Effect of allergic rhinitis on the expression of human β-defensin 2 in tonsils

BackgroundHuman β-defensins (HBDs) are a newly identified family of antimicrobial peptides that are expressed by epithelia on mucosal surfaces. Exposure of airway epithelial cells to T_H2-type cytokines results in a significant decrease in the antimicrobial activity of the cells.ObjectiveTo investigate the effect of allergic rhinitis on the expression of HBD-2 in tonsils and adenoids.MethodsPalatine tonsils and adenoids were obtained from 30 patients with no history of recurrent tonsillitis. The patients were divided into 2 groups: allergic rhinitis and nonallergic rhinitis groups. Real-time polymerase chain reaction analysis was used to measure messenger RNA (mRNA) levels of HBD-2 mRNA in tonsil and adenoid tissue samples from the 2 patient groups. Immunofluorescent staining and enzyme-linked immunosorbent assay (ELISA) were used to evaluate the expression of HBD-2 protein in tonsil and adenoid tissues. The concentration of the cytokines interleukin (IL) 4, IL-5, and interferon γ (IFN-γ) in tissue homogenates was measured by ELISA.ResultsImmunofluorescent staining data demonstrated the expression of HBD-2 protein in the surface epithelia of tonsils, and a marked difference in the staining intensity was observed the between 2 groups. HBD-2 mRNA and protein levels in the tonsils were significantly lower in the allergic rhinitis group than that in the nonallergic rhinitis group (P = .03 and P = .04, respectively). IL-5 and IFN-γ were not detected, and no significant difference was found in IL-4 concentrations in tonsil homogenates between the 2 groups.ConclusionAllergic rhinitis suppresses HBD-2, an epithelial antimicrobial peptide, in the tonsils.     

Expression of the human β-defensin-1 induced by dehydroandrographolide in human pulmonary gland epithelial cells

In this study we observed the enhanced mRNA and protein expression of human β-defensin-1 (hBD-1) induced by dehydroandrographolide (DA) in human gland epithelial cells and explored the mechanism of DA on infection treatment. Human pulmonary gland epithelial cells were incubated with DA, then were harvested for hBD-1 expression detection. The mRNA expression of hBD-1 was detected by RT-PCR while the protein expression was detected by Western blot. It was found that DA can up-regulate mRNA and protein expression of hBD-1, the optimal concentration was 80 µM, the maximal expression of hBD-1 mRNA and protein occurred after 8 h. The DA can up-regulate mRNA and protein expression of hBD-1 in human gland epithelial cells. It suggests that β-defensin-1 may play an important role in the treatment of infective diseases with DA.     

Effects of antioxidant and NF-κB on the induction of iNOS gene in rat pulmonary microvascular endothelial cells in vitro

Effects of antioxidant and NF-κB on the induction of iNOS gene in rat pulmonary microvascular endothelial cells in vitro     

Different involvement of the MAPK family in inflammatory regulation in human pulmonary microvascular endothelial cells stimulated with LPS and IFN-γ

Pulmonary endothelial injury is central in the pathogenesis of acute lung injury (ALI). The MAPK signaling cascades are generally thought to be involved in the molecular mechanism underlying the ALI development, but their roles in pulmonary endothelial injury is poorly understood. We thus examined the involvement of the MAPK family member in inflammatory responses of human pulmonary microvascular endothelial cells (HPMVECs) stimulated with LPS and IFN-γ. HPMVECs were found to exhibit the upregulation of expression of Toll-like receptor 4 by IFN-γ, resulting in potentiation of inflammatory cytokine release by LPS stimulation. All MAPKs, ERK1/2, JNK, and p38, were activated by simultaneous stimulation with LPS/IFN-γ. JNK activation in cells stimulated with LPS/IFN-γ was significantly potentiated by the two different p38 inhibitors, SB203580 and RWJ67657, suggesting the negative regulation of JNK activation by p38 in HPMVECs. The mRNA and protein expression levels of ICAM-1 were eliminated by the JNK inhibitor, suggesting that ICAM-1 expression is positively regulated by JNK. The p38 inhibitor significantly enhanced ICAM-1 expression. ERK1/2 activation was not responsible for the LPS/IFN-γ-induced ICAM-1 upregulation in HPMVECs. THP-1 monocyte adhesion to HPMVECs under LPS/IFN-γ stimulation was inhibited by the JNK inhibitor and enhanced by the p38 inhibitor. We conclude that, in HPMVECs stimulated with LPS/IFN-γ, JNK mediates ICAM-1 expression that can facilitate leukocyte adherence and transmigration, while p38 MAPK negatively regulates the upregulation of ICAM-1 through inhibition of JNK activation.     

