Interleukin-32 induced thymic stromal lymphopoietin plays a critical role in the inflammatory response in human corneal epithelium

Interleukin (IL)-32, a novel cytokine, participates in a variety of inflammatory disorders. Thymic stromal lymphopoietin (TSLP) plays important roles in mucosal epithelial cells, especially in allergy-induced inflammation, through the TSLP-TSLPR (thymic stromal lymphopoietin receptor) signalling pathway. However, the association of IL-32 with TSLP on the ocular surface remains unclear. The present work aimed to assess the functional association of IL-32 with TSLP in the control of pro-inflammatory cytokine levels in the corneal epithelium. Human corneal tissue specimens and human corneal epithelial cells (HCECs) were administered different concentrations of IL-32 in the presence or absence of various inhibitors to assess TSLP levels and localization, as well as the molecular pathways that control pro-inflammatory cytokine production. TSLP mRNA levels were determined by real time RT- PCR, while protein levels were quantitated by ELISA and immunohistochemical staining. TSLP protein expression was examined in donor corneal epithelium samples. IL-32 significantly upregulated TSLP and pro-inflammatory cytokines (TNFα and IL-6) in HCECs at the gene and protein levels. The production of pro-inflammatory molecules by IL-32 was increased by recombinant TSLP. Interestingly, both NF-κB (quinazoline) and caspase-1 (VX-765) inhibitors suppressed the IL-32-related upregulation of pro-inflammatory cytokines (TNFα and IL-6). These findings demonstrate that IL-32 and IL-32-induced-TSLP are critical cytokines that participate in inflammatory responses through the caspase-1 and NF-κB signalling pathways in the corneal epithelium, suggesting new molecular targets for inflammatory diseases of the ocular surface. The effects of IL-32 on cell proliferation and apoptosis were investigated by MTT assays and RT-PCR,respectively. The results demonstrated that IL-32 inhibits cells apoptosis in HCECs.     

Comparison of nasal and bronchial epithelial cells obtained from patients with COPD

For in vitro studies of airway pathophysiology, primary epithelial cells have many advantages over immortalised cell lines. Nasal epithelial cells are easier to obtain than bronchial epithelial cells and can be used as an alternative for in vitro studies. Our objective was to compare nasal and bronchial epithelial cells from subjects with COPD to establish if these cells respond similarly to pro-inflammatory stimuli. Cell cultures from paired nasal and bronchial brushings (21 subjects) were incubated with cigarette smoke extract (CSE) prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide. IL-6 and IL-8 were measured by ELISA and Toll-like receptor 4 (TLR-4) message and expression by RT-PCR and FACS respectively. IL-8 release correlated significantly between the two cell types. IL-6 secretion was significantly less from bronchial compared to nasal epithelial cells and secreted concentrations did not correlate. A 4 h CSE incubation was immunosuppressive for both nasal and bronchial cells, however prolonged incubation for 24 h was pro-inflammatory solely for the nasal cells. CSE reduced TLR-4 expression in bronchial cells only after 24 h, and was without effect on mRNA expression. In subjects with COPD, nasal epithelial cells cannot substitute for in vitro bronchial epithelial cells in airway inflammation studies.     

Lactoferrin downregulates pro-inflammatory cytokines upexpressed in intestinal epithelial cells infected with invasive or noninvasive Escherichia coli strains.

Intestinal epithelial cells are able to differentially interact with commensal or pathogenic microorganisms, triggering a physiological or destructive inflammation, respectively. To mimic commensal-enteroinvasive bacteria-host cell interaction, we infected Caco-2 cells with noninvasive Escherichia coli HB101 and with recombinant invasive E. coli HB101(pRI203). Using DNA microarray mRNA profiling and ELISA assays, we studied the expression of several cytokine and cytokine-related genes in infected Caco-2 cells in the absence or presence of bovine lactoferrin (bLf). Infection of Caco-2 cells with the noninvasive strain induced a slight increase in the expression of interleukin 8 (IL-8), whereas infection with invasive E. coli HB101(pRI203) induced a significant increase in the expression of IL-8 as well as other pro-inflammatory cytokines. The addition of bLf, in native- or holo-form, did not influence expression of cytokine genes by uninfected Caco-2 cells, but it decreased expression of IL-8 by cells infected with E.coli HB101. Moreover, except for IL-8, bLfs dramatically downregulated pro-inflammatory cytokines upexpressed by Caco-2 cells infected with the invasive strain. Although IL-8 was decreased by bLfs, it remained upregulated, suggesting that it could be a signal of persistence of intracellular bacteria. The bLf ability to reduce expression of some pro-inflammatory cytokines, which appears independent of its iron saturation, might represent an important natural mechanism in regulating epithelial cell responses to pathogenic bacteria and in limiting cell damage and the spread of infections.     

