol-treated DC were found to be highly efficient in regards to Ag capture via mannose receptor-mediated endocytosis. D-pinitol was also demonstrated to inhibit LPS-induced MAPKs activation and NF-κB nuclear translocation. Moreover, the D-pinitol-treated DC manifested impaired induction of Th1 responses, and normal cell-mediated immune responses. These novel findings provide new insight into the immunopharmacological role of D-pinitol in terms of its effects on DC. These findings also broaden current perspectives concerning our understanding of the immunopharmacological functions of D-pinitol, and have ramifications for the development of therapeutic adjuvants for the treatment of DC-related acute and chronic diseases. © 2007 Elsevier B.V. All rights reserved.
Keywords: D-pinitol; Dendritic cell; Th1 polarization; IFN-γ; NF-κB; IL-12
Abbreviations: DC, dendritic cells; MAPKs, mitogen-activated protein kinases; BM, bone marrow; APC, antigen-presenting cells; MHC, major histocompatibility complex; MLR, mixed lymphocyte reaction; TNCB, 2,4,6-trinitrochlorobenzene; CHS, contact hypersensitivity. ⁎ Corresponding author. Department of Microbiology and Immunology and National Research Laboratory of Dendritic Cell Differentiation & Regulation, Pusan National University College of Medicine, Ami-dong 1-10, Seo-gu, Busan 602-739, South Korea. Tel.: +82 51 240 7557; fax: +82 51 243 2259. E-mail address: [email protected] (Y.-M. Park). 1567-5769/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2007.01.018
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1. Introduction A substantial body of research is currently focused on the biology of dendritic cells (DC), specifically with regard to their possible clinical use as cellular adjuvants in the treatment of chronic infectious diseases and tumors. DC are professional antigen-presenting cells (APC) that have been determined to possess immune sentinel properties, initiating T-cell responses against microbial pathogens and tumors [1]. Immature DC capture and process exogenous agents within peripheral tissues, in which they begin to mature. Maturing DC migrate into the lymphoid organs, where they stimulate naive T cells via the signaling of both major antigen-peptide-presenting histocompatibility complex (MHC) molecules and costimulatory molecules [2,3]. Maturation and differentiation are generally understood to require the activation phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), p38 MAPK, and the transcription nuclear factor kappa B (NFκB) [4,5]. DC have also been demonstrated to be highly responsive to both inflammatory cytokines and bacterial products, such as TNF-α and lipopolysaccharides (LPS). When encountered in the peripheral organs, these products are known to induce a series of phenotypic and functional alterations in the DC. Similar changes, indicative of maturation, have also been reported after the infection of cells with mycoplasma, viruses, intracellular bacteria, and parasites [7]. DC located within the peripheral tissues generally tend to be both phenotypically and functionally immature. Immature DC do not induce primary immune responses, as they do not express the requisite costimulatory molecules. In addition, they don't express antigenic peptides as stable complexes with MHC molecules. While in an immature state, DC have been shown to effectively capture and process exogenous antigens (Ag) within the peripheral tissues, where the DC then begin to mature. DC maturation has previously been associated with decreased or absent Ag uptake, high levels of MHC class II and accessory molecule expression, as well as IL-12 generation upon stimulation [6]. The flavonoids comprise a family of common phenolic plant pigments, which have been identified as dietary anticarcinogens and antioxidants [7]. We reported previously that a variety of phytochemicals exhibit profound immunoregulatory activity, particularly in DC [8–11]. D-pinitol (3-O-methyl-chiroinositol), an active principle of the traditional antidiabetic plant, Bougainvillea spectabilis, reportedly exerts insulin-like effects [12]. Pinitol is also known to exert insulin-like
effects, via the driving of creatine and other nutrients into muscle cells [12]. D-pinitol has been suggested to possess multifunctional properties, including anti-inflammatory properties [13]. In addition, it has also been implicated in the prevention of cardiovascular diseases [14]. Until now, the cellular targets of D-pinitol in the immune system have remained enigmatic, thereby leaving the issue of the global function of D-pinitol in relation to DC maturation and immuno-regulatory activities open to discussion in terms of future research. In this study, we have attempted to characterize the effects of a noncytotoxic concentration of D-pinitol on the maturational and functional properties of murine bone marrow (BM)-derived DC. Our findings demonstrated, for the first time, that D-pinitol treatment inhibited phenotypic and functional maturation in the DC. D-pinitol treatment also suppressed the LPSinduced activation of ERK1/2, JNK, and p38 MAPK, as well as NF-κB nuclear translocation, in the DC. The in vivo data indicate that, although the D-pinitol-treated DC were observed to migrate to the T-cell areas of secondary lymphoid tissue, they did not induce normal cell-mediated contact hypersensitivity (CHS). Moreover, this readily available drug may constitute a simple, inexpensive, and highly effective means for the manipulation of the immunostimulatory capacity of DC. Considering, then, the critical functions of these professional APC in the initiation and regulation of immune responses, coupled with the ready availability of D-pinitol, our findings appear to bear important implications with regard to the manipulation of the functions of DC in potential therapeutic applications. 2. Materials and methods 2.1. Animals and chemicals Male 8–12-week-old C57BL/6 (H-2Kb and I-Ab) and BALB/c (H-2Kd and I-Ad) mice were purchased from the Korean Institute of Chemistry Technology (Daejeon, Korea). They were housed in a specific pathogen-free environment within our animal facility for at least 1 week before use. D-pinitol was purchased from Sigma. 2.2. Reagents and Abs Recombinant mouse (rm)GM-CSF and rmIL-4 were purchased from R&D Systems. D-pinitol, dextran-FITC (molecular mass, 40,000), and LPS (from Escherichia coli 055:B5) were obtained from Sigma-Aldrich. An endotoxin filter (END-X) and an endotoxin removal resin (END-X B15) were acquired from Associates of Cape Cod. Cytokine ELISA kits for murine IL-12 p70, IL-4, and IFN-γ were purchased from BD Pharmingen. FITC-conjugated or PE-conjugated mAbs used to detect the
