Claudin-1 expression is induced by tumor necrosis factor-alpha in human pancreatic cancer cells

Claudin-1 is a membrane protein with four transmembrane domains, that is exclusively localized at cellular tight junctions. Recent studies have reported that claudin-1 plays an important role in cancer invasion and metastasis. However, the significance of claudin-1 in pancreatic cancer is still unknown. In the present study, we investigated the role of claudin-1 expression in pancreatic cancer growth using the PANC-1 human pancreatic cancer cell line. Treatment with tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine, resulted in increased detection of 89 kDa products of poly-(ADP-ribose) polymerase (PARP), a marker of apoptosis, and decreased PANC-1 cell proliferation by 23%. Expression of claudin-1 was up-regulated by TNF-alpha in a concentration-dependent manner in PANC-1 cells. PANC-1 cells treated with TNF-alpha and siRNA against claudin-1 showed a 15% increase in proliferation; i.e. the cells transfected with siRNA against claudin-1 showed resistance to TNF-alpha-induced apoptosis. These results suggest that claudin-1 expression is responsible for TNF-alpha-dependent growth signals and the proliferation of pancreatic cancer cells.     

Modulation of cadherin and catenins expression by tumor necrosis factor-alpha and dexamethasone in human bronchial epithelial cells

Asthma is an inflammatory disease, and the epithelial mesenchymal unit appears to be of importance in regulating the disease mechanisms. Cell-cell adhesion plays an important role in tissue morphogenesis and homeostasis and is commonly mediated by cadherins, a family of Ca(2+)-dependent transmembrane adhesion receptors. The cadherin family is involved in control of the cellular architecture. Proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha are involved in asthma and may interfere with epithelial integrity. In the present study, we investigated the role of TNF-alpha and dexamethasone on the expression of E-cadherin, beta-catenin, and gamma-catenin. We used two bronchial epithelial cell models: primary small airway epithelial cell cultures and primary culture obtained from human bronchial tubes. After 48 h of TNF-alpha stimulation with or without dexamethasone expression of E-cadherin, beta-catenin and gamma-catenin were analyzed using Western blot analysis and immunofluorescence. This study showed a decrease in the expression of adhesion molecules in both epithelial cell cultures after stimulation. Dexamethasone and anti-TNF-alpha inhibited this effect. In unstimulated cells, E-cadherin and beta- and gamma-catenin expression was membranous, expressed only on the lateral cell wall with minimal cytoplasmic expression. Immunoreactivity was cytoplasmic in stimulated cells. We demonstrated, using Western blot analysis and immunofluorescence, that proinflammatory cytokines could be responsible for structural damage to the epithelium and that this process was potentially reversed by steroids.     

Increased tumor necrosis factor-alpha (TNF-alpha) gene expression in parainfluenza type 1 (Sendai) virus-induced bronchiolar fibrosis.

Increased airway resistance and airway hyperresponsiveness induced in rats by infection with parainfluenza type I (Sendai) virus is associated with bronchiolar fibrosis. To determine whether increased tumor necrosis factor (TNF)-alpha gene expression is an important regulatory event in virus-induced bronchiolar fibrosis, pulmonary TNF-alpha mRNA and protein expression was assessed in rat strains that are susceptible (Brown Norway; BN) and resistant (Fischer 344; F344) to virus-induced bronchiolar fibrosis. Virus-inoculated BN rats had increased TNF-alpha pulmonary mRNA levels (P < 0.05) and increased numbers of bronchiolar macrophages and fibroblasts expressing TNF-alpha protein compared with virus-inoculated F344 rats (P < 0.05). Virus inoculation also induced elevated TNF-alpha mRNA and protein levels (P < 0.05) in cultured rat alveolar macrophages (NR8383 cells). A 55-kd soluble TNF receptor-immunoglobulin G fusion protein (sTNFR-IgG) was used to inhibit TNF-alpha bioactivity in virus-inoculated BN rats. Treated rats had fewer proliferating bronchiolar fibroblasts, as detected by bromodeoxyuridine incorporation, compared with virus-inoculated control rats (P < 0.05). There was also increased mortality in p55sTNFR-IgG-treated virus-inoculated rats associated with increased viral replication and decreased numbers of macrophages and lymphocytes in bronchoalveolar lavage fluid (P < 0.05). The results of this study indicate that 1) Sendai virus can directly up-regulate TNF-alpha mRNA and protein expression in macrophages, 2) TNF-alpha is an important mediator of virus-induced bronchiolar fibrosis, and 3) TNF-alpha has a critical role in the termination of Sendai viral replication in the lung.     