Unfractionated heparin suppresses lipopolysaccharide-induced monocyte chemoattractant protein-1 expression in human microvascular endothelial cells by blocking Krüppel-like factor 5 and nuclear factor-κB pathway

Unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH), apart from anticoagulant activities, contain a variety of biological properties such as anti-inflammatory actions possibly affecting sepsis. Chemokines are vital for promoting the movement of circulating leukocytes to the site of infection and are involved in the pathogenesis of sepsis. The purpose of this study was to investigate the effects and potential mechanisms of UFH on lipopolysaccharide (LPS)-induced chemokine production in human pulmonary microvascular endothelial cells (HPMECs). HPMECs were pretreated with UFH (0.1 U/ml and 1 U/ml), 15 min prior to stimulation with LPS (10 μg/ml). Cells were cultured under various experimental conditions for 2 h and 6 h for analysis. UFH markedly decreased LPS-induced interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1) mRNA and protein expression in HPMECs. UFH also attenuated the secretion of these chemokines in culture supernatants. In addition, UFH blocked the chemotactic activities of LPS-stimulated HPMECs supernatants on monocytes migration as expected. UFH inhibited LPS-induced Krüppel-like factor 5 (KLF-5) mRNA and protein levels. Concurrently, UFH reduced nuclear factor (NF)-κB nuclear translocation. Importantly, transfection with siRNA targeting KLF-5 reduced NF-κB activation and chemokines expression. These results demonstrate that interfering with KLF-5 mediated NF-κB activation might contribute to the inhibitory effects of chemokines and monocytes migration by UFH in LPS-stimulated HPMECs.     

Activation of AMPK attenuates lipopolysaccharide-impaired integrity and function of blood–brain barrier in human brain microvascular endothelial cells

The blood–brain barrier (BBB), formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. Lipopolysaccharide (LPS) is known to alter the integrity of the BBB in sepsis, although the underlying mechanism remains unknown. The aim of this study was to elucidate the molecular mechanisms underlying the disruption of the BBB in LPS-induced sepsis and to determine whether the activation of AMP-activated protein kinase (AMPK) prevents LPS-induced BBB dysfunction. The exposure of human brain microvascular endothelial cells (HBMECs) to LPS (1 μg/ml) for 4 to 24 h a week dramatically increased the permeability of the BBB in parallel with the lowered expression levels of occludin and claudin-5, which are essential to maintain tight junctions in HBMECs. In addition, LPS significantly increased the reactive oxygen species (ROS) productions. All effects induced by LPS in HBVMCs were reversed by adenoviral overexpression of superoxide dismutase, inhibition of NAD(P)H oxidase by apocynin or gain-function of AMPK by adenoviral overexpression of constitutively active mutant (AMPK-CA) or by 5-amino-4-imidazole carboxamide riboside (AICAR). Finally, the upregulation of AMPK by either AMPK-CA or AICAR abolished the levels of LPS-enhanced NAD(P)H oxidase subunit protein expressions. We conclude that AMPK activation improves the integrity of the BBB disrupted by LPS through suppressing the induction of NAD(P)H oxidase-derived ROS in HBMECs.     

