Inactivation of Max-interacting Protein 1 Induces Renal Cilia Disassembly through Reduction in Levels of Intraflagellar Transport 20 in Polycystic Kidney

Cilia in ciliated cells consist of protruding structures that sense mechanical and chemical signals from the extracellular environment. Cilia are assembled with variety molecules via a process known as intraflagellar transport (IFT). What controls the length of cilia in ciliated cells is critical to understand ciliary disease such as autosomal dominant polycystic kidney disease, which involves abnormally short cilia. But this control mechanism is not well understood. Previously, multiple tubular cysts have been observed in the kidneys of max-interacting protein 1 (Mxi1)-deficient mice aged 6 months or more. Here, we clarified the relationship between Mxi1 inactivation and cilia disassembly. Cilia phenotypes were observed in kidneys of Mxi1-deficient mice using scanning electron microscopy to elucidate the effect of Mxi1 on renal cilia phenotype, and cilia disassembly was observed in Mxi1-deficient kidney. In addition, genes related to cilia were validated in vitro and in vivo using quantitative PCR, and Ift20 was selected as a candidate gene in this study. The length of cilium decreased, and p-ERK level induced by a cilia defect increased in kidneys of Mxi1-deficient mice. Ciliogenesis of Mxi1-deficient mouse embryonic fibroblasts (MEFs) decreased, and this abnormality was restored by Mxi1 transfection in Mxi1-deficient MEFs. We confirmed that ciliogenesis and Ift20 expression were regulated by Mxi1 in vitro. We also determined that Mxi1 regulates Ift20 promoter activity via Ets-1 binding to the Ift20 promoter. These results indicate that inactivating Mxi1 induces ciliary defects in polycystic kidney.     

HCl-induced and ATP-dependent upregulation of TRPV1 receptor expression and cytokine production by human esophageal epithelial cells

The pathogenesis of gastroesophageal reflux disease (GERD) remains elusive, but recent evidence suggests that early secretion of inflammatory cytokines and chemokines by the mucosa leads to influx of immune cells followed by tissue damage. We previously showed that exposure of esophageal mucosa to HCl causes ATP release, resulting in activation of acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (lyso-PAF AT), the enzyme responsible for the production of platelet-activating factor (PAF). In addition, HCl causes release of IL-8 from the esophageal mucosa. We demonstrate that esophageal epithelial cells secrete proinflammatory mediators in response to HCl and that this response is mediated by ATP. Monolayers of the human esophageal epithelial cell line HET-1A were exposed to acidified cell culture medium (pH 5) for 12 min, a total of seven times over 48 h, to simulate the recurrent acid exposure clinically occurring in GERD. HCl upregulated mRNA and protein expression for the acid-sensing transient receptor potential cation channel, subfamily vanilloid member 1 (TRPV1), lyso-PAF AT, IL-8, eotaxin-1, -2, and -3, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1. The chemokine profile secreted by HET-1A cells in response to repeated HCl exposure parallels similar findings in erosive esophagitis patients. In HET-1A cells, the TRPV1 agonist capsaicin reproduced these findings for mRNA of the inflammatory mediators lyso-PAF AT, IL-8, and eotaxin-1. These effects were blocked by the TRPV1 antagonists iodoresiniferatoxin and JNJ-17203212. These effects were imitated by direct application of ATP and blocked by the nonselective ATP antagonist suramin. We conclude that HCl/TRPV-induced ATP release upregulated secretion of various chemoattractants by esophageal epithelial cells. These chemoattractants are selective for leukocyte subsets involved in acute inflammatory responses and allergic inflammation. The data support the validity of HET-1A cells as a model of the response of the human esophageal mucosa in GERD.     

Differential expression and polarized secretion of CXC and CC chemokines by human intestinal epithelial cancer cell lines in response to Clostridium difficile toxin A

