Tumor necrosis factor-alpha inhibits aquaporin 5 expression in mouse lung epithelial cells

Aquaporin 5 (AQP5), the major water channel expressed in alveolar, tracheal, and upper bronchial epithelium, is significantly down-regulated during pulmonary inflammation and edema. The mechanisms that underlie this decrease in AQP5 levels are therefore of considerable interest. Here we show that AQP5 expression in cultured lung epithelial cells is decreased 2-fold at the mRNA level and 10-fold at the protein level by the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). Treatment of murine lung epithelial cells (MLE-12) with TNF-alpha results in a concentration- and time-dependent decrease in AQP5 mRNA and protein expression. Activation of the p55 TNF-alpha receptor (TNFR1) with an agonist antibody is sufficient to cause decreased AQP5 expression, demonstrating that the TNF-alpha effect is mediated through TNFR1. Inhibition of nuclear factor kappaB (NF-kappaB) translocation to the nucleus blocks the effect of TNF-alpha on AQP5 expression, indicating that activation of NF-kappaB is required, whereas inhibition of extracellular signal-regulated or p38 mitogen-activated protein kinases showed no effect. These data show that TNF-alpha decreases AQP5 mRNA and protein expression and that the molecular pathway for this effect involves TNFR1 and activated NF-kappaB. The ability of inflammatory cytokines to decrease aquaporin expression may help explain the connection between inflammation and edema.     

Aquaporin 5 increases keratinocyte-derived chemokine expression and NF-κB activity through ERK activation

Aquaporin-5 (AQP5) is a water-selective channel protein that is expressed in submucosal glands and alveolar epithelial cells in the lungs. Recent studies have revealed that AQPs regulate not only water metabolism, but also some cellular functions such as cell growth and migration. Here, we report the role of AQP5 in inflammatory responses. In MLE-12 cells, knockdown of AQP5 using siRNA (10–50 nM) attenuated TNF-α-induced expression of keratinocyte chemoattractant (KC) mRNA and protein. Conversely, in NIH-3T3 cells, overexpression of AQP5 increased KC expression, NF-κB activation, and ERK phosphorylation. The AQP5-induced increase of KC expression was diminished by treatment with ERK inhibitors. Taken together, we propose a new function of AQP5 as an inflammatory signal potentiator, which may be mediated by increased activation of ERK and NF-κB.     

Antisense-mediated inhibition of ICAM-1 expression: a therapeutic strategy against inflammation of human periodontal tissue

Periodontal diseases, such as gingivitis and periodontitis, are caused by a mixed infection by several types of bacteria in the dental plaque, causing a chronic inflammation of the gingival mucosa. Inflammatory processes in conjunction with immune responses to bacterial attacks are generally protective. In profound periodontitis, however, hyperresponsiveness and hypersensitivity of the immune system are counterproductive because of the destruction of the affected periodontal connective tissues. The intercellular adhesion molecule type 1 (ICAM-1) plays a key role in the onset and manifestation of inflammatory responses. Thus, inhibition of ICAM-1 expression could be of therapeutic relevance for the treatment of destructive periodontitis. Here, antisense oligonucleotides (AS-ON) directed against ICAM-1 suppress protein expression and mRNA levels specifically and effectively in primary human endothelial cells of different tissue origin. Moreover, downregulation of ICAM-1 expression is also observed in AS-ON-transfected inflamed gingival mucosal tissue of patients with periodontal diseases. This work strongly suggests exploiting the local topical application of ICAM-1-directed AS-ON as a therapeutic tool against inflammatory processes of the human gingiva.     

Knockdown of TNFR1 by the sense strand of an ICAM-1 siRNA: dissection of an off-target effect

