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JNK and ERK MAP kinases mediate induction of IL-1beta, TNF-alpha and IL-8 following hyperosmolar stress in human limbal epithelial cells.

ABSTRACT: Hyperosmolarity has been recognized to be a pro-inflammatory stress to the corneal epithelium. The cell signalling pathways linking hyperosmolar stress and inflammation have not been well elucidated. This study investigated whether exposure of human limbal epithelial cells to hyperosmotic stress activates the mitogen-activated protein kinase (MAPK) pathways and induces production of pro-inflammatory cytokines, interleukin (IL) -1beta, tumor necrosis factor (TNF) alpha, and the C-X-C chemokine IL-8. Primary human limbal epithelial cultures in normal osmolar media (312 mOsM) were exposed to media with higher osmolarity (400-500 mOsM) by adding 50-90 mM NaCl, with or without SB202190, an inhibitor of c-Jun N-terminal kinases (JNK) pathway, PD 98059, an inhibitor of extracellular-regulated kinase (ERK) pathway, dexamethasone or doxycycline for different lengths of time. The conditioned media were collected after 24 hr of treatment for ELISA. Total RNA was extracted from cultures treated for 6 hr for semi-quantitative RT-PCR. Cells treated for 15-60 min were lysed in RIPA buffer and subjected to Western blot with phospho (p)-specific antibodies against p-JNK and p-ERK. The concentrations of IL-1beta, TNF-alpha and IL-8 proteins in 24 hr conditioned media of limbal epithelial cells progressively increased as the media osmolarity increased from 312 to 500 mOsM. Active p-JNK-1/p-JNK-2 and p-ERK-1/p-ERK-2 were detected by Western blot and peaked at 60 min in cells exposed to hyperosmolar media. The levels of p-JNK-1/p-JNK-2 and p-ERK1/p-ERK2 were positively correlated with the medium osmolarity. SB202190, PD98059 and doxycycline markedly suppressed the levels of p-JNK-1/p-JNK-2 and/or p-ERK1/p-ERK2, as well as IL-1beta, TNF-alpha and IL-8 mRNAs and proteins stimulated by hyperosmolar media. These findings provide direct evidence that hyperosmolarity induces inflammation in human limbal epithelial cells by increasing expression and production of pro-inflammatory cytokines and chemokines, a process that appears to be mediated through activation of the JNK and ERK MAPK signalling pathways. The efficacy of doxycycline in treating ocular surface diseases may be due to its ability to suppress JNK and ERK signalling activation and inflammatory mediator production in the limbal epithelium.

SUBMITTER: Li DQ

PROVIDER: S-EPMC2198933 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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JNK and ERK MAP kinases mediate induction of IL-1beta, TNF-alpha and IL-8 following hyperosmolar stress in human limbal epithelial cells.

Li De-Quan DQ Luo Lihui L Chen Zhuo Z Kim Hyun-Seung HS Song Xiu Jun XJ Pflugfelder Stephen C SC

Experimental eye research 20051003 4

Hyperosmolarity has been recognized to be a pro-inflammatory stress to the corneal epithelium. The cell signalling pathways linking hyperosmolar stress and inflammation have not been well elucidated. This study investigated whether exposure of human limbal epithelial cells to hyperosmotic stress activates the mitogen-activated protein kinase (MAPK) pathways and induces production of pro-inflammatory cytokines, interleukin (IL) -1beta, tumor necrosis factor (TNF) alpha, and the C-X-C chemokine IL ...[more]

PMID: 16202406

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Project description:Swine influenza is an acute respiratory disease in pigs caused by swine influenza virus (SIV). Highly virulent SIV strains cause mortality of up to 10%. Importantly, pigs have long been considered "mixing vessels" that generate novel influenza viruses with pandemic potential, a constant threat to public health. Since its emergence in 2009 and subsequent pandemic spread, the pandemic (H1N1) 2009 (H1N1pdm) has been detected in pig farms, creating the risk of generating new reassortants and their possible infection of humans. Pathogenesis in SIV or H1N1pdm-infected pigs remains poorly characterized. Proinflammatory and antiviral cytokine responses are considered correlated with the intensity of clinical signs, and swine macrophages are found to be indispensible in effective clearance of SIV from pig lungs. In this study, we report a unique pattern of cytokine responses in swine macrophages infected with H1N1pdm. The roles of mitogen-activated protein (MAP) kinases in the regulation of the host responses were examined. We found that proinflammatory cytokines IL-6, IL-8, IL-10, and TNF-? were significantly induced and their induction was ERK1/2-dependent. IFN-? and IFN-inducible antiviral Mx and 2'5'-OAS were sharply induced, but the inductions were effectively abolished when ERK1/2 was inhibited. Induction of CCL5 (RANTES) was completely inhibited by inhibitors of ERK1/2 and JNK1/2, which appeared also to regulate FasL and TNF-?, critical for apoptosis in pig macrophages. We found that NF?B was activated in H1N1pdm-infected cells, but the activation was suppressed when ERK1/2 was inhibited, indicating there is cross-talk between MAP kinase and NF?B responses in pig macrophages. Our data suggest that MAP kinase may activate NF?B through the induction of RIG-1, which leads to the induction of IFN-? in swine macrophages. Understanding host responses and their underlying mechanisms may help identify venues for effective control of SIV and assist in prevention of future influenza pandemics.

Project description:BackgroundAnopheles gambiae is the primary mosquito vector of human malaria parasites in sub-Saharan Africa. To date, three innate immune signaling pathways, including the nuclear factor (NF)-kappaB-dependent Toll and immune deficient (IMD) pathways and the Janus kinase/signal transducers and activators of transcription (Jak-STAT) pathway, have been extensively characterized in An. gambiae. However, in addition to NF-kappaB-dependent signaling, three mitogen-activated protein kinase (MAPK) pathways regulated by JNK, ERK and p38 MAPK are critical mediators of innate immunity in other invertebrates and in mammals. Our understanding of the roles of the MAPK signaling cascades in anopheline innate immunity is limited, so identification of the encoded complement of these proteins, their upstream activators, and phosphorylation profiles in response to relevant immune signals was warranted.ResultsIn this study, we present the orthologs and phylogeny of 17 An. gambiae MAPKs, two of which were previously unknown and two others that were incompletely annotated. We also provide detailed temporal activation profiles for ERK, JNK, and p38 MAPK in An. gambiae cells in vitro to immune signals that are relevant to malaria parasite infection (human insulin, human transforming growth factor-beta1, hydrogen peroxide) and to bacterial lipopolysaccharide. These activation profiles and possible upstream regulatory pathways are interpreted in light of known MAPK signaling cascades.ConclusionsThe establishment of a MAPK "road map" based on the most advanced mosquito genome annotation can accelerate our understanding of host-pathogen interactions and broader physiology of An. gambiae and other mosquito species. Further, future efforts to develop predictive models of anopheline cell signaling responses, based on iterative construction and refinement of data-based and literature-based knowledge of the MAP kinase cascades and other networked pathways will facilitate identification of the "master signaling regulators" in biomedically important mosquito species.

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JNK and ERK MAP kinases mediate induction of IL-1beta, TNF-alpha and IL-8 following hyperosmolar stress in human limbal epithelial cells.

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JNK and ERK MAP kinases mediate induction of IL-1beta, TNF-alpha and IL-8 following hyperosmolar stress in human limbal epithelial cells.

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