Intestinal epithelial cells are the initial sites of host response to Clostridium difficile infection and can play a role in signaling the influx of inflammatory cells. To further explore this role, the regulated expression and polarized secretion of CXC and CC chemokines by human intestinal epithelial cells were investigated. An expression of the CXC chemokines, including IL-8 and growth-related oncogene (GRO)-alpha, and the CC chemokine monocyte chemoattractant protein (MCP)-1 from HT-29 cells increased in the 1-6 hr following C. difficile toxin A stimulation, assessed by quantitative RT-PCR. In contrast, the expression of neutrophil activating protein-78 (ENA-78) was delayed for 18 hr. The up-regulated mRNA expression of chemokines was paralleled by the increase of protein levels. However, the expression of macrophage inflammatory protein (MIP)-1alpha, RANTES (regulated on activation normal T cells expressed and secreted), and interferon-gamma-inducible protein-10 (IP-10) was not changed in HT-29 or Caco-2 cells stimulated with toxin A. Upon stimulation of the polarized Caco-2 epithelial cells in a transwell chamber with toxin A, CXC and CC chemokines were released predominantly into the basolateral compartment. Moreover, the addition of IFN-gamma and TNF-alpha to toxin A stimulated Caco-2 cells increased the basolateral release of CC chemokine MCP-1. In contrast, IFN-gamma and TNF-alpha had no effect on the expression of the CXC chemokines IL-8 and GRO-alpha. These results suggest that a CXC and CC chemokine expression from epithelial cells infected with C. difficile may be an important factor in the mucosal inflammatory response.     

Gastrin induces CXC chemokine expression in gastric epithelial cells through activation of NF-kappaB

Although hypergastrinemia is frequently observed in individuals with a chronic Helicobacter pylori infection, its pathophysiological significance in gastric mucosal inflammation is unclear. The present study was designed to determine if gastrin induces the expression of CXC chemokines in gastric epithelial cells. Human and rat gastric epithelial cells, transfected with gastrin receptor, were stimulated with gastrin. The expression of mRNAs for human interleukin-8 (IL-8) and rat cytokine-induced neutrophil chemoattractant-1 and release of human IL-8 protein were then determined by Northern blot analysis and ELISA, respectively. Gastrin not only induced the expression of mRNAs for these chemokines but also stimulated IL-8 protein release. A luciferase assay using IL-8 promoter genes showed that nuclear factor (NF)-kappaB is absolutely required and activator protein-1 (AP-1) is partly required for the maximum induction of IL-8 by gastrin. An electrophoretic mobility shift assay revealed that gastrin is capable of activating both NF-kappaB and AP-1. In addition, the inhibition of NF-kappaB abrogated gastrin-induced chemokine expression. These results suggest that gastrin is capable of upregulating CXC chemokines in gastric epithelial cells and therefore may contribute to the progression of the inflammatory process in the stomach.     

Effect of proinflammatory cytokines on interleukin-8 mRNA expression and protein production by isolated human alveolar epithelial cells type II in primary culture

Alveolar epithelial cells type II (AEC-II) are ideally situated to regulate the recruitment and activation of different types of cells through the production of chemokines in response to inflammatory stimulation from the alveolar space. We hypothesized that these cells are important producers of interleukin-8 (IL-8) in the lung. This lead us to investigate the capacity of isolated human AEC-II cells to release IL-8 and whether this IL-8 release is regulated by proinflammatory cytokines, i.e. IL-1 beta, TNF-alpha and IFN-gamma. We isolated AEC-II from tumor-free sections of human lungs obtained by pneumectomy and purified the cells by magnetic activated cell sorting. For control experiments the AEC-II-like cell line A549 was used. IL-8 concentration was measured by ELISA in supernatants of unstimulated and LPS-, IL-1 beta-, TNF-alpha- and IFN-gamma- stimulated cells. IL-8 mRNA expression was evaluated by RT-PCR. Spontaneous IL-8 mRNA expression and protein secretion by AEC-II were significantly higher in comparison with A549 cells. TNF-alpha increased both IL-8 mRNA expression and protein production, whereas IL-1 beta slightly increased IL-8 release but did not change mRNA expression in AEC-II. LPS and IFN-gamma did not influence IL-8 expression in AEC-II and A549 cells. These results show considerable differences between A549 cell and AEC-II. The latter are capable of producing IL-8 under the control of proinflammatory cytokines. Our findings demonstrate that the modulation of IL-8 release in AEC-II may have an important impact on the immunoreactivity of these cells during pulmonary inflammation in vivo.     

Identification of apolipoproteinA1 reduction in the polycystic kidney by proteomics analysis of the Mxi1‐deficient mouse

Autosomal dominant polycystic kidney disease is one of the most common human monogenic diseases in which extensive epithelial‐lined cysts develop in kidney and other organs. Affected kidneys are not only characterized by the formation of cysts, but also by changes associated with the extracellular matrix and interstitial inflammation, which can progress to fibrosis and loss of renal function. Mxi1 protein, which is a c‐myc antagonist, may be essential in controlling cellular growth and differentiation. Previously, multiple tubular cysts were observed in kidney of Mxi1‐deficient mice aged 6 months and more. Presently, 2‐DE and MALDI‐TOF MS was employed to identify the differentially expressed proteins in the kidney. Several proteins were identified, among them, apolipoproteinA1 which is a major component of the high‐density lipoprotein complex and has anti‐inflammation effects, was significantly decreased in the Mxi1‐deficient mouse. We confirm the development of inflammation and renal fibrosis and the expression of extracellular matrix molecules including transforming growth factor were also increased in cystic kidney. These results indicate that expression of proteins related with inflammation and renal fibrosis changes by Mxi1 inactivation in polycystic kidney.     