Tumor necrosis factor (TNF) initiates local inflammation by triggering endothelial cells (EC) to express adhesion molecules for leukocytes such as intercellular adhesion molecule-1 (ICAM-1 or CD54). A prior study identified siRNA molecules that reduce ICAM-1 expression in cultured human umbilical vein EC (HUVEC). One of these, ISIS 121736, unexpectedly inhibits TNF-mediated up-regulation of additional molecules on EC, including E-selectin (CD62E), VCAM-1 (CD106) and HLA-A,B,C. 736 siRNA transfection was not toxic for EC nor was there any evidence of an interferon response. 736 Transfection of EC blocked multiple early TNF-related signaling events, including activation of NF-κB. IL-1 activation of these same pathways was not inhibited. A unifying explanation is that 736 siRNA specifically reduced expression of mRNA encoding tumor necrosis factor receptor 1 (TNFR1) as well as TNFR1 surface expression. A sequence with high identity to the 736 antisense strand (17 of 19 bases) is present within the 3′UTR of human TNFR1 mRNA. An EGFP construct incorporating the 3′UTR of TNFR1 was silenced by 736 siRNA and this effect was lost by mutagenesis of this complementary sequence. Chemical modification and mismatches within the sense strand of 736 also inhibited silencing activity. In summary, an siRNA molecule selected to target ICAM-1 through its antisense strand exhibited broad anti-TNF activities. We show that this off-target effect is mediated by siRNA knockdown of TNFR1 via its sense strand. This may be the first example in which the off-target effect of an siRNA is actually responsible for the anticipated effect by acting to reduce expression of a protein (TNFR1) that normally regulates expression of the intended target (ICAM-1).     

Haemophilus influenzae stimulates ICAM-1 expression on respiratory epithelial cells

Epithelial cells interact directly with bacteria in the environment and play a critical role in airway defense against microbial pathogens. In this study, we examined the response of respiratory epithelial cells to infection with nontypable Haemophilus influenzae. Using an in vitro cell culture model, we found that epithelial cell monolayers released significant quantities of IL-8 and expressed increased levels of ICAM-1 mRNA and surface protein in response to H. influenzae. In contrast, levels of IL-1beta, TNF-alpha, and MHC class I were not significantly affected, suggesting preferential activation of a specific subset of epithelial genes directed toward defense against bacteria. Induction of ICAM-1 required direct bacterial interaction with the epithelial cell surface and was not reproduced by purified H. influenzae lipooligosaccharide. Consistent with a functional role for this response, induction of ICAM-1 by H. influenzae mediated increased neutrophil adherence to the epithelial cell surface. Furthermore, in an in vivo murine model of airway infection with H. influenzae, increased epithelial cell ICAM-1 expression coincided with increased chemokine levels and neutrophil recruitment in the airway. These results indicate that ICAM-1 expression on human respiratory epithelial cells is induced by epithelial cell interaction with H. influenzae and suggest that an ICAM-1-dependent mechanism can mediate neutrophil adherence to these cells independent of inflammatory mediator release by other cell types. Direct induction of specific epithelial cell genes (such as ICAM-1 and IL-8) by bacterial infection may allow for rapid and efficient innate defense in the airway.     

The presence of chemokine (MCP-1, MIP-1alpha,MIP-1beta,IP-10, RANTES)-positive cells and chemokine receptor (CCR5, CXCR3)-positive cells in inflamed human gingival tissues

Chemokines are said to be small peptides that are chemoattractants for leukocyte subpopulations within local inflammation sites. Gingival inflammation is characterized by infiltration of inflammatory mononuclear cells. The point of this study was to examine the presence or absence of chemokine-positive cells and chemokine receptor-positive cells by means of immunohistochemical methods in samples of gingival tissues obtained from patients with marginal periodontitis. Macrophage chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, (IFN-gamma)-inducible protein-10 (IP-10) and RANTES-producing cells were found to be present in inflamed human gingival tissues. In addition, CCR5- and CXCR3-positive cells were present. In contrast, no factor expression was observed in periodontally healthy gingival tissue. Our findings suggest that these chemokines may be responsible for modulating the process of infectious disease such as marginal periodontitis.     

Porphyromonas gingivalis-mediated shedding of extracellular matrix metalloproteinase inducer (EMMPRIN) by oral epithelial cells: a potential role in inflammatory periodontal disease