Increased expression of tumor necrosis factor-alpha in diabetic macrovasculopathy.

Large vessel disease, a common feature of diabetes mellitus, appears to run an aggressive course, but its cellular and molecular aspects remain partially elucidated. Although in common atherosclerosis and especially in other forms of accelerated vasculopathy, immunoinflammatory mechanisms participate in the disease process, it is unclear whether this is present in diabetic vasculopathy. We hypothesized that diabetic macrovasculopathy, compared with classical atherosclerosis, is associated with increased immunoinflammatory features and matrix accumulation. In this study, vessel segments obtained after lower-limb amputation for advanced atherosclerotic disease, from type 2 diabetic patients (n = 20; 68.9+/-10.9 years) and nondiabetic patients (n = 16; 67.1+/-14.6 years) were analyzed. Histological characteristics (extent of intimal proliferation, cellularity, and fibrosis) were semiquantitatively graded in the two lesion types. Using immunohistochemistry, the presence of T cells and macrophages, accumulation of fibronectin, and expression of tumor necrosis factor-alpha was also assessed. Histological features of these advanced atherosclerotic lesions were similar in the two lesions examined. By immunohistochemistry, a similar pattern of T-cell and macrophage infiltration and fibronectin accumulation was observed. Nevertheless, increased expression of tumor necrosis factor-alpha was observed in diabetic lesions (13/19 patients had positive staining), whereas only 2 of 16 lesions from nondiabetic patients had positive staining (p < 0.003), with an odds ratio of 15.17 (confidence interval 2.12-139.5). These data suggest that increased expression of tumor necrosis factor-alpha observed in the diabetic lesions may reflect an enhanced inflammatory activity associated with the development of vascular lesions in type 2 diabetic patients.     

Differential inhibition of human secretory and cytosolic phospholipase A2

The roles and relative contributions of secretory and cytosolic phospholipases A₂ in physiology and pathology are not precisely known. In a search for differential inhibitors of these enzymes, which could serve as tools to clarify this issue, we evaluated the potencies of reference compounds and three series of new compounds, viz. substrate analogues, 1,2-amino alcohols and enolized -tricarbonyl derivatives, as inhibitors of secretory phospholipase A₂ from human polymorphonuclear leukocytes (sPLA₂) and of cytosolic phospholipase A₂ from human U937 cells (cPLA₂). With few exceptions, the compounds selected are potent inhibitors of sPLA₂ with IC₅₀ values (concentration inhibiting 50%) in the low micromolar range. Inhibition of cPLA₂ was only observed with some phosphate-free substrate analogues, with 1,2-amino alcohols and two of seven reference compounds. These results suggest that inhibition of secretory and of cytosolic phospholipases A₂ are independent effects. Several inhibitors could be identified with a marked selectivity for sPLA₂.This work has been presented in part at the Symposium Phospholipase A₂, Basic and Clinical Aspects in Inflammatory Diseases Reisensburg Castle (FRG), November 18–20, 1992.     

Interleukin-4 differentially regulates tumor necrosis factor-alpha gene expression by human T lymphocytes and monocytes.