Galectin-4 controls intestinal inflammation by selective regulation of peripheral and mucosal T cell apoptosis and cell cycle

Galectin-4 is a carbohydrate-binding protein belonging to the galectin family. Here we provide novel evidence that galectin-4 is selectively expressed and secreted by intestinal epithelial cells and binds potently to activated peripheral and mucosal lamina propria T-cells at the CD3 epitope. The carbohydrate-dependent binding of galectin-4 at the CD3 epitope is fully functional and inhibited T cell activation, cycling and expansion. Galectin-4 induced apoptosis of activated peripheral and mucosal lamina propria T cells via calpain-, but not caspase-dependent, pathways. Providing further evidence for its important role in regulating T cell function, galectin-4 blockade by antisense oligonucleotides reduced TNF-alpha inhibitor induced T cell death. Furthermore, in T cells, galectin-4 reduced pro-inflammatory cytokine secretion including IL-17. In a model of experimental colitis, galectin-4 ameliorated mucosal inflammation, induced apoptosis of mucosal T-cells and decreased the secretion of pro-inflammatory cytokines. Our results show that galectin-4 plays a unique role in the intestine and assign a novel role of this protein in controlling intestinal inflammation by a selective induction of T cell apoptosis and cell cycle restriction. Conclusively, after defining its biological role, we propose Galectin-4 is a novel anti-inflammatory agent that could be therapeutically effective in diseases with a disturbed T cell expansion and apoptosis such as inflammatory bowel disease.     

IL-10 protects mouse intestinal epithelial cells from Fas-induced apoptosis via modulating Fas expression and altering caspase-8 and FLIP expression

We have previously shown that the absence of Fas/Fas ligand significantly reduced tissue damage and intestinal epithelial cell (IEC) apoptosis in an in vivo model of T cell-mediated enteropathy. This enteropathy was more severe in IL-10-deficient mice, and this was associated with increased serum levels of IFN-gamma and TNF-alpha and an increase in Fas expression on IECs. In this study, we investigated the potential of IL-10 to directly influence Fas expression and Fas-induced IEC apoptosis. Mouse intestinal epithelial cell lines MODE-K and IEC4.1 were cultured with IFN-gamma, TNF-alpha, or anti-Fas monoclonal antibody (mAb) in the presence or absence of IL-10. Fas expression and apoptosis were determined by FACScan analysis of phycoerythrin-anti-Fas mAb staining and annexin V staining, respectively. Treatment with a combination of IFN-gamma and TNF-alpha induced significant apoptosis. Anti-Fas mAb alone did not induce much apoptosis unless cells were pretreated with IFN-gamma and TNF-alpha. These IECs constitutively expressed low levels of Fas, which significantly increased by preincubation of the cells with IFN-gamma and TNF-alpha. Treatment with cytokine or cytokine plus anti-Fas mAb increased apoptosis, which correlated with a decreased Fas-associated death domain IL-1-converting enzyme-like inhibitory protein (FLIP) level, increased caspase-8 activity, and subsequently increased caspase-3 activity. IL-10 diminished both cytokine- and anti-Fas mAb-induced apoptosis, and this was correlated with decreased cytokine-induced Fas expression, increased FLIP, and decreased caspase-8 and caspase-3 activity. In conclusion, IL-10 modulated cytokine induction of Fas expression on IEC cell lines and regulated IEC susceptibility to TNF-alpha, IFN-gamma, and Fas-mediated apoptosis. These findings suggest that IL-10 directly modulates IEC responses to T cell-mediated apoptotic signals.     