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expression of CD11c (HL3), CD80 (16-10A1), CD86 (GL1), CD54 (1C10), IAb β-chain (AF-120.1), H2Kb (AF6-88.5), CD3, and CD4 (L3T4), or the intracellular expression of IL-12 p40/ p70 (C15.6), and IL-10 (JESS-16E3) by flow cytometry, as well as isotype-matched control mAbs, biotinylated anti-CD11c (N418) mAb, Cytofix/Cytoperm kit and FITC-annexin V in combination with a propidium iodine kit were purchased from BD Pharmingen. To detect protein levels, anti-phospho-ERK, anti-ERK, anti-phospho-p38, anti-p38, anti-p-IκB, anti-β-actin anti-phospho-JNK, anti-JNK, and anti-p65 Ab from Santa Cruz. Bead-conjugated anti-CD11c mAb (Miltenyi Biotec) and paramagnetic columns (LS columns) were purchased from Miltenyi Biotec. 2.3. Generation and culture of DC DC was generated from murine BM cells, as described by Inaba et al. [15] with modifications. Briefly, BM was flushed from the tibiae and femurs of C57BL/6 and depleted of red cells with ammonium chloride. The cells were plated in sixwell culture plates (106 cells/ml; 3 ml/well) in OptiMEM (Invitrogen Life Technologies) supplemented with 10% heatinactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 × 10− 5 M 2-ME, 10 mM HEPES (pH 7.4), 20 ng/ml rmGM-CSF and rmIL-4 at 37 °C, 5% CO2. On day 3 of the culture, floating cells were gently removed and fresh medium was added. On day 6 or 7 of the culture, nonadherent cells and loosely adherent proliferating DC aggregates were harvested for analysis or stimulation. In some experiments, they were replated in 60-mm dishes (106 cells/ml; 5 ml/dish). On day 6, 80% or more of the nonadherent cells expressed CD11c. In certain experiments, in order to obtain highly purified populations for subsequent analyses, the DC were labeled with bead-conjugated antiCD11c mAb. This was followed by positive selection through paramagnetic columns (LS columns) according to the manufacturer's instructions. The purity of the selected cell fraction was N 95%.
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serum and 0.1% sodium azide in PBS). Cells were first blocked with 10% (v/v) normal goat serum for 15 min at 4 °C and stained with phycoerythrin (PE)-conjugated anti-H-2Kb [major histocompatibility complex (MHC) class I], anti-I-Ab (MHC class II), anti-CD80, and anti-CD86 with fluorescein isothiocyanate (FITC)-conjugated anti-CD11c for 30 min at 4 °C. The stained cells were analysed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). 2.6. Quantitation of Ag uptake Endocytosis was quantitated, as described by Lutz et al. [16,17]. In brief, 2 × 105 cells were equilibrated at 37 or 4 °C for 45 min and then pulsed with fluorescein-conjugated dextran at a concentration of 1 mg/ml. Cold staining buffer was added to stop the reaction. The cells were washed three times and stained with PE-conjugated anti-CD11c Abs, and then analyzed by FACSCalibur. Nonspecific binding of dextran to DC, determined by incubation of DC with FITC-conjugated dextran at 4 °C, was subtracted. The medium used in the culture, to stimulate DC with D-pinitol, was supplemented with GM-CSF, because the ability of DC to capture Ag is lost if DC are cultured without GM-CSF [18]. 2.7. Cytokines assay Cells were first blocked with 10% (v/v) normal goat serum for 15 min at 4 °C and then stained with FITC-conjugated CD11c+ antibody for 30 min at 4 °C. Cells stained with the appropriate isotype-matched Ig were used as negative controls. The cells were fixed and permeated with the Cytofix/ Cytoperm kit according to the manufacturer's instructions. Intracellular IL-12p40/p70, IL-10, and IFN-γ were stained with fluorescein R-phycoerythrin (PE)-conjugated antibodies in a permeation buffer. The cells were analyzed on a FACSCalibur flow cytometer using the CellQuest program. Furthermore, murine IL-12p70, IL-4, and IFN-γ from DC were measured using an ELISA kit according to the manufacturer's instructions.
2.4. Stimulation of DC by D-pinitol 2.8. Mixed lymphocyte reaction D-pinitol
was dissolved in DMSO, or DMSO alone (0.01% v/v) was added to cultures of isolated DC in six-well plates (106 cells/ml; 3 ml/well). DMSO (0.1%) alone was used as a control due to the lack of no cytotoxicity in DC. For the analysis of apoptosis, DC were stimulated with LPS or left without any stimuli. Additionally, apoptosis was analyzed over time by staining of phosphatidylserine translocation with FITC-annexin V in combination with a propidium iodine kit according to the manufacturer's instructions. 2.5. Flow cytometric analysis On day 6, BM-DC were harvested, washed with phosphate buffered saline (PBS) and resuspended in fluorescence activated cell sorter (FACS) washing buffer (2% fetal bovine
Responder T cells, which participate in allogeneic T-cell reactions, were isolated from spleen of BALB/c mice with a MACS column. Staining with fluorescein isothiocyanate (FITC)-conjugated anti-CD3 antibodies revealed that they consisted mainly of CD3+ cells (N 95%). The lymphocyte population (95% of CD3+ cells) was then washed twice in PBS and labeled with CFSE, as previously described [19]. The cells were resuspended in 5 μM CFSE in phosphate-buffered saline (PBS). After being shaken for 8 min at room temperature, the cells were washed once in pure fetal bovine serum (FBS) and twice in PBS with 10% FBS. DC (1 × 104) or DC exposed to D-pinitol (80 μM/ml) or LPS (100 ng/ml) for 24 h were cocultured with 1 × 105 allogenic CFSE-labeled T lymphocytes in 96-well, U-bottom plates (Nunc). A negative control (CD3+
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BCA assay kit (Pierce, Rockford, IL). Equivalent amounts of proteins were separated by SDS-10% PAGE and analyzed by Western blotting using an antiphospho-ERK1/2 (p-ERK), antiphospho-JNK (p-JNK), or anti-phospho-p38 (p-p38) MAPK mAb for 2 h, as described by the manufacturer of the antibodies. Following washing three times with TBST, membranes were incubated with secondary HRP-conjugated anti-mouse IgG for 1 h. After washing, the blots were developed using the ECL system (Amersham) by following the manufacturer's instructions. DC nuclear extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL), according to the manufacturer's instructions. NF-κB p65 subunits in the nuclear extracts were determined by Western blot analysis with anti-NF-κB p65 subunit Ab. 2.10. Generation of DC from spleen and culture
Fig. 1. D-pinitol has no cytotoxicity to BM-DC up to 80 μM. BMderived DC were generated as was described in the Materials and methods section, and analyzed via flow cytometry on day 7. The DC were stained for CD11c and annexin-V and PI. The percentage within each positive cell represents the incidence of annexin-V+ PI+. DC were generated as described in the Materials and methods section of this article.