Tumor necrosis factor-alpha (TNF-alpha), a product of both mononuclear phagocytes and T lymphocytes, is an important proximal mediator of a number of acute and chronic inflammatory disease states. In this investigation we examine the regulatory effects of the lymphocyte product interleukin-4 (IL-4) on the gene expression of TNF-alpha from stimulated human peripheral blood monocytes (PBM) and T lymphocytes. We demonstrated the dose-dependent suppression of TNF-alpha mRNA and protein synthesis from lipopolysaccharide-treated PBM by IL-4. The suppressive effects of IL-4 appear to be dependent upon de novo protein synthesis, as cycloheximide abrogated the IL-4-induced reduction in TNF-alpha mRNA levels from PBM. In contrast to the suppressive effects of IL-4 on PBM-derived cytokine expression, IL-4 did not alter TNF-alpha mRNA expression from alpha-Cd3 or PMA + alpha-CD-28-treated T lymphocytes. Moreover, IL-2 mRNA expression from similarly treated T lymphocytes was unaltered by IL-4. Our findings demonstrate that disparity exists in the regulation of TNF-alpha gene expression from different immune cell populations which may have important implications in the evolution of acute and chronic inflammatory responses.     

Labour is associated with increased expression of type-IIA secretory phospholipase A2 but not type-IV cytosolic phospholipase A2 in human myometrium

Human labour is associated with increased prostaglandin synthesis within the uterus. The aim of this study was to examine the expression of the type-IV cytosolic phospholipase A2 (cPLA2-IV) and the type IIA secretory phospholipase A2 (sPLA2-IIA) in myometrium in association with labour onset at term and preterm deliveries. These enzymes are important for the release of the prostaglandin precursor, arachidonic acid, from phospholipid membrane stores. RT-PCR was used to determine differences in gene expression between non-labour and labour groups. Expression of sPLA2-IIA in human myometrium was significantly increased with pregnancy, and with labour, both at term and preterm. Expression of cPLA2-IV in myometrium was not significantly altered with respect to pregnancy or labour. Immunohistochemical analysis demonstrated differences in the spatial localization of cPLA2-IV and sPLA2-IIA protein in upper and lower segment myometrium. cPLA2-IV was predominantly in vascular endothelial cells, while sPLA2-IIA was observed in vascular, endothelial and smooth muscle cells. In addition, sPLA2-IIA showed a distinct nuclear or perinuclear localization in myometrial smooth muscle cells of the lower segment. We postulate that the increased expression of sPLA2-IIA rather than cPLA2-IV in the myometrium may play a role in the onset and/or maintenance of human parturition.     

Monocyte-macrophage differentiation induced by human upper airway epithelial cells.

We examined the ability of conditioned medium (CM) generated by human upper airway epithelial (Ep) cells from normal (NN) and inflamed, allergic rhinitis (AR) and nasal polyp (NP) tissues to induce monocytic differentiation of hemopoietic progenitors of the HL-60 myeloid leukemia cell line in vitro. In HL-60 cells cultured in RPMI with 10% FBS, there was differentiation to 0.4 +/- 0.4% monocytic cells. NN-, AR-, and NP-EpCM induced differentiation to 23 +/- 6%, 42 +/- 11%, and 71 +/- 10% monocytic cells, respectively. EpCM also induced isolated peripheral blood nonadherent mononuclear cells to express monocyte/macrophage-specific antigens as detected by immunohistochemistry using FMC-32 monoclonal antibodies (anti-CD14). We also examined the cytokine content of these EpCMs and found that they contained granulocyte/macrophage colony-stimulating factor (GM-CSF): 126 +/- 35, 198 +/- 22, and 489 +/- 118 pg/ml for NN-, AR-, and NP-EpCM, respectively. These CMs also contained granulocyte-CSF (G-CSF) and interleukin-6 (IL-6), but there were no significant differences between normal and inflamed tissue-derived cell supernatants. No macrophage-CSF (M-CSF) was detected in these EpCMs. Recombinant human GM-CSF, G-CSF, and IL-6, alone and in combinations, at doses similar to or greater than those found in the EpCMs, did not induce comparable monocytic differentiation of HL-60 cells. Preincubation of the EpCM with neutralizing anti-GM-CSF, anti-G-CSF, or anti-IL-6 antibodies did not significantly inhibit the monocytic differentiation induced by the EpCM.(ABSTRACT TRUNCATED AT 250 WORDS)     

