Lymphocyte–stromal cell interaction induces IL-7 expression by interferon regulatory factors

https://doi.org/10.1016/j.molimm.2013.01.002Get rights and content

Abstract

The interaction between lymphocytes and stromal cells plays important roles in coordinated development of early lymphocytes. IL-7 is an essential cytokine for early lymphocyte development produced by stromal cells in the thymus and bone marrow. Although IL-7 is induced by interaction of early lymphocytes and stromal cells, its molecular basis is still unknown. To address this question, we employed co-culture system with an IL-7-dependent pre-B cell line, DW34, and a thymic stromal cell line, TSt-4. Co-culture with DW34 cells enhanced the levels of IL-7 transcripts in TSt-4 cells. Interestingly, the co-culture also induced transcripts of IFN-α and IFN-β but not of IFN-γ. In addition, exogenous IFN-β stimulation increased the levels of IL-7 transcripts in TSt-4 cells. Next, to elucidate the molecular mechanism of IL-7 induction, we analyzed the IL-7 promoter activity by reporter assay. The IL-7 promoter showed specific transcriptional activity in TSt-4 cells. An interferon-stimulated response element (ISRE) in the IL-7 promoter was essential for the induction of IL-7 transcription by both co-culture and IFN-β stimulation. Finally, overexpression of wild-type and dominant-negative forms of interferon regulatory factors (IRFs) activated and repressed, respectively, the IL-7 promoter in TSt-4 cells. Collectively, these results suggested that IRFs activated by lymphocyte adhesion induce IL-7 transcription through ISRE in stromal cells and that type I IFNs may be involved in the activation of IRFs. Thus, this study implied a physiological function of the IFN/IRF signal during lymphocyte development.

Highlights

► IFN-α/β and interferon regulatory factors (IRFs) are induced in stromal cells by lymphocyte adhesion. ► IRFs activate IL-7 transcription through interferon-stimulated response element in the IL-7 promoter. ► Type I IFNs may be involved in the IL-7 induction by lymphocyte–stromal cell interaction.

Introduction

The cell interaction between lymphocytes and stromal cells plays a critical role in development and maintenance of the immune system. Bone marrow contains several types of mesenchymal stromal cells, which support the expansion of early B cells (Whitlock et al., 1985) and the homing and survival of memory CD4 T cells, plasma cells, and plasmacytoid dendritic cells (Kohara et al., 2007, Tokoyoda et al., 2004, Tokoyoda et al., 2009). Bone marrow stromal cells produce growth factors, cytokines, and chemokines such as SCF, IL-7, and CXCL12 (Tokoyoda et al., 2010). Early B cells adhere to stromal cells by interaction between two adhesion molecules, VLA-4 and VCAM-1, and receive growth and survival signals of these factors. Remarkably, the cell interaction stimulates the stromal cells to produce higher levels of IL-7 (Sudo et al., 1989). On the other hand, thymic microenvironment is mainly composed of epithelial and mesenchymal cells, both of which express SCF and IL-7 for T cell proliferation and survival (Rodewald et al., 1995, von Freeden-Jeffry et al., 1995). Thymic epithelial cells also produce a Notch ligand, Dll-4, for T cell lineage commitment (Hozumi et al., 2008, Koch et al., 2008). Moreover, they express MHC class I and class II molecules for positive and negative selection (Starr et al., 2003) and autoimmune regulator for self-tolerance (Anderson et al., 2002). In contrast, mesenchymal stromal cells in the thymus are important for differentiation and proliferation of epithelial cells in thymic organogenesis (Itoi et al., 2007). Interestingly, CD4+CD8 or CD4CD8+ single positive thymocytes are responsible for the maintenance of medullary thymic epithelial cells (Akiyama et al., 2008, Hikosaka et al., 2008). Therefore, not only lymphocytes depend on intact microenvironments for development, but microenvironments themselves depend on lymphoid cells to differentiate and maintain their integrity.

IL-7 is an essential cytokine for development and maintenance of early and mature lymphocytes, produced by mesenchymal stromal cells and epithelial cells in bone marrow, thymus, lymph nodes, skin, and intestines. During early development, IL-7 transmits two kinds of signal in lymphocyte progenitors of bone marrow and thymus. One is for cell survival and proliferation, and the other is for V(D)J recombination in the IgH and TCRγ loci (Corcoran et al., 1998, Maki et al., 1996). In addition, IL-7 stimulates differentiation of post-selected CD8 T cells in the thymus by inducing Runx3 transcription factor (Park et al., 2010). In periphery, IL-7 is produced by fibroblastic reticular cells in T cell zones of lymph nodes and plays an important role in survival and homeostatic proliferation of naive and memory T cells (Link et al., 2007). Furthermore, IL-7 is induced in intestinal epithelial cells by IFN-γ and deteriorates inflammatory bowel diseases (Oshima et al., 2004, Totsuka et al., 2007).