Interleukin (IL)-4/IL-9 and exogenous IL-16 induce IL-16 production by BEAS-2B cells, a bronchial epithelial cell line

Previous studies have suggested that bronchial epithelial cells may perpetuate airway inflammation. We have reported that the bronchial epithelial cell line BEAS-2B can produce interleukin (IL)-16, a potent chemoattractant for CD4+ T cells. IL-16 is thought to regulate airway inflammation in asthmatics. Recent studies showed that IL-4 induces inflammatory cytokines in bronchial epithelial cells and that IL-9 is a candidate gene for development of asthma. The present study demonstrated that BEAS-2B cells produced specifically IL-16 by synergistic effects of IL-4 + IL-16, or IL-9 + IL-16, and that the synthesized IL-16 induced migration of CD4+ T cells. This study is a first report indicating that IL-16 production may be maintained by an autocrine machinery by epithelial cell-derived IL-16 with IL-4 and IL-9 in asthma.     

IL-12 plays a significant role in the apoptosis of human T cells in the absence of antigenic stimulation

Interleukin-12 (IL-12) is an immunoregulatory cytokine that plays an essential role in cell-mediated immunity. It is known to induce T cell apoptosis in in vivo systems such as graft-versus-host disease (GVHD) and experimental autoimmune uveitis (EAU). However, the role of IL-12 in T cell apoptosis in the absence of antigenic stimulation has not been clearly defined. This study was conducted to investigate whether IL-12, in the absence of an antigen, is able to induce T cell apoptosis, and also, which signalling pathways utilized by IL-12 are involved in this process. Our data clearly showed that IL-12 in the absence of an antigen induces apoptosis in T cells. Flow cytometry and ELISA showed FasL up-regulation and increased IFN-gamma synthesis in IL-12 treated T cells, while Fas and TNF-R1 showed little change. Semi-quantitative RT-PCR demonstrated that IL-12 was able to up-regulate TNF-alpha and FasL mRNA expression. Furthermore, IL-12 induced apoptosis was associated with caspase-3, caspase-2, caspase-7, DNA fragmentation factor 45 (DFF45) and Fas associated death domain (FADD) whereas TNF receptor associated death domain (TRADD) and receptor interacting protein (RIP) were not. Inhibition of Janus tyrosine kinase (JAK) was able to suppress IL-12 induced T cell apoptosis. Anti-FasL antibody was able to block IL-12 induced T cell apoptosis. In conclusion, our findings suggest that IL-12 is able to induce T cell apoptosis in the absence of an antigen. In addition, the present data suggest that this process is FasL mediated and caspase-3 dependent. Furthermore, JAK was shown to be involved in this process. These results may have significant implications in the understanding of IL-12 mediated T cell apoptosis.     

Toll-like receptor 2 is upregulated by hog confinement dust in an IL-6-dependent manner in the airway epithelium

Hog confinement workers are at high risk to develop chronic bronchitis as a result of their exposure to organic dust. Chronic bronchitis is characterized by inflammatory changes of the airway epithelium. A key mediator in inflammation is Toll-like receptor 2 (TLR2). We investigated the role of TLR2 in pulmonary inflammation induced by hog confinement dust. Normal human bronchial epithelial cells (NHBE) were grown in culture and exposed to hog confinement dust extract. Hog confinement dust upregulated airway epithelial cell TLR2 mRNA in a concentration- and time-dependent manner using real-time PCR. There was a similar increase in TLR2 protein at 48 h as shown by Western blot. TLR2 was upregulated on the surface of airway epithelial cells as shown by flow cytometry. A similar upregulation of pulmonary TLR2 mRNA and protein was shown in a murine model of hog confinement dust exposure. Hog confinement dust is known to stimulate epithelial cells to produce IL-6. To determine whether TLR2 expression was being regulated by IL-6, the production of IL-6 was blocked using an IL-6-neutralizing antibody. This resulted in attenuation of the dust-induced upregulation of TLR2. To further demonstrate the importance of IL-6 in the regulation of TLR2, NHBE were directly stimulated with recombinant human IL-6. IL-6 alone was able to upregulate TLR2 in airway epithelial cells. Hog confinement dust upregulates TLR2 in the airway epithelium through an IL-6-dependent mechanism.     