Bronchial epithelial cells release IL-6, CXCL1 and CXCL8 upon mast cell interaction

Although mast cells have been found in increased numbers in bronchial epithelium in asthma patients, the pathogenic role of the interaction of mast cells with bronchial epithelial cells in the development of local inflammation in asthma is not well understood. In this study, primary human bronchial epithelial cells and a human mast cell line (HMC-1) were cultured either together or separately in the presence or absence of various signaling molecule inhibitors or dexamethasone. Cytokine IL-6, and chemokines including CXCL1 and CXCL8 in cell culture supernatant were assayed by enzyme-linked immunosorbent assay (ELISA), and the activity of mitogen-activated protein kinases (MAPKs), or nuclear factor-κB (NF-κB) in co-culture system was analyzed by ELISA. Co-culture of bronchial epithelial cells and mast cells induced a significant elevation of IL-6, CXCL1 and CXCL8 in bronchial epithelial cells, and both IL-17A and IL-17F could further enhance the release of these inflammatory mediators from co-culture. The induction of IL-6, CXCL1 and CXCL8 upon the interaction of bronchial epithelial cells with mast cells was mediated by MAPKs and NF-κB signaling pathways. These data indicate that the interaction of mast cells with bronchial epithelial cells may represent a crucial mechanism of regulating local inflammatory response in allergic asthma.     

Interleukin-35 Inhibits Endothelial Cell Activation by Suppressing MAPK-AP-1 Pathway

Vascular response is an essential pathological mechanism underlying various inflammatory diseases. This study determines whether IL-35, a novel responsive anti-inflammatory cytokine, inhibits vascular response in acute inflammation. Using a mouse model of LPS-induced acute inflammation and plasma samples from sepsis patients, we found that IL-35 was induced in the plasma of mice after LPS injection as well as in the plasma of sepsis patients. In addition, IL-35 decreased LPS-induced proinflammatory cytokines and chemokines in the plasma of mice. Furthermore, IL-35 inhibited leukocyte adhesion to the endothelium in the vessels of lung and cremaster muscle and decreased the numbers of inflammatory cells in bronchoalveolar lavage fluid. Mechanistically, IL-35 inhibited the LPS-induced up-regulation of endothelial cell (EC) adhesion molecule VCAM-1 through IL-35 receptors gp130 and IL-12Rβ2 via inhibition of the MAPK-activator protein-1 (AP-1) signaling pathway. We also found that IL-27, which shares the EBI3 subunit with IL-35, promoted LPS-induced VCAM-1 in human aortic ECs and that EBI3-deficient mice had similar vascular response to LPS when compared with that of WT mice. These results demonstrated for the first time that inflammation-induced IL-35 inhibits LPS-induced EC activation by suppressing MAPK-AP1-mediated VCAM-1 expression and attenuates LPS-induced secretion of proinflammatory cytokines/chemokines. Our results provide insight into the control of vascular inflammation by IL-35 and suggest that IL-35 is an attractive novel therapeutic reagent for sepsis and cardiovascular diseases.     

CXC chemokine receptor 4 expression and function in human astroglioma cells

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-gamma-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-alpha and IL-1beta. The peak of CXCR4 expression in response to TNF-alpha and IL-1beta was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-alpha and IL-1beta (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1alpha (SDF-1alpha), resulted in an elevation in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1alpha treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-gamma-inducible protein-10. SDF-1alpha-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.     

TNF-induced IL-8 and MCP-1 production in the eosinophilic cell line, EOL-1

The role of eosinophils in inflammation and their mode of activation is not well understood. Eosinophil accumulation and subsequent expression of cytokines at the site of inflammation may play a role in exacerbation of inflammatory responses. In the present study, we have examined the role of TNF-alpha in eosinophil activation and chemokine production using a human leukaemic eosinophil cell line, EOL-1. Initial studies demonstrated that TNF-alpha induced the upregulation of IL-8 and MCP-1 mRNA and protein. Kinetic studies indicated production of chemokines, IL-8 and MCP-1, as early as 4 h post-activation, with peak levels of chemokine produced at 8 h, and decreasing by 24 h post-TNF-alpha activation. When IL-10, a suppressive cytokine, was incubated with TNF-alpha and EOL-1 cells, no effect was observed on IL-8 and MCP-1 production. However, dexamethasone, a glucocorticoid, demonstrated potent inhibitory effects on the EOL-1-derived chemokines. These studies indicate that eosinophils may be a significant source of chemokines capable of participating in, and maintaining, leukocyte recruitment during inflammatory responses, such as asthma.     

