IL is not expressed in tissues constitutively
IL-8 is not expressed in tissues constitutively because of its strong chemoattractant, proinflammatory, and angiogenic activities. Rather, it is an inducible factor, produced by a variety of cell types, including leukocytes, fibroblasts, endothelial and epithelial cells, in response to proinflammatory cytokines (such as IL-1 and TNF-α), or in the case of microbial infection . There is an emerging literature, however, which suggests that IL-8 expression may play an essential role in disease states, particularly tumor development and metastasis. For example, the expression of IL-8 by melanoma EHop-016 australia has been shown to regulate growth and metastasis in nude mice , as well as being a paracrine factor for melanoma cell chemotaxis . Furthermore, substantial evidence exists that IL-8 is a critical angiogenic factor in a variety of human cancers .
In the colonic mucosa, upregulation of IL-8 occurs in proportion to the degree of inflammation in Crohn\'s disease and ulcerative colitis (UC) , , . Moreover, CXCR-1 (but not CXCR-2) receptor expression is strongly upregulated in the mucosal epithelium of UC . IL-8 expression also correlates with tumor cell growth and vascularity in gastric carcinoma , . With regard to colon cancer, constitutive expression of IL-8 has been linked to metastatic potential in human colon carcinoma cell lines , and has been suggested to play a role in the development of distant metastases from colorectal tumors .
We characterized a novel model of EMT in colon carcinoma, in which the inflammatory cytokine TNF-α augmented this TGF-β-directed process . In the current study, we investigated the signaling pathways that are specifically stimulated by TNF-α during the EMT, and we identify NF-κB activation as a critical component of this process. Furthermore, we defined transcriptional targets of the TNF-α or NF-κB pathway, and found that increased expression of IL-8 is characteristic of the post-EMT phenotype. During this EMT, a concomitant increase in CXCR-1, but not CXCR-2, also occurred. From a functional perspective, we demonstrate that the IL-8–CXCR-1 interaction induces chemotaxis in invasive colon carcinoma cells, thereby linking this signaling pathway to a more invasive tumor cell phenotype.
Material and methods
Discussion The mechanism of IL-8 induction because of the EMT is of interest because it bears on the mechanisms that underlie the EMT itself and it likely serves as a paradigm for the regulation of other proteins associated with the EMT. In response to EMT induction by stimulation of organoids with both TNF-α and TGF-β, we observed NF-κB activation and the NF-κB-dependent expression of IL-8. These findings per se are not unexpected because TNF-α is known to activate NF-κB, and IL-8 transcription is dependent on the cooperative action of the two transcription factors, NF-κB and AP-1 . The novelty of our results concerning mechanism lies in the finding that that the induction of the EMT by TGF-β stimulation alone, a process that requires much longer times (5–7 days) than does combined stimulation with TGF-β and TNF-α (24 h), also resulted in induction of IL-8 expression. The ability of TGF-β to trigger an EMT has been shown in numerous cell types, and presumably, it relates to stromal influences on the EMT in vivo. Thus, IL-8 expression is not simply a consequence of stimulation with the pro-inflammatory cytokine TNF-α, but it appears to be intrinsic to the EMT itself. An intriguing issue that emerges from these findings is the mechanism by which TGF-β stimulates IL-8 expression. In this regard, it is interesting to note that there are reports of cooperativity and cross-talk interactions between NF-κB and the Smad proteins, which are the intracellular signaling molecules involved in mediating the cellular effects of TGF-β , . The central role of TNF-α in inflammation and the immune system is established (reviewed in Ref. ) where it regulates such cellular processes as cytokine induction, proliferation, differentiation, and apoptosis. In contrast to these roles, however, several studies have suggested a function for TNF-α in tumor progression that is consistent with our findings. First and foremost is the widespread observation that macrophages, which are a rich source of TNF-α, are abundant in most solid tumors, and the emerging consensus is that such macrophages promote tumor growth and spread. This hypothesis is substantiated by our recent work demonstrating that activated macrophages, and more specifically, their production of TNF-α, facilitate the EMT . Moreover, our finding that TNF-α stimulates IL-8 expression in the context of the EMT provides one mechanism for its contribution to tumor progression. These in vitro studies are substantiated by clinical observations. For example, TNF-α mRNA transcripts are more abundant in colorectal tumor cells than in their normal epithelial counterparts, and that there is a positive correlation between TNF-α expression and Dukes\' stages . A similar finding for TNF-α expression and tumor grade was previously reported for serous ovarian tumors , and it has been postulated that autocrine production of TNF-α by ovarian tumor cells in situ was the result of paracrine stimulation by infiltrating monocytes . Finally, for lung cancer, it has been reported that a high density of tumor-associated macrophages is associated with angiogenesis and poor prognosis , and it has been proposed that macrophage-dependent upregulation of IL-8 expression is responsible for these adverse outcomes . Clearly, these results indicate that secretion of IL-8 by tumor cells can promote progression through both autocrine and paracrine signaling.