Regulation of granulocyte colony-stimulating factor gene expression by interleukin-17

Abstract

Interleukin-17 (IL-17) has been previously reported to induce stromal cells to produce a number of hematopoietic and proinflammatory cytokines, including granulocyte colony-stimulating factor (G-CSF). Here, we have evaluated the mechanisms responsible for the augmentation of G-CSF gene expression by IL-17, using the murine 3T3 fibroblast cell line. Treatment of 3T3 cells, but not primary bone marrow-derived macrophages or murine monocyte/macrophage cell lines, resulted in increased steady-state G-CSF mRNA levels within 2–4 h and augmented G-CSF protein production. The combination of IL-17 and LPS enhanced G-CSF expression in an additive fashion. Stability studies revealed that IL-17 stabilized G-CSF mRNA levels, with a t_1/2 of 4 h, compared to a t_1/2 of less than 2 h in medium or LPS-treated cells. Induction of G-CSF expression in 3T3 cells by IL-17 did not appear to require tyrosine kinase activation or de novo protein synthesis. These studies indicate that post-transcriptional mechanisms play an important role in IL-17-induced G-CSF expression in fibroblasts and suggest that IL-17 may be useful for further delineating mechanisms of G-CSF gene regulation.

Introduction

Murine IL-17 was originally cloned by Rouvier et al. by subtractive hybridization from an activated T cell library and named cytotoxic T lymphocyte associated antigen-8 (CTLA-8) [1]. The murine CTLA-8 gene showed 57% homology with the predicted amino acid sequence of open reading frame 13 of Herpesvirus saimiri (HSV13) 1, 2. The predicted amino acid sequence of both CTLA-8 and HSV13 indicated these molecules contained signal sequences and potential N-linked glycosylation sites [3]. Moreover, the 3′ untranslated region of CTLA-8 contained nucleotide sequences which appear to be associated with mRNA instability, like those seen in many cytokines, growth factors and proto-oncogenes 4, 5. Both human and murine IL-17, as well as the protein product encoded by HSV13, are produced predominantly by activated T cells and have been shown to stimulate IL-6, IL-8, G-CSF and prostaglandin E2 production from epithelial, fibroblast and endothelial cells, enhance ICAM-1 expression and stimulate T cell proliferation 6, 7. In addition, co-culture of CD34⁺ hematopoietic progenitors and primary synovial fibroblasts with human IL-17 resulted in prolonged proliferation of the progenitor cells and their selective maturation toward the neutrophil lineage, suggesting that IL-17, either directly or indirectly via the induction of specific growth factors, may be able to promote granulopoiesis [7]. An IL-17 receptor (IL-17R) which is ubiquitously expressed has been described, and a soluble IL-17R-fusion protein has been shown to inhibit T cell proliferation and IL-2 production [8]. At least a portion of the biological activity of IL-17 may be mediated by the activation of the NFκB transcription factor [8].

That IL-17 significantly induces G-CSF production and causes preferential differentiation of progenitor cells to neutrophils suggests that IL-17 or an analog [9]might be useful for exploring mechanisms of G-CSF gene expression, and may be beneficial for restoring peripheral neutrophil numbers and/or activity in myeloablated patients, who are at risk for infection [10]. Moreover, IL-17 may be useful as an anti-infective agent in these individuals. Because of the central role played by G-CSF in these processes, we have further examined the ability of murine IL-17 to regulate G-CSF gene expression in the mouse 3T3 fibroblast line. Our results indicate that post-transcriptional mechanisms, possibly involving a critical RNase activity, are important for IL-17-induced G-CSF expression in these cells.