Extracellular matrix metalloproteinase inducer (EMMPRIN) or CD147 is a transmembrane glycoprotein expressed by various cell types, including oral epithelial cells. Recent studies have brought evidence that EMMPRIN plays a role in periodontitis. In the present study, we investigated the effect of Porphyromonas gingivalis, a major pathogen in chronic periodontitis, on the shedding of membrane-anchored EMMPRIN and on the expression of the EMMPRIN gene by oral epithelial cells. A potential contribution of shed EMMPRIN to the inflammatory process of periodontitis was analyzed by evaluating the effect of recombinant EMMPRIN on cytokine and matrix metalloproteinase (MMP) secretion by human gingival fibroblasts. ELISA and immunofluorescence analyses revealed that P. gingivalis mediated the shedding of epithelial cell-surface EMMPRIN in a dose- and time-dependent manner. Cysteine proteinase (gingipain)-deficient P. gingivalis mutants were used to demonstrate that both Arg- and Lys-gingipain activities are involved in EMMPRIN shedding. Real-time PCR showed that P. gingivalis had no significant effect on the expression of the EMMPRIN gene in epithelial cells. Recombinant EMMPRIN induced the secretion of IL-6 and MMP-3 by gingival fibroblasts, a phenomenon that appears to involve mitogen activated protein kinases. The present study brought to light a new mechanism by which P. gingivalis can promote the inflammatory response during periodontitis.     

Proinflammatory cytokines induce proteinase 3 as membrane-bound and secretory forms in human oral epithelial cells and antibodies to proteinase 3 activate the cells through protease-activated receptor-2

Anti-neutrophil cytoplasmic Abs targeting proteinase 3 (PR3) have been detected in relation to a wide range of inflammatory conditions such as periodontitis, and interaction of anti-PR3 Abs with endothelial and epithelial cells provokes cell activation, although the underlying mechanism has been unclear. The present study showed that human oral epithelial cells expressed PR3 mRNA after treatment with proinflammatory cytokines such as IL-1alpha, TNF-alpha, IFN-alpha, IFN-beta, and IFN-gamma. A 29-kDa PR3 was expressed on the cell surface and released into culture supernatants by the cells upon stimulation with these cytokines. The membrane and supernatant fractions of oral epithelial cells exhibited enzymatic activity, which was inhibited by serine proteinase inhibitors, but not by a cysteine proteinase inhibitor or secretory leukocyte protease inhibitor. Addition of anti-PR3 Abs to cytokine-primed oral epithelial cells in culture induced remarkable secretion of IL-8 and monocyte chemoattractant protein 1 and aggregation of PR3 on the cells. RNA interference targeted to protease-activated receptor-2 mRNA and intracellular Ca2+ mobilization assays revealed that anti-PR3 Abs activated the epithelial cells through protease-activated receptor-2, a family of G protein-coupled receptors. The anti-PR3 Ab-mediated cell activation was completely abolished by RNA interference targeted to PR3 mRNA and by inhibition of phospholipase C and NF-kappaB. Immunohistochemistry showed that inflamed oral epithelium actually expresses PR3 protein. These results suggest that oral epithelial cells express functional PR3 in the inflamed sites and respond to anti-PR3 Abs detected in diseased sera, and that these mechanisms may actively participate in the inflammatory process, including periodontitis.     

TNF-alpha activation of arterioles and venules alters distribution and levels of ICAM-1 and affects leukocyte-endothelial cell interactions

The observation that leukocyte-endothelial cell (EC) interactions are localized to specific regions on the microvessel wall suggests that adhesion molecule distribution is not uniform. We investigated ICAM-1 distribution and leukocyte-EC interactions in blood-perfused microvessels (<80 mum) in cremaster muscle of anesthetized mice, using intravital confocal microscopy and immunofluorescent labeling. Variability of ICAM-1 expression directly determines leukocyte adhesion distribution within the venular microcirculation and contributes to leukocyte rolling in arterioles during inflammation. The number of rolling interactions increased with ICAM-1 intensity (r(2) = 0.69, P < 0.05), and rolling velocity was lower in regions of higher ICAM-1 intensity. In controls, venular ICAM-1 expression was approximately twofold higher than in arterioles. After TNF-alpha treatment, ICAM-1 expression was significantly increased, 2.8 +/- 0.2-fold in arterioles and 1.7 +/- 0.2-fold in venules (P < 0.05). ICAM-1 expression on activated arteriolar ECs only reached the level of control venular ICAM-1. Arteriolar but not venular ECs underwent redistribution of ICAM-1 among cells; some cells increased and some decreased ICAM-1 expression, magnifying the variability of ICAM-1. TNF-alpha treatment increased the length of bright fluorescent regions per unit vessel length (42%, control; 70%, TNF-alpha) along the arteriolar wall, whereas no significant change was observed in venules (60%, control; 63%, TNF-alpha). The spatial distribution and expression levels of adhesion molecules in the microcirculation determine the timing and placement of leukocyte interactions and hence significantly impact the inflammatory response. That arteriolar ECs respond to TNF-alpha by upregulation of ICAM-1, although in a different way compared with venules, suggests an explicit role for arterioles in inflammatory responses.     