Subcellular expression pattern and role of IL-15 in pneumococci induced lung epithelial apoptosis

Streptococcus pneumoniae is the leading causative agent of community-acquired pneumonia. Induction of apoptosis in pulmonary epithelial cells by bacteria during pneumonia might be harmful to the host. Interleukin-15 (IL-15) has been demonstrated as an effective inhibitor of apoptosis and is expressed in lung epithelium on the mRNA and protein level. Therefore, we characterized the sub-cellular expression pattern of the short and long IL-15 isoforms in lung epithelial cells in vitro as well as its role in pneumococci-related lung epithelial cell apoptosis. We found an expression pattern for both IL-15 signal peptides in the pulmonary epithelial cell lines A549 and Beas-2B. Moreover, a strong co-localization of IL-15 and IL-15Ralpha was detected on cell surfaces. Compared to pro-inflammatory cytokine stimulation, neither IL-15 nor its trimeric receptor complex was up-regulated after pneumococcal infection. However, overexpression of IL-15 isoforms revealed IL-15LSP and IL-15Vkl as inhibitors of pneumococci induced apoptosis in pulmonary epithelial cells. Thus, IL-15 may act as an anti-apoptotic molecule in pneumococci infection, thereby suggesting IL-15 as a benefical cytokine in pulmonary host defense against infection.     

Up-regulation of production of TGF-beta and IL-4 and down-regulation of IL-6 by apoptotic human bronchial epithelial cells

Human bronchial epithelial cells secrete cytokines that play a role in immune responses in the lung. However, the roles of these cytokines in regulating epithelial repair following acute lung injury are largely unknown. Responses to injury include hyperplasia of epithelial cells and squamous metaplasia. The resolution stage is characterized by discontinuation of hyperplasia. Apoptosis is considered to be the most efficient mechanism of removal of unwanted cells without causing inflammation. The presence of TGF-beta1 increases apoptosis, induces squamous metaplasia and inhibits proliferation of airway epithelial cells. Interleukin-4 increases the ability of macrophages to phagocytose epithelial cells and produce inflammatory cytokines. The purpose of this study was to investigate the hypothesis that apoptotic lung epithelial cells produce cytokines, which could act in an autocrine manner to control hyperplasia and induce squamous differentiation following acute lung injury. A bronchial epithelial cell line (16 HBE) was used as an in vitro model, to study the production of TGF-beta, IL-4 and IL-6 by lung epithelial cells undergoing apoptosis. Apoptotic and live cells were sorted on the basis of bright and negative staining with FITC-conjugated Annexin V, respectively. Intracellular IL-6, TGF-beta and IL-4 was measured using flow cytometric techniques. Electron microscopy, immunohistochemistry and ELISA were used as supportive techniques. Apoptotic cells produced significantly more TGF-beta and IL-4 (but less IL-6) than viable cells. Increased production of TGF-beta and IL-4 by epithelial cells undergoing apoptosis may contribute to the inhibition of proliferation, squamous metaplasia, and reduction of the inflammatory response in acute lung injury.     

Apoptosis induced by filarial parasitic sheath protein in HEp 2 cell lines blocked by ectopic expression of bcl 2.

Chronic filarial patients exhibit an occult manifestation, Tropical Pulmonary Eosinophilia, (TPE), caused by an exaggerated immune response to shed and circulating filarial antigens, leading to extensive lung damage. We have attempted to examine the disease in vitro using the human epithelial cell line, HEp2. Filarial sheath proteins induce apoptosis in HEp2 cells characterized by chromatin condensation, internucleosomal DNA cleavage, positive staining for TUNEL assay and shows a sub-G1 peak on FACS analysis. In order to understand subcellular events and to analyse the protective role of bcl2, we engineered HEp2 to overexpress Bcl2 protein. HEp2 bcl2 cells do not undergo apoptosis on exposure to filarial sheath protein, indicating that filarial protein-induced apoptosis in epithelial cells proceeds via a pathway, inhibitable by overexpression of bcl 2.     