Effects of fostriecin on β2-adrenoceptor-driven responses in human mast cells

As part of the intracellular processes leading to mast cell and basophil activation, phosphorylation of key substrates is likely to be important. These processes, mediated by phosphatases, are responsible for regulating phosphorylation. The aim of the present study was to determine effects fostriecin – a selective inhibitor of PP2A (protein phosphatase-2) – on β₂-adrenoceptor-driven responses in human mast cells. Here, the effects of fostriecin (PP inhibitors) on the inhibition of histamine release from HLMC, on β-adrenoceptor-driven responses in mast cells and on desensitization were investigated. Long-term incubation (24?h) of mast cells with fostriecin (10^?6 M) resulted in a significant (p 2-adrenoceptors.     

Effect of the antioxidant α-lipoic acid on apoptosis in human umbilical vein endothelial cells induced by high glucose

High glucose plays an important role in the pathogenesis of atherosclerosis. In this study, we assessed the effects of high glucose on human umbilical vein endothelial cell (HUVEC) apoptosis. Additionally, we investigated whether α-lipoic acid, an antioxidant, prevents high glucose-induced apoptosis of HUVECs. HUVECs were treated with high glucose in the presence or absence of α-lipoic acid. Treatment of HUVECs with high glucose changed cell morphology and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by high glucose in a dose-and time-dependent fashion. High glucose markedly elevated Bax, and decreased NF-κB and Bcl-2 expression. Most importantly, pretreatment with α-lipoic acid protected against high glucose-induced apoptosis in the endothelial cells. α-Lipoic acid significantly promoted the expression of NF-κB while decreasing the expression of Bax and the activities of caspase-3 and-9 without significantly affecting the Bcl-2 level. Our data suggest that high glucose induces apoptosis in endothelial cells. α-Lipoic acid effectively attenuates high glucose-induced endothelial cell apoptosis. These findings provide new perspectives on the role of α-lipoic acid in cardiovascular disease.     

Effect of PKC signalling pathway and aldose reductase on expression of fibronectin induced by transforming growth factor-β1 in human mesangial cells

Objective To study the effect of PKC signalling pathway and aldose reductase (AR) on the expression of fibronectin (FN) induced by transforming growth factor-β1 (TGF-β1). Methods Human mesangial cells (HMCs) were cultured and transfected with pcDNA3-AR, and subject to AR gene silencing with small interfering RNA (siRNA) and then the cell was treated with recombinant human TGF-β1. The AR mRNA expression in the HMCs was examined using real time RT-PCR and protein expression of AR and FN was detected by Western blotting. Results The cultured HMC treated with TGF-$l showed increased expression of AR and FN,the normal HMC showed not reduced expression of FN after incubation with single inhibitors of AR. Pre-incubation of cells with inhibitors of AR and PKC, then the different groups of cells were treated with TGF-$l ,and the induction effect on FN expression was suppressed (34%) in HMC. HMCs transfected with AR showed a strong protein expression of FN, which was increased by 3. 6-fold after treatment with TGF-pl (P <0. 05) , and the induction effect on FN expression was suppressed by G(O)6983 (42%) in HMCs (P < 0. 05) . The HMC with AR gene knock-down by siRNA showed a decreased expression of AR and 90% decrease of FN protein in HMCs(P <0. 01) , and TGF-β1-induced up-regulation of FN was significantly suppressed by siRNA (12%) in HMCs (P <0. 01). Conclusions AR is capable of regulating FN expression only in the presence of TGF-β1, and this reaction is possibly accomplished through the activation of PKC signalling pathway.     