The mechanism underlying IL-7 expression is only partially understood. It was reported that the transcription of IL-7 gene starts from two distinct regions: one from 480 to 640 bp upstream and the other from 200 to 220 bp upstream of the coding sequence (Ariizumi et al., 1995, Lupton et al., 1990). IL-7 transcripts were induced in bone marrow stromal cells by co-culture with pre-B cells as well as by stimulation with IL-1 (Sudo et al., 1989). Furthermore, IFN-γ treatment induces IL-7 transcription in keratinocytes and intestinal epithelial cells (Ariizumi et al., 1995, Oshima et al., 2004). The IL-7 promoter contains an interferon-stimulated response element (ISRE) at 270 bp upstream of the coding sequence. The ISRE is important for the transcription mediated by IFN-γ, and interferon regulatory factor (IRF) 1 and IRF2 activate the IL-7 promoter via the ISRE (Ariizumi et al., 1995, Oshima et al., 2004). Therefore, IFN-γ and IRFs play a crucial role in IL-7 induction during inflammation. However, the molecular basis of IL-7 induction by lymphocyte–stromal cell interaction is still unknown.

To define molecular mechanisms enhancing IL-7 transcription by cell interactions, we employed a co-culture system with mesenchymal stromal cells and pre-B cells. The co-culture induced expression of IFN-α and IFN-β in the stromal cells. The IFNs then activated IRFs, which transactivated the IL-7 promoter via the ISRE. Thus, this study suggests that direct cell interaction between lymphocytes and stromal cells induces IL-7 transcription through type I IFNs and IRFs, implying a physiological function of the IFN/IRF signal in lymphocyte development.

Section snippets

Cells

A thymic mesenchymal stromal cell line, TSt-4 (Watanabe et al., 1992), is maintained in RPMI-1640 medium containing 5% FBS, 50 μM 2-mercaptoethanol, 2 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. A stromal cell-dependent pre-B cell line, DW34 (Nishikawa et al., 1988), is cultured on TSt-4 cell layer with the same medium. For co-culture, DW34 cells (4 × 106) were seeded on TSt-4 cell layer in T75 flask. After 5 days, DW34 cells were separated from TSt-4 cell layer by gentle

IL-7 is induced by co-culture

The induction of IL-7 was previously reported in a co-culture system with a bone marrow-derived stromal cell line, ST2, and a pre-B cell line, DW34 (Sudo et al., 1989). To investigate the molecular mechanism of IL-7 induction from stromal cells, we first checked the combination of stromal cells and lymphoid cells. We tested ST2 and TSt-4 (a thymic mesenchymal stromal line) as stromal cells, and DW34, preBRI (an IL-7-dependent pre-B cell line), and Jurkat lines as lymphoid cells. We found that

Discussion

In this study, we employed the co-culture system with the thymic stromal cell line, TSt-4, and the pre-B cell line, DW34, to analyze the molecular mechanism of IL-7 production from stromal cells. We first showed that the co-culture with DW34 cells induces IL-7 transcription in TSt-4 cells (Fig. 1, Fig. 2). Direct cell interaction is crucial for the induction. We also demonstrated that the ISRE in the IL-7 promoter is essential for basal and inducible transcription (Fig. 3). In addition, the

Acknowledgments

We thank Dr. Tetsuo Sudo for recombinant mouse IFN-β and human IL-6; Mses. S. Kamioka and S. Hayashi for excellent technical assistance; and members of the Ikuta lab for discussion. This study was supported by Grants-In-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References (29)

  • A.E. Corcoran et al.

    Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor

    Nature

    (1998)
  • K. Hozumi et al.

    Delta-like 4 is indispensable in thymic environment specific for T cell development

    Journal of Experimental Medicine

    (2008)
  • M. Itoi et al.

    Mesenchymal cells are required for functional development of thymic epithelial cells

    International Immunology

    (2007)
  • U. Koch et al.

    Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment

    Journal of Experimental Medicine

    (2008)
  • 1

    Present address: Department of Immunology and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.

    2

    Present address: Division of Molecular Immunology, Medical Mycology Research Center, Chiba University, Chiba 260-8673, Japan.

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