Effect of cytokines on ICAM-1 and ZO-1 expression on human airway epithelial cells

The presence of adhesion molecules on airway epithelial cells may be important in recruiting leukocytes to the epithelium. The study aimed at investigating the effects of interleukin (IL)-4, IL-8, IL-13 and interferon-gamma (IFN-gamma) on cell viability and intracellular adhesion molecule (ICAM)-1 and zonula occludens protein (ZO)-1 expression on cultured human basal and columnar airway epithelial cells. Cycloheximide (CHX) induced cell death in both cell lines. The cytokines IL-4, IL-8, IL-13 and IFN-gamma had only minor effects on cell proliferation in the columnar 16HBE14o-cells, and inhibited the effects of CHX on cell death. IFN-gamma increased ICAM-1 expression in both cell lines. Western blot analysis showed that CHX inhibited both ICAM-1 and ZO-1 expression in the basal cell line. A combination of IL-4 and IFN-gamma appeared to break the tight junctions. IL-4 and IL-13 potentiated CHX-induced apoptosis in basal cells but not in columnar cells, possibly due to low levels of the IL-4 receptor. It is concluded that cytokines produced by airway epithelium may have a role in regulating sequestering of leukocytes to the airways during airway inflammation.     

Inhibition of Nox2 oxidase activity ameliorates influenza A virus-induced lung inflammation

Influenza A virus pandemics and emerging anti-viral resistance highlight the urgent need for novel generic pharmacological strategies that reduce both viral replication and lung inflammation. We investigated whether the primary enzymatic source of inflammatory cell ROS (reactive oxygen species), Nox2-containing NADPH oxidase, is a novel pharmacological target against the lung inflammation caused by influenza A viruses. Male WT (C57BL/6) and Nox2(-/y) mice were infected intranasally with low pathogenicity (X-31, H3N2) or higher pathogenicity (PR8, H1N1) influenza A virus. Viral titer, airways inflammation, superoxide and peroxynitrite production, lung histopathology, pro-inflammatory (MCP-1) and antiviral (IL-1β) cytokines/chemokines, CD8(+) T cell effector function and alveolar epithelial cell apoptosis were assessed. Infection of Nox2(-/y) mice with X-31 virus resulted in a significant reduction in viral titers, BALF macrophages, peri-bronchial inflammation, BALF inflammatory cell superoxide and lung tissue peroxynitrite production, MCP-1 levels and alveolar epithelial cell apoptosis when compared to WT control mice. Lung levels of IL-1β were ～3-fold higher in Nox2(-/y) mice. The numbers of influenza-specific CD8+D(b)NP(366)+ and D(b)PA(224)+ T cells in the BALF and spleen were comparable in WT and Nox2(-/y) mice. In vivo administration of the Nox2 inhibitor apocynin significantly suppressed viral titer, airways inflammation and inflammatory cell superoxide production following infection with X-31 or PR8. In conclusion, these findings indicate that Nox2 inhibitors have therapeutic potential for control of lung inflammation and damage in an influenza strain-independent manner.     

Apoptosis resistance in epithelial tumors is mediated by tumor-cell-derived interleukin-4

We investigated the mechanisms involved in the resistance to cell death observed in epithelial cancers. Here, we identify that primary epithelial cancer cells from colon, breast and lung carcinomas express high levels of the antiapoptotic proteins PED, cFLIP, Bcl-xL and Bcl-2. These cancer cells produced interleukin-4 (IL-4), which amplified the expression levels of these antiapoptotic proteins and prevented cell death induced upon exposure to TRAIL or other drug agents. IL-4 blockade resulted in a significant decrease in the growth rate of epithelial cancer cells and sensitized them, both in vitro and in vivo, to apoptosis induction by TRAIL and chemotherapy via downregulation of the antiapoptotic factors PED, cFLIP, Bcl-xL and Bcl-2. Furthermore, we provide evidence that exogenous IL-4 was able to upregulate the expression levels of these antiapoptotic proteins and potently stabilized the growth of normal epithelial cells rendering them apoptosis resistant. In conclusion, IL-4 acts as an autocrine survival factor in epithelial cells. Our results indicate that inhibition of IL-4/IL-4R signaling may serve as a novel treatment for epithelial cancers.     