TNF is a key mediator of septic encephalopathy acting through its receptor, TNF receptor-1

In this study, we demonstrate that mice deficient in TNFR1 (TNFR1(-/-)) were resistant to LPS-induced encephalopathy. Systemic administration of lipopolysaccharide (LPS) induces a widespread inflammatory response similar to that observed in sepsis. Following LPS administration TNFR1(-/-) mice had less caspase-dependent apoptosis in brain cells and fewer neutrophils infiltrating the brain (p<0.039), compared to control C57Bl6 (TNFR1(+/+)) mice. TNFR1-dependent increase in aquaporin (AQP)-4 mRNA and protein expression was observed with a concomitant increase in water content, in brain (18% increase in C57Bl6 mice treated with LPS versus those treated with saline), similar to cerebral edema observed in sepsis. Furthermore, absence of TNFR1 partially but significantly reduced the activation of astrocytes, as shown by immunofluorescence and markedly inhibited iNOS mRNA expression (p<0.01). Septic encephalopathy is a devastating complication of sepsis. Although, considerable work has been done to identify the mechanism causing the pathological alterations in this setting, the culprit still remains an enigma. Our results demonstrate for the first time that endotoxemia leads to inflammation in brain, with alteration in blood-brain barrier, up-regulation of AQP4 and associated edema, neutrophil infiltration, astrocytosis, as well as apoptotic cellular death, all of which appear to be mediated by TNF-alpha signaling through TNFR1.     

Retinoic acid potentiates TNF-alpha-induced ICAM-1 expression in normal human epidermal keratinocytes

ICAM-1 protein in keratinocytes is thought to contribute to cutaneous inflammatory reactions. Its induction depends-among others-on cytokines such as TNF-alpha, IFN-gamma, IL-1 or on retinoic acid (RA), a key regulator of epidermal homeostasis. We investigated the effect of treatments with TNF-alpha, RA or their combination on ICAM-1 expression on proliferative or differentiating keratinocytes over an 8 day culture period. Basal ICAM-1 levels were undetectable at low (30 microM) and standard (88 microM) Ca2+ and RA alone did not induce ICAM-1. However, at high Ca2+ (1500 microM), ICAM-1 levels were augmented in response to RA-treatment. TNF-alpha induced a transient ICAM-1 increase in NHK, which reached peak-levels 2-4 days post cytokine stimulus. RA potentiated the TNF-alpha-induced ICAM-1 response in all Ca2+-concentrations. This potentiating effect of RA was confirmed at the mRNA level. In summary, our results establish retinoic acid as an enhancer of TNF-alpha-induced ICAM-1 levels in NHK.     

TNF-α Mediates PKCδ/JNK1/2/c-Jun-Dependent Monocyte Adhesion via ICAM-1 Induction in Human Retinal Pigment Epithelial Cells

Retinal inflammatory diseases induced by cytokines, such as tumor necrosis factor-α (TNF-α) are associated with an up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the retinal pigment epithelial cells (RPECs). Retinal pigment epithelium (RPE) is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). However, the detailed mechanisms of TNF-α-induced ICAM-1 expression are largely unclear in human RPECs. We demonstrated that in RPECs, TNF-α could induce ICAM-1 protein and mRNA expression and promoter activity, and monocyte adhesion. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of PKCs (Ro318220), PKCδ (Rottlerin), MEK1/2 (U0126), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of TNFR1, TRAF2, JNK2, p42, or c-Jun. We showed that TNF-α could stimulate the TNFR1 and TRAF2 complex formation. TNF-α-stimulated JNK1/2 was also reduced by Rottlerin or SP600125. However, Rottlerin had no effect on TNF-α-induced p42/p44 MAPK phosphorylation. We observed that TNF-α induced c-Jun phosphorylation which was inhibited by Rottlerin or SP600125. On the other hand, TNF-α-stimulated ICAM-1 promoter activity was prominently lost in RPECs transfected with the point-mutated AP-1 ICAM-1 promoter plasmid. These results suggest that TNF-α-induced ICAM-1 expression and monocyte adhesion is mediated through a TNFR1/TRAF2/PKCδ/JNK1/2/c-Jun pathway in RPECs. These findings concerning TNF-α-induced ICAM-1 expression in RPECs imply that TNF-α might play an important role in ocular inflammation and diseases.     