TNF-alpha induces the late-phase airway hyperresponsiveness and airway inflammation through cytosolic phospholipase A(2) activation

BACKGROUND: Late-phase airway hyperresponsiveness (AHR) in asthma is considered the event leading to persistent inflammation in the lungs, but the molecular mechanisms involved in this process are poorly understood. OBJECTIVE: To examine the role of TNF-alpha in the development of a late AHR and airway inflammation in asthma. METHODS: We established a murine model of asthma with not only biphasic AHR to methacholine but also airway eosinophilia. The effect of TNF-alpha blockade was determined by using anti-TNF-alpha antibody and TNF-alpha knockout mice. Cytosolic phospholipase A(2) (cPLA(2)) mRNA expression and activity were assessed by using RT-PCR and 1-stearoyl-2-[1-(14)C] arachidonyl-sn-glycero-3-phosphocholine as the substrate, respectively. RESULTS: TNF-alpha blockade resulted in significant inhibition of the late AHR without affecting the early AHR, and reduction in airway eosinophilia and inflammation. cPLA(2) activity was increased in asthmatic lungs in a TNF-alpha-dependent way, and cPLA(2) inhibitor blocked late AHR and airway eosinophilia. TNF-alpha also stimulated the synthesis of cPLA(2) metabolites such as leukotriene B(4) and platelet-activating factor in the airway. Specific inhibitors of cPLA(2) metabolites inhibited the late AHR and airway eosinophilia. CONCLUSIONS: TNF-alpha is the proximal key cytokine capable of developing late-phase AHR and subsequent airway inflammation through expression/activation of cPLA(2).     

Neuronal damage by secretory phospholipase A2: modulation by cytosolic phospholipase A2, platelet-activating factor,and cyclooxygenase-2 in neuronal cells in culture

Activation of cytosolic phospholipase A(2) (cPLA(2)) is an early event in brain injury, which leads to the formation and accumulation of bioactive lipids: platelet-activating factor (PAF), free arachidonic acid, and eicosanoids. A cross-talk between secretory PLA(2) (sPLA(2)) and cPLA(2) in neural signal transduction has previously been suggested (J Biol Chem 271:32722; 1996). Here we show, using neuronal cell cultures, an up-regulation of cPLA(2) expression and an inhibition by the selective cPLA(2) inhibitor AACOCF3 after exposure to neurotoxic concentrations of sPLA(2)-OS2. Pretreatment of neuronal cultures with recombinant PAF acetylhydrolase (rPAF-AH) or the presynaptic PAF receptor antagonist, BN52021, partially blocked neuronal cell death induced by sPLA(2)-OS2. Furthermore, selective COX-2 inhibitors ameliorated sPLA(2)-OS2-induced neurotoxicity. We conclude that sPLA(2)-OS2 activates a neuronal signaling cascade that includes activation of cPLA(2), arachidonic acid release, PAF production, and induction of COX-2.     

Detection of in vivo production of tumour necrosis factor-alpha by human thyroid epithelial cells.

We have established previously that human thyroid epithelial cells (TEC) from patients with autoimmune thyroiditis are able to synthesize cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6). This paper examines TEC in sections from autoimmune thyroiditis for the in vivo production of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) using the combined techniques of in situ hybridization and immunohistochemistry. Thyroid tissue from patients with Graves' disease, Hashimoto's disease and non-toxic goitre was examined and both mRNA and the protein of TNF-alpha were detected in TEC on frozen sections. Representative figures of only Graves' samples are illustrated in this paper. In contrast, using the same methods, IFN-gamma was detected only in the infiltrating cells and not in TEC of thyroid tissue from the patients.