Effect of PKC signalling pathway and aldose reductase on expression of fibronectin induced by transforming growth factor-β1 in human mesangial cells

Objective To study the effect of PKC signalling pathway and aldose reductase (AR) on the expression of fibronectin (FN) induced by transforming growth factor-β1 (TGF-β1). Methods Human mesangial cells (HMCs) were cultured and transfected with pcDNA3-AR, and subject to AR gene silencing with small interfering RNA (siRNA) and then the cell was treated with recombinant human TGF-β1. The AR mRNA expression in the HMCs was examined using real time RT-PCR and protein expression of AR and FN was detected by Western blotting. Results The cultured HMC treated with TGF-$l showed increased expression of AR and FN,the normal HMC showed not reduced expression of FN after incubation with single inhibitors of AR. Pre-incubation of cells with inhibitors of AR and PKC, then the different groups of cells were treated with TGF-$l ,and the induction effect on FN expression was suppressed (34%) in HMC. HMCs transfected with AR showed a strong protein expression of FN, which was increased by 3. 6-fold after treatment with TGF-pl (P <0. 05) , and the induction effect on FN expression was suppressed by G(O)6983 (42%) in HMCs (P < 0. 05) . The HMC with AR gene knock-down by siRNA showed a decreased expression of AR and 90% decrease of FN protein in HMCs(P <0. 01) , and TGF-β1-induced up-regulation of FN was significantly suppressed by siRNA (12%) in HMCs (P <0. 01). Conclusions AR is capable of regulating FN expression only in the presence of TGF-β1, and this reaction is possibly accomplished through the activation of PKC signalling pathway.     

Microgravity inhibition of lipopolysaccharide-induced tumor necrosis factor-α expression in macrophage cells

Objective and design

Microgravity environments in space can cause major abnormalities in human physiology, including decreased immunity. The underlying mechanisms of microgravity-induced inflammatory defects in macrophages are unclear.

Material or subjects

RAW264.7 cells and primary mouse macrophages were used in the present study. Lipopolysaccharide (LPS)-induced cytokine expression in mouse macrophages was detected under either simulated microgravity or 1g control.

Methods

Freshly isolated primary mouse macrophages and RAW264.7 cells were cultured in a standard simulated microgravity situation using a rotary cell culture system (RCCS-1) and 1g control conditions. The cytokine expression was determined by real-time PCR and ELISA assays. Western blots were used to investigate the related intracellular signals.

Results

LPS-induced tumor necrosis factor-α (TNF-α) expression, but not interleukin-1β expression, in mouse macrophages was significantly suppressed under simulated microgravity. The molecular mechanism studies showed that LPS-induced intracellular signal transduction including phosphorylation of IKK and JNK and nuclear translocation of NF-κB in macrophages was identical under normal gravity and simulated microgravity. Furthermore, TNF-α mRNA stability did not decrease under simulated microgravity. Finally, we found that heat shock factor-1 (HSF1), a known repressor of TNF-α promoter, was markedly activated under simulated microgravity.

Conclusions

Short-term treatment with microgravity caused significantly decreased TNF-α production. Microgravity-activated HSF1 may contribute to the decreased TNF-α expression in macrophages directly caused by microgravity, while the LPS-induced NF-κB pathway is resistant to microgravity.     

The role of interferon β in human cytomegalovirus-mediated inhibition of HLA DR induction on endothelial cells

Summary Human cytomegalovirus (HCMV), a member of the virus familyHerpesviridae that is associated with extensive worldwide morbidity and mortality in immunocompromised hosts, inhibits interferon- (IFN)-mediated induction of human leukocyte antigen (HLA) class II antigens on endothelial cells. In this study, the ability of HCMV-infected endothelial cells to synthesize interferon- (IFN), and the role of IFN in HCMV-mediated inhibition of HLA class II induction, was investigated. As detemined by an encephalomyocarditis virus protection assay, HCMV-infected endothelial cell culture supernatants contained 240 IU/ml of IFN type I activity, of which 99.9% was IFN, as compared to the absence of IFN in mock-infected culture supernatants. UV-irradiated supernatants from HCMV-infected cultures inhibited induction of HLA class II in noninfected cultures by 24%. This inhibition could be abolished with 500 NU/ml of anti-IFN antibody. Addition of anti-IFN antibody directly to HCMV-infected cultures mitigated but did not abolish HLA class II antigen inhibition. Dual immunohistochemistry for HCMV and HLA DR demonstrated that infected cells, in contrast to noninfected cells, were rarely induced to express HLA class II even in the presence of anti-IFN antibody. These findings suggest that HCMV inhibits induction of HLA class II antigens by IFN dependent and independent mechanisms.     