Increased Eotaxin and MCP-1 Levels in Serum from Individuals with Periodontitis and in Human Gingival Fibroblasts Exposed to Pro-Inflammatory Cytokines

Periodontitis is a chronic inflammatory disease of tooth supporting tissues resulting in periodontal tissue destruction, which may ultimately lead to tooth loss. The disease is characterized by continuous leukocyte infiltration, likely mediated by local chemokine production but the pathogenic mechanisms are not fully elucidated. There are no reliable serologic biomarkers for the diagnosis of periodontitis, which is today based solely on the degree of local tissue destruction, and there is no available biological treatment tool. Prompted by the increasing interest in periodontitis and systemic inflammatory mediators we mapped serum cytokine and chemokine levels from periodontitis subjects and healthy controls. We used multivariate partial least squares (PLS) modeling and identified monocyte chemoattractant protein-1 (MCP-1) and eotaxin as clearly associated with periodontitis along with C-reactive protein (CRP), years of smoking and age, whereas the number of remaining teeth was associated with being healthy. Moreover, body mass index correlated significantly with serum MCP-1 and CRP, but not with eotaxin. We detected higher MCP-1 protein levels in inflamed gingival connective tissue compared to healthy but the eotaxin levels were undetectable. Primary human gingival fibroblasts displayed strongly increased expression of MCP-1 and eotaxin mRNA and protein when challenged with tumor necrosis factor-α (TNF-α and interleukin-1β (IL-1β), key mediators of periodontal inflammation. We also demonstrated that the upregulated chemokine expression was dependent on the NF-κΒ pathway. In summary, we identify higher levels of CRP, eotaxin and MCP-1 in serum of periodontitis patients. This, together with our finding that both CRP and MCP-1 correlates with BMI points towards an increased systemic inflammatory load in patients with periodontitis and high BMI. Targeting eotaxin and MCP-1 in periodontitis may result in reduced leukocyte infiltration and inflammation in periodontitis and maybe prevent tooth loss.     

Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through protease-activated receptor 2

Protease-activated receptors (PARs) compose a family of G protein-coupled receptors activated by proteolysis with exposure of their tethered ligand. Recently, we reported that a neutrophil-derived serine proteinase, proteinase 3 (PR3), activated human oral epithelial cells through PAR-2. The present study examined whether other neutrophil serine proteinases, human leukocyte elastase (HLE), and cathepsin G (Cat G) activate nonepithelial cells, human gingival fibroblasts (HGF). HLE and Cat G as well as PR3 activated HGF to produce IL-8 and monocyte chemoattractant protein 1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor, an inhibitor of HLE and Cat G, and recombinant secretory leukocyte protease inhibitor clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HGF express PAR-1 and PAR-2 mRNA in the cells and the proteins on the cell surface. HLE and Cat G cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca(2+) mobilization and rendered cells refractory to subsequent stimulation with HLE and Cat G. The production of cytokine induced by HLE and Cat G and the PAR-2 agonist peptide was completely abolished by inhibition of phospholipase C. These findings suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines and may control a number of inflammatory processes such as periodontitis.     

Enhanced expressions of endometrial tumour necrosis factor alpha and its receptors during early pregnancy in bonnet monkeys

Tumour necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine may play an active role in stimulating inflammatory reactions during pregnancy. However, the expression of endometrial TNF-alpha has not been investigated especially during early pregnancy, a phenomenon invariably accompanied by inflammatory reaction. In the present study, the endometrial expressions of TNF-alpha and its receptors (TNFR1 and TNFR2) during early pregnancy, when the embryo lies free in the zona hatched state in the uterine lumen, were analyzed by immunohistochemistry. The endometrial expressions of TNF-alpha, TNFR1 and TNFR2 were found to be significantly up-regulated (p < 0.05) in the glandular epithelium on day 6 post-ovulation in pregnant animals. The alteration in the expression of these molecules may contribute to the induction of local inflammatory reactions during implantation.