Paracellular permeability regulation by cytokines

Background: Paracellular barrier permeability is controlled by the junctional complexes. These multiprotein complexes located at the cell to cell contact sites have been recognized as highly dynamic structures responsive to intracellular signaling. Many pharmaceuticals and toxins exhibit the adverse effect of increasing paracellular permeability. In addition, paracellular permeability is subject to dysregulation in various disease states. In many instances, this is mediated by pro-inflammatory and inflammatory cytokines. The role and mode of action of the cytokine induced intracellular signaling pathways in the control of the paracellular permeability have yet to be elucidated. Aims: We propose to perform investigations on these signaling mechanisms. We previously discovered that the "mitogen activated protein kinase (MAPK)" intracellular signaling module is utilized to transduce the interferon alpha (IFNalpha) initiated signaling to the junctional complexes, resulting in increased paracellular permeability. Based on this finding we propose to address three major questions important for the further elucidation of paracellular permeability regulation: How can such a universal signaling pathway like the MAPK pathway provide specific cellular responses: i.e. how does the cell discriminate between MAPK induced by IFNalpha that results in enhanced paracellular permeability, and MAPK activated by a growth factor, that induces cell division? How is the MAPK signal transduced to the junctional complexes? Is the IFNalpha induced MAPK dependent change in junctional permeability a specific mechanism for IFNalpha or is this mechanism operative for regulatory responses in epithelia in general? Methodology: Our model systems will be cell cultures of renal and intestinal origin (LLC-PK1, HK-2 and Caco2) stimulated with cytokines (IFNalpha IFNgamma and TNFalpha). These in vitro systems shall enable us to perform analyses of the intracellular signaling processes using microscopic methods (immunofluorescence combined with deconvolution analysis and confocal microscopy, fluorescence resonance energy transfer, immuno electronmicroscopy), subcellular fractionation, immunoprecipitation, Western blotting and in vitro kinase assays. Implications: An improved understanding of cytokine induced paracellular permeability regulation has major implications in the treatment of many chronic inflammatory diseases and tumor extravasation and metastasis.

Type I interferons, like IFNalpha, are major regulators of the immune system. Release of cytokines by activated immune cells affects not only the immune response, but also influences normal tissue functions. We have found that IFNalpha induces impairment of renal epithelial barrier function in vitro. In addition, IFNalpha treatment induced apoptosis in proximal tubular cells (Lechner et al., Am J Physiol Cell Physiol 294, C153-60, 2008). Barrier destabilization by IFNalpha was found to be independent of apoptotic cell death. A mitogen-activated protein kinase pathway involving ERK1/2 was necessary to mediate the IFNalpha-induced changes in epithelial barrier function. EGF, a growth factor implicated in renal repair processes, was also shown to affect barrier function, however in the opposite way, causing increased barrier tightness. Therefore, we questioned if barrier stabilization by EGF could counteract barrier permeabilization by IFNalpha. As a result, EGF was not only unable to prevent, but even exacerbated IFNalpha-induced epithelial barrier destabilization. ERK1/2-signaling was necessary for this effect linking it to enhanced cell proliferation. In contrast to its damage-intensifying effect, EGF was also able to accelerate epithelial barrier re-establishment when administered after IFNalpha withdrawal. This action correlated with an anti-apoptotic mechanism induced by EGF and was independent of proliferation. We concluded that the time point, when EGF treatment was initiated, determined whether tissue repair was achieved or deleterious effects were intensified. Thus, timing of growth factor release appears to be a crucial parameter determining tissue fate (Lechner et al., Am J Physiol Cell Physiol 293, C1843-50, 2007). Similar processes might be operative in vivo during inflammatory reactions subjecting epithelial tissue to high local concentrations of cytokines released by activated immune cells. A misbalanced onset of tissue repair processes by growth factors may result in further exacerbation of deteriorating effects. The affected sites might represent trigger points for fibrotic tissue remodeling and the development of tumor growth may be facilitated by the continuous presence of proliferative stimuli. Epithelial barrier defects may furthermore contribute to metastatic dissemination by promoting the escape of tumor cells from their primary sites. Therefore, our findings not only provide a better understanding of cytokine actions, but may have implications for improving diagnosis, prognosis and treatment of diseases involving inflammatory reactions, toxic organ failure and cancer.