lymphocytes alone) and a positive control (CD3+ lymphocytes in 5 μg of Concanavalin A) were created for each experiment. After 4 days, the cells were harvested and washed in PBS. CFSE dilution optically gated lymphocytes were assessed by flow cytometry. 2.9. Nuclear and cytoplasmic extracts and Western blot The cells were exposed to LPS (200 ng/ml) in the absence or presence of 80 μM in a D-pinitol pretreatment. Following 15 or 30 min of incubation at 37 °C, cells were washed twice with cold PBS and lased with modified RIPA buffer (1.0% NP-40, 1.0% sodium deoxycholate, 150 nM NaCl, 10 mM Tris–HCl [pH 7.5], 5.0 mM sodium pyrophosphate, 1.0 mM NaVO4, 5.0 mM NaF, 10 mM/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride) for 15 min at 4 °C. Lysates were cleared by centrifuging at 14,000 ×g for 20 min at 4 °C. The protein content of cell lysates was determined using the Micro
Mice were injected intraperitoneally with D-pinitol (10 mg/kg) 3 times (1 time/day) for 3 days before the administration of 1 mg/kg of LPS in a lateral vein of the tail. Twenty-four hours after the LPS challenge, they were scarified, their spleens were disrupted and the cells were centrifuged at 400 ×g for 5 min, resuspended in OptiMem supplemented with 10% heatinactivated FBS, L-glutamine, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, HEPES and 2-ME for 2 h. Finally, the non-adherent cells were washed out. The residual adherent cells were maintained in the culture medium and incubated overnight at 37 °C in a 5% CO2 atmosphere. After incubation, DC (which exhibit adherence capacity in the first hours of culture) became non-adherent and floated in the medium. The cells were gated on CD11c+ for DC. 2.11. Isolation of CD4+ T cell from spleen and culture Mice were injected intraperitoneally with D-pinitol (10 mg/kg) every 3 days before the administration of 1 mg/kg of LPS in a lateral vein of the tail. Twenty-four hours after the LPS challenge, they were killed, their spleens were disrupted and the cells were centrifuged at 400 ×g for 5 min, resuspended in OptiMem supplemented with 10% heat-inactivated FBS, Lglutamine, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, HEPES and 2-ME for 4 h. The residual adherent cells were maintained in the culture medium and incubated overnight at 37 °C in a 5% CO2 atmosphere. The cells were fixed and permeated with the Cytofix/Cytoperm kit according to manufacturer's instructions. Intracellular IFN-γ was stained with fluorescein R-phycoerythrin (PE)-conjugated antibodies in a permeation buffer. The cells were gated on CD4+ T cell for T cell. 2.12. 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced CHS In the sensitization phase, bead-sorted CD11c+ DC were pulsed with 0.1% (w/v) TNBS (Sigma Aldrich) in PBS for
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Fig. 2. D-pinitol suppresses the expression of CD80, CD86, MHC class I, and MHC class II in a dose-dependent manner during DC maturation. DC were generated as described in the Materials and methods section of this article. On day 6, the cells were harvested and analyzed using two-color flow cytometry. The cells were gated on CD11c+. D-pinitol was added for 24 h to the DC at concentrations of 20, 40, and 80 μM. Surface molecule expression was then analyzed. A, The DC were left untreated (control) or stimulated for 24 h with 200 ng/ml LPS, in the absence or presence (gray line, control isotype; thin line, D-pinitol with LPS; thick line, LPS) of 80 μM D-pinitol on day 6. B, Overlay of histogram A in one box for clarification, respectively. UN represents the chemically untreated control group. The histogram is from one out of four representative experiments.
15 min at 37 °C. After three washes in PBS, the cells were counted, and their viability was assessed by trypan blue exclusion. One million cells were injected s.c. into the abdomen of animals shaved and painted with 7% w/v 2,4,6trinitrochlorobenzene (TNCB; Sigma-Aldrich) diluted in acetone-olive oil, 4/1 (v/v) (vehicle). Negative controls included animals injected with unpulsed DC (without hapten) and animals treated with the vehicle alone. Seven days after
sensitization, the mice were painted on the dorsal and ventral side of the left ear with 10 μl of 1% TNCB in vehicle. The thickness of the left (challenged) and the right (control) ear were measured after 48 h by using an engineer's spring-loaded micrometer (Mitutoyo). The percentage increase in ear thicknesses was calculated using the following formula: 100 × ((thickness of challenged ear-thickness of unchallenged ear) / thickness of unchallenged ear).
