Role of mitogen-activated protein kinase and PI3K pathways in the regulation of IL-12-family cytokines in dendritic cells and the generation of T H-responses.

Mitogen-activated protein kinases (MAPK) are targets for the immune-modulation of dendritic cells (DC). However, our knowledge of their role in the regulation of IL-12-family cytokines is limited. This study investigated the roles of p38, JNK, p44/42 and PI3K pathways in IL-12/23/27 production by human DC, and their impact on naïve T(H)-responses. We first identified TOP and UBC as robust DC housekeeping genes. Peak transcription of p35 and p40 occurred by 12h, p19 and p28 by 8h and EBI3 by 12-24h. Using selective antagonists, we showed that p38 was a positive regulator of IL-12, 23 and 27, JNK positively regulated IL-12 and IL-27, and inhibition of MEK1/2 had no marked effect. In contrast, the PI3K pathway markedly attenuated IL-23 responses and, to a lesser extent, IL-12, but not IL-27. To identify the role of these soluble factors, we co-stimulated naïve CD4+ T-cells in the presence of DC supernatant. The presence of mature DC supernatant induced not only strong IFNγ responses, but also IL-10 and IL-17A. Inhibition of p38 ablated T(H1), and IL-10 and IL-17A responses, whilst modestly enhancing IL-5 secretion. In contrast, inhibition of MEK1/2 abolished IL-17A production, whilst leaving other responses unaffected, whereas inhibition of JNK or PI3K had no discernable effect. In summary, we describe the expression of IL-12-family cytokines from DC and propose a modified model for their regulation. This study further clarifies the potential for therapeutic modulation through these mediators.

IL-17 expression by breast-cancer-associated macrophages: IL-17 promotes invasiveness of breast cancer cell lines.

INTRODUCTION: IL-17 plays an important role in autoimmunity, promoting autoimmunity, inflammation and invasion in multiple sclerosis, rheumatoid arthritis and type I diabetes. The role of IL-17 in cancer is unclear, however, as there are few studies examining IL-17 protein expression in cancer. We therefore examined IL-17 protein expression in human breast cancer and modelled its potential biological significance in vitro. METHODS: Immunohistochemistry was used to determine IL-17 expression in breast cancers. Matrigel invasion assays were employed to examine the effect of IL-17 on cancer cell invasion by a panel of breast cancer cell lines. The role of matrix metalloproteinases (MMPs) was investigated with selective antagonists and immunoassays for MMP-2, MMP-3, MMP-9 and tissue inhibitor of MMP. RESULTS: IL-17-expressing cells with macrophage morphology were identified in the peritumoural area of a proportion of patients (8/19 patients). Macrophages were confirmed by CD68 staining on serial sections. With the exception of occasional lymphocytes, one patient with rare multinucleate giant cells and one patient with occasional expression of IL-17 in tumour cells, no other IL-17-positive cells were detected. Addition of IL-17 to cell lines in vitro stimulated marked invasion of Matrigel. In contrast, IL-17 did not promote the invasion of MCF7 or T47D cell lines. Invasion was initially thought to be dependent on MMPs, as evidenced by the broad-spectrum MMP inhibitor GM6001 and selective antagonists of MMP-2/MMP-9 and MMP-3. Measurement of MMP-2, MMP-3 and MMP-9, and tissue inhibitor of MMP 1 secretion, failed to reveal any changes in expression following IL-17 exposure. In contrast, TNF promoted secretion of MMPs but IL-17 did not augment TNF, indicating that IL-17 acts via an independent mechanism. CONCLUSIONS: The present study is the first to describe in situ expression of IL-17 protein in human breast tumours and to propose a direct association between IL-17 and breast cancer invasion. The precise effectors of IL-17-dependent invasion remain to be characterised but could include a range of proteases such as a disintegrin and metalloproteinase protein or astacins. Nevertheless, this work identifies a novel potential mechanism for breast cancer invasion and tumour progression, the prognostic implication of which is currently under investigation.