Inhibitory effect of β-elemene on human breast cancer cells

It has been approved for the clinical application of β-elemene to treat various cancers mainly brain tumors in China. In the present study, we found that β-elemene significantly inhibited the in vitro growth of human breast cancer cells by inducing apoptosis. In addition, β-elemene also induced the conversion of LC3-I into LC3-II as well as the formation of autolysosomes, indicating the activation of autophagy. Interestingly, inhibition of autophagy significantly potentiated the growth-inhibitory effect of β-elemene on breast cancer cells. In summary, β-elemene induced cytoprotective autophagy in human breast cancer cells in addition to apoptosis. Inhibition of autophagy significantly enhanced the cytotoxicity of β-elemene to human breast cancer cells. Therefore, combination of β-elemene with autophagy inhibitors could be a promising strategy for the treatment of breast cancer.     

Enhancement of activated β1-integrin expression by prostaglandin E2 via EP receptors in isolated human coronary arterial endothelial cells: implication for the treatment of Kawasaki disease

Objective:  Plasma prostaglandin E₂ (PGE₂) levels are markedly elevated in acute Kawasaki disease (KD). We evaluated the function of the EP receptors in the expression of activated β₁-integrin stimulated by PGE₂ in human coronary arterial endothelial cells (HCAEC). Methods:  We determined the mRNA expression of the PGE₂ receptors, EP receptors (EP_1-4) in HCAEC by RT-PCR and protein expression by Western blotting. We evaluated the function of the EP receptors in the expression of activated β₁-integrin stimulated by PGE₂ in HCAEC, using antagonists and agonists of the EP receptors, by flow cytometry. Results:  RT-PCR revealed mRNAs for all four EP receptors in HCAEC. Western blotting demonstrated EP₁, EP₂ and EP₃ expression in HCAEC. The EP₂ and EP₃ agonists enhanced the expression of activated β₁-integrin in HCAEC. The potency of the EP₂ agonist was significantly greater than that of the EP₃ agonist. Pretreatment with the EP₁, EP₂ and EP₃ antagonists inhibited the expression of activated β₁-integrin induced by PGE₂ in HCAEC. The potency of the EP₂ antagonist was significantly greater than that of the EP₁ and EP₃ antagonists. Conclusions:  Our results suggest that PGE₂ mainly induces the activation of β₁-integrins via the EP₂ receptor in HCAEC. Our results further suggest that the EP₂ antagonist modulates the inflammatory response during KD vasculitis. Received 13 August 2008; returned for revision 15 September 2008; returned for final revision 28 October 2008; accepted by G. Wallace 24 November 2008     

Transforming growth factor β1 acts as an inducer of matrix metalloproteinase expression and activity in human bone-metastasizing cancer cells