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Table 1 D-pinitol markedly inhibits the expression of costimulatory molecules (CD80, CD86 and CD54), MHC class I and II on LPS-stimulated CD11c + DC % of positive cell a (MFI) b Surface Ag
Medium
D-pinitol
LPS
D-pinitol + LPS
CD80 CD86 MHC I MHC II CD54
71 ± 2 (263 ± 32) 72 ± 1 (167 ± 21) 74⁎ ± 3 (168 ± 17) 76 ± 4 (395 ± 43) 74⁎ ± 4 (315 ± 24)
72⁎ ± 3 (270 ± 17) 73 ± 4 (154 ± 21)⁎ 72⁎ ± 3 (151 ± 17)⁎ 74 ± 2 (263 ± 42) 73 ± 1 (310 ± 25)⁎
86⁎ ± 2 (596 ± 42) 87 ± 1 (582 ± 47) 88⁎ ± 2(286 ± 12) 84⁎ ± 2 (721 ± 52) 89 ± 1 (893 ± 95)
77 ± 4 (420 ± 23)⁎⁎ 79 ± 2 (379 ± 42)⁎⁎ 82 ± 2 (215 ± 7)⁎⁎ 81 ± 3 (559 ± 36)⁎⁎ 83 ± 1 (712 ± 71)
⁎, ⁎⁎, The statistical significance between samples with and without D-pinitol is indicated (⁎, P b 0.05 vs unstimulated DC (medium); ⁎⁎, P b 0.01 vs LPS-stimulated DC). a BM-derived DC were cultured in the absence or presence of D-pinitol (80 μM) following the LPS (200 ng/ml) stimulation for 24 h. The expression of surface molecules was analyzed by FACSCalibur. A two-color flow cytometry was used to determine the level of surface molecules expression on CD11c + DC. b MFI, (mean fluorescence intensity). The results are from one experiment of three performed.
2.13. Statistics All results were expressed as the means ± SD of the indicated number of experiments. Statistical significance was estimated using a Student's t-test for unpaired observations. Afterwards the differences were compared with regard to statistical significance by one-way ANOVA, followed by the Bonferroni's post hoc test. The categorical data from the fertility test were subjected to statistical analysis via a Chisquare test. A P of b 0.05 was considered significant. 3. Results 3.1. D-pinitol inhibits the maturation of BM-derived murine DC In the initial series of experiments, we attempted to determine whether D-pinitol influences DC maturation. BMDC were cultured for 6 days in OptiMEM supplemented with 20 ng/ml of granulocyte-macrophage-colony stimulating factor (GM-CSF) and 20 ng/ml of IL-4. Different concentrations of D-pinitol were added to the cultures on day 6, with or without 200 ng/ml of LPS. D-pinitol was determined to be noncytotoxic to BM-DC at concentrations of 80 μM. There were no marked differences in the percentage of dead cells, as evidenced by CD11c+ cell and annexin-V/propidium iodide (PI) staining (Fig. 1); for this reason, it was adjusted to concentrations below 80 μM. We then investigated the effects of a range of D-pinitol concentrations on DC maturation. BMDC were cultured for 24 h in the presence of 0 to 80 μM D-pinitol, as is described in the Materials and methods section of this article. As is shown in Fig. 2A and Table 1, D-pinitol 80 μM significantly attenuated CD80, CD86, CD54, and MHC class I and II expression on the surface of the CD11c+, in contrast to LPS. These inhibitory effects occurred in a dosedependent manner, and were most pronounced with regard to the expression of CD80, CD86, CD54, MHC class I and MHC class II molecules. These molecules were also upregulated within 24 h of LPS exposure (Fig. 2B, thick lines). Exposure to 80 μM D-pinitol in the presence of LPS was accompanied by an impaired expression of MHC class I and MHC class II molecules.
Interestingly, a significant downregulation of the costimulatory molecules, CD80 and CD86, was also observed under these conditions (Fig. 2B, thin lines). 3.2. D-pinitol impairs IL-12 secretion, and does not influence IL-10 production during LPS-induced DC maturation It has been previously hypothesized that DC, as well as macrophages and monocytes, function as sources of proinflammatory molecules [18,20]. We assessed the ability of the BM-DC to generate pro-inflammatory cytokines. IL-12 expression was identified previously as a specific marker of DC activity. It is also known to be an important marker for DC maturation, and can be used to select Th1-dominant adjuvant. The secretion of bioactive IL-12 p70 requires the coordinated expression of two of its subunits, namely p35 and p40, which are encoded by two separate genes, and regulated independently [16]. We analyzed the production of both intracellular IL-12p40/p70 and bioactive IL-12p70 in D-pinitol-treated DC. As is shown in Fig. 3A, the intracellular staining of FITClabeled anti-CD11c+ DC with PE-labeled anti-IL-12 p40/p70 or anti-IL-10 mAbs revealed that DC stimulated with 80 μM D-pinitol expressed small amounts of IL-12 p40/p70, in contrast to unstimulated DC (D-pinitol-untreated, i.e., LPStreated DC), whereas IL-10 was merely detected. When the supernatants were analyzed via ELISA, IL-10 was also barely detectable 24 h after stimulation with 200 ng/ml of LPS. As is shown in Fig. 3B, ELISA analysis revealed high levels of IL12 p70 upon the stimulation of DC with LPS (95.4 ± 12.2 ng/ ml) for 24 h. D-pinitol (54.2 ± 7.9 ng/ml) was shown to alleviate the effects of LPS. These results indicated that exposure to D-pinitol impairs the ability of DC to generate large amounts of IL-12 p70 and pro-inflammatory cytokines. The findings also indicate that D-pinitol suppresses the functional maturation of LPS-stimulated DC. 3.3. D-pinitol suppresses endocytic activity of DC The expression of surface molecules on DC and the observed changes in IL-12 production show that D-pinitol exposure leads
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Fig. 3. D-pinitol impairs IL-12 secretion and has no influence on IL-10 in LPS-induced DC maturation. A, BM-DC was stimulated by 80 μM D-pinitol for 24 h with or without LPS. This figure also represents the analysis of IL-12 p40/p70 and IL-10 expression in CD11c+ DC by intracellular cytokine staining after 24 h of LPS stimulation. The numbers indicate the percentages of CD11c+ cells expressing IL-12 p40/p70 or IL-10. The results are representative of four separate experiments. B, Analysis of IL-12 p70 production by magnetic bead-purified DC (1 × 106 cells) over time (12–48 h) after LPS stimulation (200 ng/ml) using ELISA. The data represent the means (± SD) of four separate experiments. (⁎⁎, P b.01 vs DC-stimulated with D-pinitol + LPS).