Release of cytokines/chemokines and cell death in UVB-irradiated human keratinocytes, HaCaT

Ultraviolet (UV) B can lead to inflammatory responses such as sunburn, which involves the production of various inflammatory cytokines and chemokines, and the induction of cell death. Keratinocytes in the skin has one of the highest risks of exposure to UV. However, the detailed mechanisms underlying UVB irradiation-induced inflammation and cell death are not well known. Thus, we investigated the effect of UVB irradiation on the production of various cytokines/chemokines and the induction of cell death in UVB-irradiated human keratinocytes (HaCaT cells). We evaluated 11 cytokines/chemokines in cell culture supernatants from HaCaT cells exposed to 0-400 mJ/cm(2) UVB irradiation. UVB at a dose 400 mJ/cm(2) induced the release of various cytokines; interleukin (IL)-1beta, IL-6, IL-8, interferon (IFN)-gamma, granulocyte-colony stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, and tumor necrosis factor (TNF)-alpha. These results suggest that UVB irradiation-induced the release of several cytokines/chemokines and led to cell death in human keratinocytes. UV exposure may be associated with multiple physiological events in the human skin.     

Toll-like receptor 4 and cytokine expression involved in functional immune response in an originally established porcine intestinal epitheliocyte cell line

To study the immune responses of porcine intestinal epithelial cells to gram-negative bacteria via toll-like receptors (TLRs), originally established porcine intestinal epitheliocyte (PIE) cells were treated with lipopolysaccharide (LPS) or swine-specific enterotoxigenic Escherichia coli (ETEC). Real-time quantitative PCR revealed that PIE cells expressed TLR1-9 and MD-2 mRNAs, preferentially expressed TLR4/MD-2. Immunostaining of PIE cells revealed that TLR4 was precisely expressed in PIE cells at the protein level. PIE cells treated with LPS had up-regulated expression of several TLRs (TLR2, 3, 4, 5 and 8), type 1 helper T (Th1) cytokines (interleukin (IL)-1alpha, IL-1beta, IL-6, IL-15, 18, leukemia inhibitory factor (LIF), and interferon (IFN)-beta), and chemokines (monocyte chemoattractant protein (MCP)-1 and IL-8). ETEC enhanced the expression of TLR2, Th1 type cytokines (IL-1alpha, IL-12p35 and IL-6) and chemokines (MCP-1 and IL-8). These results indicate that PIE induces inflammatory responses by up-regulating Th1 cytokines and chemokines in response to LPS or ETEC, suggesting that PIE is a useful cell line for studying inflammatory responses via TLR4/MD-2 in intestinal epithelial cells.     

Leptin enhances survival and induces migration, degranulation, and cytokine synthesis of human basophils

Basophils are the rarest leukocytes in human blood, but they are now recognized as one of the most important immunomodulatory as well as effector cells in allergic inflammation. Leptin, a member of the IL-6 cytokine family, has metabolic effects as an adipokine, and it is also known to participate in the pathogenesis of inflammatory reactions. Because there is an epidemiologic relationship between obesity and allergy, we examined whether basophil functions are modified by leptin. We found that human basophils express leptin receptor (LepR) at both the mRNA and surface protein levels, which were upregulated by IL-33. Leptin exerted strong effects on multiple basophil functions. It induced a strong migratory response in human basophils, similar in potency to that of basophil-active chemokines. Also, leptin enhanced survival of human basophils, although its potency was less than that of IL-3. Additionally, CD63, a basophil activation marker expressed on the cell surface, was upregulated by leptin, an effect that was neutralized by blocking of LepR. Assessments of basophil degranulation and cytokine synthesis found that leptin showed a strong priming effect on human basophil degranulation in response to FcεRI aggregation and induced Th2, but not Th1, cytokine production by the cells. In summary, the present findings indicate that leptin may be a key molecule mediating the effects of adipocytes on inflammatory cells such as basophils by binding to LepR and activating the cellular functions, presumably exacerbating allergic inflammation.