Bone metastases are a common complication in prostate and breast cancer patients. It leads to extensive morbidity and eventually mortality. Matrix metalloproteinases (MMPs) are known to be involved in the metastatic process. MMP activity can be down-regulated by transforming growth factor 1 (TGF-1), a growth-modulating factor, found in high concentrations in the bone. TGF-1 acts through the TGF-1 inhibitory element (TIE) element, a cis-acting element found in the promoter region of most MMP genes, with the exception of MMP-2. We used three human cell lines relevant for bone metastases, namely prostate adenocarcinoma PC-3, breast adenocarcinoma MDA-MB-231, and adenocarcinoma cells of unknown origin, Hs696, and one human osteosarcoma cell line, SAOS-2, and showed that in these cell lines TGF-1 partially lost its repressing action on MMP expression. TGF-1 was able to induce MMP-9 activity and protein expression in all three bone-metastatic tumour cell types, whereas MMP-9 protein levels were repressed in SAOS-2 cells. In PC-3 cells, TGF-1 repressed MMP-1 expression, whereas in MDA-MB-231 and SAOS-2 cells, an increase in the expression of MMP-1 protein was detected. Additionally, an increase in MMP-3 expression was observed in Hs696 cells. Expression and activity of the tissue inhibitors of matrix metalloproteinases, TIMP-1 and TIMP-2, were found increased in both PC-3 and MDA-MB-231 cells. With respect to cell proliferation, TGF-1 was able to induce a dose-dependent growth inhibition of up to 50% in primary human mammary epithelial cells. However, in none of the tumour cell lines was TGF-1 able to suppress growth substantially. Data presented in this paper support the hypothesis that TGF-1 can potentially disrupt the balance existing between osteoclast- and osteoblast-derived MMP activity by inducing altered expression of matrix metalloproteinases and their tissue inhibitors derived from bone-metastasizing cancer cells. This could eventually lead to skeletal destruction in patients with advanced metastatic disease.     

Suppressive influences of IFN-α on IL-17 expression in human CD4+ T cells

We examined the direct effects of IFN-α on the development of Th17 with a system using immobilized anti-CD3, which permits activation of CD4+ T cells in the complete absence of accessory cells. Highly purified CD4+ T cells obtained from healthy donors were stimulated with immobilized anti-CD3 with or without IFN-α. IFN-α suppressed the production of IL-17 of immobilized anti-CD3-stimulated CD4+ T cells in a dose–response manner. Accordingly, IFN-α inhibited IL-17 mRNA expression in immobilized anti-CD3-stimulated CD4+ T cells. IFN-α did not affect the production of TGF-β or IL-6, but inhibited RORC mRNA expression of anti-CD3-stimulated CD4+ T cells. These results indicate that IFN-α suppresses IL-17 expression and Th17 differentiation through down-regulation of RORC mRNA expression. It is therefore suggested that these effects might play a role in the mode of action of IFN-α in the treatment of various inflammatory diseases.     

Cytohesin-1 regulates human blood neutrophil adhesion to endothelial cells through β2 integrin activation

Cytohesin-1 is a guanine nucleotide exchange factor for ADP ribosylation factor 6 (Arf6) in human blood neutrophils and differentiated PLB-985 neutrophil-like cells. Cytohesin-1 regulates adhesion and the transendothelial migration of monocytes, dendritic cells and T lymphocytes through activation of the β2 integrin LFA-1. In this study we investigated the role of cytohesin-1 in neutrophil and neutrophil-like cell adhesion to HUVECs, immobilized ICAM-1, and the α4β1 and α5β1 integrin extracellular matrix ligand fibronectin. We show that cytohesin-1 knockdown or inhibition with secinH3 inhibits fMLF-mediated cell adhesion to HUVECs and immobilized ICAM-1, whereas cytohesin-1 over-expression has the opposing effect. Binding of PLB-985 cells to HUVECs correlated with expression of the high-affinity β2 integrin epitope recognized by mAb24. Adhesion to HUVECs was inhibited by soluble ICAM-1, anti-ICAM-1, anti-CD11a and anti-CD18, but not anti-CD11b, blocking antibodies. We also demonstrate that cytohesin-1 knockdown promotes fMLF-mediated cell adhesion to fibronectin whereas cytohesin-1 over-expression has the opposing effect. Crosstalk between β1 and β2 integrins also exists since inhibition of β1 integrin functions with blocking antibodies enhanced adhesion of PLB-985 over-expressing cytohesin-1 to ICAM-1. We suggest that cytohesin-1 is a key regulator of neutrophil adhesion to endothelial cells and to components of extracellular matrix, which may influence cell emigration through its dual opposing effect on β2 and β1 integrin activation.