to a profound inhibition of the phenotypic and functional maturation of DC in vitro. However, these results did not permit us to exclude the possibility that D-pinitol may induce a general inhibition of the physiological functions of DC. Thus, we attempted to determine whether the stimulation of DC with D-pinitol alters the ability of the cells to capture antigens. We exposed the DC to D-pinitol, with or without LPS, adding dextran-FITC to the culture media. The percentage of doublepositive cells (CD11c+ + dextran-FITC) in the D-pinitol-treated and non-treated DC was identical. The percentage of LPSstimulated DC was lower than the percentage of untreated DC. The D-pinitol-treated DC exhibited a higher capacity for endocytosis of dextran-FITC than did the LPS-stimulated DC (Fig. 4A, B). Again, this shows that the D-pinitol-treated DC were both phenotypically and functionally immature. A set of identical experiments were also performed at 4 °C, and the results of these indicated that the uptake of dextran-FITC by DC
was inhibited at low temperatures. This data indicates that D-pinitol induces immaturity in the DC. 3.4. D-pinitol impairs the allostimulatory capacity of DC The fluorescein-based dye, CFSE, has biochemical properties which render it particularly appropriate for this application. Specifically, CFSE dye is loaded into cells in vitro and the CFSE in a given cell is monitored over time. Upon division, the CFSE segregates equally between the daughter cells in such a way that the intensity of cellular fluorescence decreases twofold with each successive generation. This property of CFSE allows for the accurate tracking of the number of divisions that a given cell has undergone, either in vitro or following transfer in vivo [19]. In order to determine whether D-pinitol exerts detectable effects on allogeneic T-cell stimulation, we subjected the DC to 24 h of stimulation with
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Fig. 4. D-pinitol-treated DC exhibit enhanced endocytic capacity. DC (1 × 105 cells) were treated with 80 μM D-pinitol, with or without LPS (200 ng/ml), for 24 h. The endocytic activities of the DC were assessed via flow cytometry after FITC-dextran treatment. The cells were then washed twice in cold HBSS and stained with a PE-conjugated anti-CD11c+ antibody. The endocytic activity of the controls was determined after exposure to FITC-dextran at 4 °C. A, The numbers represent the percentages of FITC-dextran-/CD11c+-PE double positive cells. B, The profile of endocytic capacity was also demonstrated as a histogram by mean fluorescence intensity (MFI). Medium designates the chemically untreated control group. The results represent the means (± SD) of three separate experiments, all of which yielded similar results. D-pinitol. As is shown in Fig. 4, the LPS-treated DC exhibited a far more profound rate of proliferation than did the controls, whereas D-pinitol appeared to impair proliferation responses in the allogeneic T cells elicited by the LPS-stimulated DC. Importantly, the maturation induced via LPS stimulation (24 h at 200 ng/ml) profoundly promoted the allostimulatory capacity of the untreated DC, whereas D-pinitol exposure significantly impaired their allostimulatory capacity (Fig. 5B). We also attempted to determine the potency of DC in terms of their ability to adhere to T cells, and their ability to form
clusters. The size of the DC/T-cell clusters decreased with exposure to D-pinitol in contrast to the LPS-treated group. The cluster number of LPS-treated DC in the presence of D-pinitol was markedly inhibited when compared with LPS-stimulated DC in the absence of D-pinitol (Fig. 5A). Considering the inhibitory effects of D-pinitol on the IL-12 (a Th1-inducing cytokine) production in DC, we attempted to characterize the quality of the primary T-cell response in DC that had matured in the presence of D-pinitol. Naive allogenic T cells primed with mature DC were observed to differentiate into Th1
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Fig. 5. D-pinitol treated DC impair cluster formation and proliferation of allogeneic T-cells, and inhibit Th1 responses in vitro. The DC were incubated for 24 h in medium alone, in 80 μM D-pinitol, 200 ng/ml LPS, or in D-pinitol coupled with LPS. The DC were washed and cocultured with T cells. A, Cluster formation was assessed after 72 h. One representative experiment of three is also included in the figure, showing a representative field in a culture well photographed using an inverted phase contrast microscope. B, DC were cultured in medium with or without D-pinitol for 24 h. The treated DC were harvested and thoroughly washed to remove the D-pinitol. A mixed lymphocyte reaction was allowed to proceed for 3 days, as described in the Materials and methods section of this article. T-cell proliferation was analyzed by flow cytometry and presented as a percentage of dividing cells. C, Cells were then examined for cytokine release after 48 h. IFN-γ and IL-4 were measured by ELISA in culture supernatants. Data are expressed as ng/ml/106 cells ± SD of triplicate cultures (⁎⁎, P b.01 vs T-cells primed with LPS-treated DC). Medium represents the chemically untreated control group. Similar results were obtained and expressed as the means (±SD) from four separate experiments.
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immature DC to D-pinitol, prior to LPS stimulation. Pretreatment with 80 μM D-pinitol resulted in a marked inhibition of the LPS-induced upregulation of p-p38, p-ERK1/2, and pJNK. The total ERK1/2 proteins were constitutively expressed (Fig. 6A). Furthermore, other researchers have reported that LPS signal transduction activates a variety of signal pathways, including the NF-κB pathway [18]. These data indicate that Dpinitol inhibits MAPKs expression, which is clearly relevant to the regulation of LPS-induced DC maturation. To characterize the role of NF-κB translocation more precisely, we stimulated immature DC with LPS prior to exposing them to D-pinitol. Then, in order to determine whether D-pinitol affects the blockade of LPS-induced NF-κB translocation, we prepared nuclear extracts from DC that had been treated with both LPS and D-pinitol. Nuclear translocation of the NF-κB p65 subunit was then evaluated via Western blotting. LPS was determined to enhance the nuclear translocation of the NF-κB p65 subunit within 45 min of exposure. Conversely, pretreatment with 80 μM D-pinitol resulted in suppression of the LPS-enhanced NF-κB p65 nuclear translocation (Fig. 6B). 3.6. Intraperitoneal administration of LPS-induced DC maturation
D-pinitol
inhibits
In order to determine whether the apparent inhibitory effects of D-pinitol on splenic DC maturation in vivo was mediated solely by drug toxicity or by interference with DC Fig. 6. D-pinitol suppresses MAPKs and NF-κB activation in LPSstimulated DC. The DC were pretreated with 80 μM D-pinitol for 15, 30 and 45 min prior to LPS stimulation (200 ng/ml). The cell lysates were prepared and blotted with anti-phospho-ERK1/2 (p-p44/42), antiERK1/2 (p44/42), anti-phospho-JNK1/2, anti-JNK1/2, anti-phosphop38 (p-p38), and anti-p38 (p38) Abs (A). LPS-induced NF-κB nuclear translocation was inhibited by D-pinitol (B). The DC were pretreated for 30 min with D-pinitol, and stimulated with 200 ng/ml LPS for the indicated lengths of time. The nuclear extracts were blotted with antip65 Ab. The bound Abs were visualized using biotinylated goat antirabbit IgG. The results shown represent three independent experiments. (−), Represents the chemically untreated control group.
