NDRG2 is one of novel intrinsic factors for regulation of IL-10 production in human myeloid cell

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Abstract

N-myc downstream-regulated gene 2 (NDRG2) implicated in cellular growth and differentiation was previously reported as it is specifically expressed in primary and in vitro-differentiated dendritic cells (DCs) from monocytes and CD34+ progenitor cells. However, its function has yet to be investigated in DCs. Here, the novel NDRG2 function about modulation of cytokines in DC was observed in this study. The secretion of IL-10 was not found in the monocyte-derived DC cells with high level of NDRG2 expression, but IL-10 was abundantly secreted up to 1 ng/ml in the monocyte-derived macrophages with low level of NDRG2 expression, and further confirmed that the expression of IL-10 was dramatically increased in NDRG2-silenced DCs under presence of LPS, and significantly reduced in the NDRG2-overexpressed U937 cells under stimulation of PMA. The secretion of IL-12p70 was significantly reduced in the siNDRG2 introduced DC cells. The intracellular signaling of IL-10 secretion was markedly inhibited by SB203580, inhibitor of p38 MAPK, in the LPS-activated DCs and phosphorylation of p38 MAPK was decreased in the NDRG2 introduced U937 cells under PMA-stimulation. Taken together, NDRG2 might have a pivotal role as one of intrinsic factors for the modulation of p38 MAPK phosphorylation, and subsequently involve in controlling of IL-10 production.

Introduction

N-myc downstream-regulated gene 2 (NDRG2), a member of a new family of differentiation-related genes, is highly expressed in adult brain, salivary gland, and skeletal muscle [1], [2]. NDRG2 has been implicated in cell growth [3], [4], differentiation [4], and apoptosis [5] and is rapidly responsive to mineral corticoid-stimulation in the kidney [2]. Recently, it has been shown that NDRG2 contains several phosphorylation sites, and its phosphorylation is mediated through insulin-stimulated AKT activation [6], [7]. It was also reported that dendritic cells (DCs) express NDRG2 during the differentiation from monocytes or CD34+ cord blood cells [4].

DCs are the most potent antigen-presenting cells (APCs) whose primary function is to capture, process, and present antigens for initiation of the immune response [8], [9], [10], [11]. Immature DCs reside in almost all tissues where they can capture and process antigens. Thereafter, DCs migrate to lymphoid organs where they undergo a complex maturation process and become specialized in antigen presentation. Fully mature DCs express high levels of MHC and co-stimulatory molecules and they also produce high levels of cytokines such as IL-6, IL-8, IL-10, and IL-12 [10], [11]. IL-10 among the cytokines is a representative immunosuppressive cytokine suppressing both macrophage and DCs function, including antigen-presenting function and the production of pro-inflammatory cytokines. IL-10 is produced by a variety of mammalian cells including T cells, B cells, eosinophils, mast cells, and monocytes/macrophages [12], [13], [14]. The dominant function of IL-10 is reinforced by IL-10 knockout mice models. The mice develop spontaneous inflammatory colitis in the presence of normal flora and clear intracellular pathogens more effectively with immunopathologic injury which often induces death of the host [13], [15], [16], [17], [18]. In intestinal immune system of mouse, IL-10-producing macrophages of lamina propria contribute to the balance between immune activation and tolerance through dynamic interaction with CD11b+ DCs [19]. Although the functions of IL-10 in immune system were known well, the regulatory machinery for IL-10 production remains undefined.

In this report, we confirmed that macrophages produced IL-10 unlike immature DCs and revealed that down-regulation of IL-10 in DCs was controlled by the expression of NDRG2, which was expressed during differentiation of DCs. Intracellular function of NDRG2 showed the inhibition of p38 MAPK activation related with IL-10 production in the NDRG2-overexpressed U937 cell. These results suppose that NDRG2 regulates IL-10 production in myeloid cell through down-regulation of p38 MAPK.

Section snippets

Reagents for cell culture, cytokines, and antibodies

All cultures were performed in RPMI 1640 medium (Sigma, St. Louis, MO, USA) supplemented with l-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), HEPES (10 mM), and 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA). The growth factors used in the primary cultures of DC precursors were rhGM-CSF (kindly provided by LG LS, Iksan, South Korea) and rh-IL-4 (BD Biosciences, San Jose, CA, USA). Phorbol 12-myristate 13-acetate (PMA) and kinase inhibitors, PD98059 and SB203580

Macrophages do not express NDRG2 but produce IL-10, whereas immature DCs express NDRG2 but do not produce IL-10

Differentiation of monocytes to either DCs or macrophages depends on the culture conditions. Monocytes cultured with M-CSF for 6 days were firmly attached to the plate, but monocytes cultured with GM-CSF/IL-4 showed characteristics of dendritic morphology displaying ruffled membranes. The immunophenotypes of macrophages or DCs were determined by surface markers that characterized as CD1aCD14+CD80dimHLA-DR+ or CD1a+ CD14CD80brightHLA-DR+, respectively (Fig. 1A). Northern and Western blot

Discussion

In this paper we demonstrate for the first time a NDRG2 function in DCs as cytokine regulator reducing of IL-10. NDRG2-silenced DCs by transfection with NDRG2-siRNA produced a significantly increased IL-10. To test whether NDRG2 can affect IL-10 production directly, when NDRG2-negative U937 cells were transfected with NDRG2 and then stimulated with PMA, IL-10 production from NDRG2-U937 cells also decreased. When NDRG2-U937 cells were stimulated with PMA, phosphorylation of p38 MAPK was

Authorship

J.W.K. and J.-S.L. have overall contributed to the study’s conception and design. S.-C.C. and K.D.K. have mainly contributed to design, experimental performance, and the preparation of manuscript. J.-T.K. and S.Y.Y. were contributed to some experimental performance or the preparation of manuscript. Other authors (S.-S.O., E.Y.S., H.G.L., Y.-K.C., I.C.) were supported to us the experimental materials and devices.

Acknowledgments

This work was supported by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A090509) (to J.W.K.), grant from the Research center for women’s Diseases of the KOSEFR11-2005-017-03007 (to J.S.L.), and the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2008-331-E00090) (to K.D.K.).

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