lymphocytes when they generated high levels of INF-γ and low levels of IL-4 (Fig. 5C). By way of contrast, T cells primed with DC that had matured in the presence of D-pinitol inhibited INF-γ production. These results show that the majority of D-pinitol's effects on the T-cell-differentiating properties of DC are a consequence of the inhibition of IL-12 production. 3.5. D-pinitol suppresses LPS-induced activation of MAPKs and nuclear translocation of the NF-κB in DC LPS stimulation has been shown to affect MAPK and NFκB signal pathway activation in DC. MAPK and NF-κB activation are important events in DC maturation [21]. LPS activates p-p38 MAPK, p-ERK1/2, and p-JNK (Fig. 6A). In order to characterize the effects of D-pinitol on the expression of p-p38 MAPK, p-ERK1/2, and p-JNK in DC, we exposed
Fig. 7. In vivo D-pinitol administration suppresses the phenotypic maturation of LPS-challenged splenic DC. Mice were intraperitoneally injected with 10 mg/kg D-pinitol 3 times (1 time/day) for 3 days. One hour after the final injection, the mice were injected in a lateral tail vein with 1 mg/kg LPS. At 24 h, the mice were killed and the splenic DC were generated, as was described in the Materials and methods section of this article. The cells were harvested and we analyzed the expression of CD80, CD86, MHC I, and MHC II via two-color flow cytometry. The cells were firstly gated for CD11c+, after which we analyzed the expression of CD80, CD86 and MHC class II by flow cytometry as histogram with mean fluorescence intensity (MFI) (A) and distribution of positive populations (B). The histogram shown is from one of three representative experiments. All data points represent the average (±SD) of three separate experiments, which were conducted with five mice per group. (⁎⁎, P b 0.01 vs LPS-treated mice).
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Fig. 8. D-pinitol impaired INF-γ production in the CD4+ T-cells of LPS-treated mice. Mice were injected i.p. with 10 mg/kg of D-pinitol 3 times (1 time/day) for 3 days. One hour after the final injection, the mice were injected with 1 mg/kg of LPS in a lateral tail vein (this trial was conducted with five mice per group). The mice were killed at 24 h, and T-cells were generated as was described in the Materials and methods section of this article. The CD4+ T-cells were subsequently analyzed using an intracellular cytokine staining technique. Data are presented as a dotted plot (% of positive cells) and as a histogram mean fluorescence intensity (MFI) (A) and also presented as a histogram with MFI (B). The histogram (B) is from one representative experiment out of 3 (⁎⁎, P b 0.01 vs LPS-treated mice).
production, we assessed the effects of D-pinitol on phenotypic characteristics in LPS-stimulated mice. We isolated spleenderived DC from all experimental groups (5 mice per group), and confirmed the phenotypic characteristics of the mice using flow cytometry. We determined that 95% of the evaluated DC expressed CD11c+ molecules (Fig. 7A, B). Representative FACS
histograms showed that only the LPS-exposed splenic DC had expressed detectable levels of costimulatory and MHC molecules. However, in cells receiving 3 days of D-pinitol pretreatment, CD80, CD86, and MHC class I and II molecules were found to have been markedly downregulated 24 h after LPS challenge. These in vivo results showed that D-pinitol
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challenged with a hapten. By way of contrast, D-pinitol-treated DC, regardless of tested D-pinitol concentration, failed to elicit significant immune responses under identical conditions (Fig. 9). Additionally, the responses of animals that had been sensitized with TNBS-pulsed, D-pinitol-treated DC were shown to be comparable with those evidenced by the unsensitized animals. The results for the control group, which received injections with unpulsed DC (negative control), and for the group sensitized via epicutaneous hapten applications (positive control), confirmed that the observed immune responses were antigen-specific.
4. Discussion Fig. 9. D-pinitol-treated DC impairs cell-mediated immune response. A culture consisting of approximately 106 purified DC in the presence or absence of 80 μM. D-pinitol were pulsed with 0.1% (w/v) TNBS and injected with SC on day 0, as described in the Materials and methods section of this article. In the control groups, DC were either not TNBSpulsed ([−] control) and injected s.c., or the animals were shaved and the skin of their abdomen was painted with 7% w/v TNCB ([+] control). After 7 days, ear thickness was measured in the experimental mice. The results are expressed as the mean ± SD percentage increase in ear swelling for the 5 treatment group animals and the 5 control group animals. Treatment with vehicle alone induced no detectable swelling. (⁎⁎, P b 0.01 vs D-pinitol-treated DC or [−] control).
pretreatment inhibits the phenotypic maturation of LPSexposed DC. 3.7. D-pinitol impairs the production of INF-γ by CD4+ T cells in LPS-treated mice Naive allogenic T cells that had been primed with mature DC were shown to differentiate into Th1 lymphocytes when they produced high levels of INF-γ. In addition, high levels of IFN-γ in CD4+ T cells induce the production of IL-12 in DC. In an effort to characterize the effects of D-pinitol on INF-γ production in the CD4+ T cells of LPS-exposed mice, we isolated spleen-derived CD4+ T cells from all of the experimental mice and determined INF-γ production via flow cytometry. We determined that 95% of the evaluated T cells expressed CD4+ molecules. Representative FACS histograms showed that only the LPS-exposed splenic CD4+ T cells expressed detectable INF-γ levels. However, in cells pretreated for 3 days with D-pinitol, INF-γ production was markedly downregulated 24 h after LPS challenge (Fig. 8A, B). These in vivo data indicated that pretreatment with D-pinitol impaired INF-γ production in LPS-stimulated splenic CD4+ T cells. These results show that the majority of the effects of D-pinitol on the T-cell-differentiating properties of DC occur via INF-γ inhibition. 3.8. D-pinitol-treated DC fail to induce normal cell-mediated immune responses A single s.c. injection of 106 TNBS-pulsed purified DC was demonstrated to induce a significant CHS response, which was visualized 7 days after injection when the animals were
This is, to the best of our knowledge, the first report concerning the effects of D-pinitol on the phenotypic and functional maturation of murine BM-derived DC. This is also the first study in which D-pinitol-exposed DC have been analyzed with regard to their capacity to sensitize recipients for cell-mediated immune responses. Many researchers have previously shown that D-pinitol exerts a variety of biological effects, including anti-diabetic [12] and anti-inflammatory properties [13]. Nevertheless, the exact modulation pathways of D-pinitol remain unclear. Also, the effects of D-pinitol on the maturation and immunological responses of DC remain largely unknown. DC are also considered to play important roles in the establishment of hypersensitivity and transplantation tolerance [22]. We conducted a series of functional assays in order to ascertain the phenotype and function of D-pinitol-treated DC, and also conducted in vivo assessments of their capacity for hypersensitivity. Our findings indicate that the effects of D-pinitol principally involved the suppression of MAPKs and NF-κB activation. To ensure that the observed effects of D-pinitol could be attributed to DC and not to contaminating cells persisting in the BM-derived cell cultures, the DC were purified (N 95%) prior to analysis in each of the conducted assays. Our findings conclusively indicated that D-pinitol is a potent inhibitor of LPS-induced DC maturation. These findings provide new insights into the immunopharmacology of D-pinitol. D-pinitol inhibited DC maturation in a dose-dependent manner. The profound suppressive effects of D-pinitol on DC maturation can be safely attributed to a nonspecific inhibitory effect. Therefore, we also assessed the ability of D-pinitol-treated DC to internalize FITC-dextran via mannose receptor-mediated endocytosis. This mechanism is a defining characteristic of mature DC, as opposed to immature DC [17]. The endocytic capacities of the D-pinitol-treated DC were found to be profoundly elevated, thereby suggesting that D-pinitol inhibited both the phenotypic and functional maturation of DC. We conducted an evaluation of MAPKs and NF-κB
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signal pathways, in order to characterize the mechanism underlying the preventive effects of D-pinitol on LPS-induced DC maturation. The MAPKs and NF-κB signaling pathways appear to be extremely relevant to the process of DC maturation [21]. Within the MAPKs and NF-κB signaling pathways, several molecules, including ERK, JNK, p38 and IκB, have been shown to be crucial. In this study, D-pinitol exerted a detectable suppressive effect on the phenotypic and functional maturation of murine BM-DC, via ERK1/2 and JNK inhibition. This mechanism may also be relevant to the observed protective effects of D-pinitol against certain autoimmune diseases, including arthritis, allergy, and diabetes. We also found that the NF-κB signaling pathway may be closely involved in the DC maturation-inhibitory effects of D-pinitol. Recently collected evidence suggests that DC cytokine production is dependent on either the particular subset of DC, or on the stimuli received by the DC [23]. IL-12, in particular, exerts multiple immunoregulatory functions, inducing the activation of the Th1 subset, which performs a pivotal role in the induction of inflammation. A growing body of evidence suggests that development toward Th1 cells is primarily regulated by DC-derived cytokines, including IL-12 [24] and IFN-γ [25]. The results obtained in this report indicate that D-pinitol caused CD11c+ DC to generate IL-12 in the presence of LPS, and also confirm that D-pinitol exerts inhibitory effects on the expression of intracellular IL-12 p40/p70 and IL-12 p70. Additionally, this study demonstrates that LPS-stimulated CD11c+ DC skews naive T cells toward development into IFN-γ-producing T cells. D-pinitol was determined to significantly impair the capacity of these cells to proliferate and initiate Th1 responses, under both in vitro and in vivo conditions. Naive T cells stimulated with D-pinitol-treated DC generated lower IFN-γ levels, but did not exhibit significantly altered abilities to generate IL-4. These results indicated that D-pinitol is a potent DC maturation inhibitor. Because Th1 cells exert functionally immunogenic or protective effects against invading pathogens, the inhibition of DC-mediated Th1 polarization might constitute a D-pinitol-associated immunosuppressive mechanism. However, the inhibition of Th1 development exerts negative regulatory roles on a wide variety of immune cells [24]. Consequently, D-pinitol-mediated inhibition of IL-12 generation in LPS-stimulated CD11c+ DC may also contribute to the induction of an immunosuppressive state. Based on observations in our in vitro and in vivo experiments, we hypothesized that D-pinitol-treated DC
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should exhibit deficiencies in their ability to stimulate naive T cells in vivo, in addition, they should initiate cellmediated immune responses. It was demonstrated recently that as few as 105 TNBS pulsed murine BM-derived DC (TNBS-DC) induced a profound CHS response [26]. The ability of the DC to induce T-cell-mediated immune responses was evaluated using TNBS-DC to sensitize for CHS in naive syngeneic recipients. CHS sensitization was verified in recipients of s.c. injections containing 105 TNBS-DC, but was not observed in recipients of D-pinitol-pretreated TNBS-DC. This shows that the decreased T-cell-stimulatory capacity of the D-pinitoltreated, BM-derived DC cannot be readily reversed after the removal of D-pinitol is sustained in vivo [27]. In conclusion, we have characterized a variety of effects exerted by D-pinitol on BM-derived DC. D-pinitol was demonstrated to inhibit phenotypic maturation and modulate cytokine production in these DC, resulting in a significant inhibition of Th1 development. Moreover, our experimental data showed that D-pinitol suppressed LPSinduced costimulatory molecules/MHC class types and IFN-γ, under in vivo experimental conditions. These findings illustrate the immunopharmarcological functions of D-pinitol. Moreover, exposure to this readily available drug may constitute a nontoxic and highly effective means for the modulation of DC immunoregulatory capacity. Acknowledgement This work was supported by the Korea Science and Engineering Foundation through National Research Laboratory Program Grant M1050000000806J00 0000810. References [1] Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991;9:271–96. [2] Rock KL, Clark K. Analysis of the role of MHC class II presentation in the stimulation of cytotoxic T lymphocytes by antigens targeted into the exogenous antigen-MHC class I presentation pathway. J Immunol 1996;156(10):3721–6. [3] Austyn JM. Dendritic cells. Curr Opin Hematol 1998;5(1):3–15. [4] Cella MR, Del Prete A, Vecchi A, Corada M, Martin-Padura I, Motoike T, et al. Increased DC trafficking to lymph nodes and contact hypersensitivity in junctional adhesion molecule-Adeficient mice. J Clin Invest 2004;114(5):729–38. [5] Salio M, Cerundolo V, Lanzavecchia A. Dendritic cell maturation is induced by mycoplasma infection but not by necrotic cells. Eur J Immunol 2000;30(2):705–8. [6] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392(6673):245–52. [7] Chen JW, Zhu ZQ, Hu TX, Zhu DY. Structure-activity relationship of natural flavonoids in hydroxyl radical-scavenging effects. Acta Pharmacol Sin 2002;23(7):667–72.
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[8] Kim GY, Kim KH, Lee SH, Yoon MS, Lee HJ, Moon DO, et al. Curcumin inhibits immunostimulatory function of dendritic cells: MAPKs and translocation of NF-kappa B as potential targets. J Immunol 2005;174(12):8116–24. [9] Kim SH, Park HJ, Lee CM, Choi IW, Moon DO, Roh HJ, et al. Epigallocatechin-3-gallate protects toluene diisocyanate-induced airway inflammation in a murine model of asthma. FEBS Lett 2006;580(7):1883–90. [10] Lee JS, Kim SG, Kim HK, Lee TH, Jeong YI, Lee CM, et al. Silibinin polarizes Th1/Th2 immune responses through the inhibition of immunostimulatory function of dendritic cells. J Cell Physiol 2006;210(2):385–97. [11] Kim GY, Lee MY, Lee HJ, Moon DO, Lee CM, Jin CY, et al. Effect of water-soluble proteoglycan isolated from Agaricus blazei on the maturation of murine bone marrow-derived dendritic cells. Int Immunopharmacol 2005;5(10):1523–32. [12] Davis A, Christiansen M, Horowitz JF, Klein S, Hellerstein MK, Ostlund Jr RE. Effect of pinitol treatment on insulin action in subjects with insulin resistance. Diabetes Care 2000;23(7): 1000–5. [13] Singh RK, Pandey BL, Tripathi M, Pandey VB. Anti-inflammatory effect of (+)-pinitol. Fitoterapia 2001;72(2): 168–70. [14] Kim JI, Kim JC, Kang MJ, Lee MS, Kim JJ, Cha IJ. Effects of pinitol isolated from soybeans on glycaemic control and cardiovascular risk factors in Korean patients with type II diabetes mellitus: a randomized controlled study. Eur J Clin Nutr 2005;59(3):456–8. [15] Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/ macrophage colony-stimulating factor. J Exp Med 1992;176 (6): 1693–702. [16] Lutz MB, Assmann CU, Girolomoni G, Ricciardi-Castagnoli P. Different cytokines regulate antigen uptake and presentation of a precursor dendritic cell line. Eur J Immunol 1996;26(3):586–94. [17] Sallusto F, Cella M, Danieli C, Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J Exp Med 1995;182(2):389–400.
[18] Rescigno M, Martino M, Sutherland CL, Gold MR, RicciardiCastagnoli P. Dendritic cell survival and maturation are regulated by different signaling pathways. J Exp Med 1998;188(11): 2175–80. [19] Lyons AB. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. J Immunol Methods 2000;243(1–2):147–54. [20] Mosca PJ, Hobeika AC, Clay TM, Nair SK, Thomas EK, Morse MA, et al. A subset of human monocyte-derived dendritic cells expresses high levels of interleukin-12 in response to combined CD40 ligand and interferon-gamma treatment. Blood 2000;96(10):3499–504. [21] An H, Yu Y, Zhang M, Xu H, Qi R, Yan X, et al. Involvement of ERK, p38 and NF-kappaB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells. Immunology 2002;106(1):38–45. [22] Shlomchik WD, Couzens MS, Tang CB, McNiff J, Robert ME, Liu J, et al. Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 1999;285(5426):412–5. [23] Brinkmann V, Geiger T, Alkan S, Heusser CH. Interferon alpha increases the frequency of interferon gamma-producing human CD4+ T cells. J Exp Med 1993;178(5):1655–63. [24] Kadowaki N, Antonenko S, Ho S, Rissoan MC, Soumelis V, Porcelli SA, et al. Distinct cytokine profiles of neonatal natural killer T cells after expansion with subsets of dendritic cells. J Exp Med 2001;193(10):1221–6. [25] Rissoan MC, Soumelis V, Kadowaki N, Grouard G, Briere F, de Waal Malefyt R, et al. Reciprocal control of T helper cell and dendritic cell differentiation. Science 1999;283(5405):1183–6. [26] Lappin MB, Weiss JM, Delattre V, Mai B, Dittmar H, Maier C, et al. Analysis of mouse dendritic cell migration in vivo upon subcutaneous and intravenous injection. Immunology 1999;98(2): 181–8. [27] Yoon MS, Lee JS, Choi BM, Jeong YI, Lee CM, Park JH, et al. Apigenin inhibits immunostimulatory function of dendritic cells: implication of immunotherapeutic adjuvant. Mol Pharmacol 2006;70(3):1033–44.