Differential effect of IL-18 on endothelial cell apoptosis mediated by TNF-alpha and Fas (CD95)

Interleukin-18 (IL-18) is a newly identified cytokine with proinflammatory activity. Numerous studies have shown that proinflammatory cytokines may regulate endothelial cells (EC) apoptosis mediated by members of the tumor necrosis factor (TNF) family, such as TNF-alpha and Fas. In this study we hypothesized that IL-18 may regulate the susceptibility of liver endothelial cells (LEC) to apoptosis induced by TNF and Fas. IL-18 increased the susceptibility of LEC to undergo apoptosis mediated by TNF but not by Fas. Since TNF-induced apoptosis is mediated by the type I TNF receptor (TNFRI), we investigated up-regulation of this receptor in IL-18-treated LEC. IL-18 induced up-regulation of the TNFRI on the surface of LEC. Partial blocking of LEC apoptosis induced by IL-18 and TNF was observed when the cells were pretreated with the broad-spectrum inhibitor of caspases z-VAD-fmk, suggesting involvement of the caspase pathway in apoptosis induced by these cytokines in these cells. Our results show that IL-18 differentially regulates apoptosis mediated by the death-inducing factors, TNF and Fas. To our knowledge, this is the first report that IL-18 may regulate endothelial cell apoptosis mediated by TNF. These results may have clinical implications in those clinical hepatic conditions associated with high levels of IL-18 and TNF.     

TWEAK/Fn14 interaction stimulates human bronchial epithelial cells to produce IL-8 and GM-CSF

TNF-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) family, is a multifunctional cytokine that regulates cellular proliferation, angiogenesis, inflammation, and apoptosis. In this study, we investigated the effect of TWEAK on human bronchial epithelial cells. A human bronchial epithelial cell line, BEAS2B, expressed a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14), and produced IL-8 and GM-CSF upon TWEAK stimulation in a dose-dependent manner, which was abrogated by anti-Fn14 blocking antibody. TWEAK induced phosphorylation of IkappaBalpha and BAY11-7082, a selective inhibitor of IkappaBalpha phosphorylation, inhibited the TWEAK-induced IL-8 and GM-CSF production by BEAS2B cells. Moreover, primary cultured human bronchial epithelial cells also expressed Fn14 and produced IL-8 and GM-CSF upon TWEAK stimulation. Collectively, TWEAK stimulated human bronchial epithelial cells to produce IL-8 and GM-CSF through Fn14. Because IL-8 and GM-CSF are associated with inflammatory conditions, these results suggest that TWEAK/Fn14 interaction may play some roles in airway inflammatory responses.     

Seminal plasma induces the expression of IL-1α in normal and neoplastic cervical cells via EP2/EGFR/PI3K/AKT pathway

Background

Cervical cancer is a chronic inflammatory disease of multifactorial etiology usually presenting in sexually active women. Exposure of neoplastic cervical epithelial cells to seminal plasma (SP) has been shown to promote the growth of cancer cells in vitro and tumors in vivo by inducing the expression of inflammatory mediators including pro-inflammatory cytokines. IL-1α is a pleotropic pro-inflammatory cytokine induced in several human cancers and has been associated with virulent tumor phenotype and poorer prognosis. Here we investigated the expression of IL-1α in cervical cancer, the role of SP in the regulation of IL-1α in neoplastic cervical epithelial cells and the molecular mechanism underlying this regulation.

Methods and results

Real-time quantitative RT-PCR confirmed the elevated expression of IL-1α mRNA in cervical squamous cell carcinoma and adenocarcinoma tissue explants, compared with normal cervix. Using immunohistochemistry, IL-1α was localized to the neoplastically transformed squamous, columnar and glandular epithelium in all cases of squamous cell carcinoma and adenocarcinomas explants studied. We found that SP induced the expression of IL-α in both normal and neoplastic cervical tissue explants. Employing HeLa (adenocarcinoma) cell line as a model system we identified PGE₂ and EGF as possible ligands responsible for SP-mediated induction of IL-1α in these neoplastic cells. In addition, we showed that SP activates EP2/EGFR/PI3kinase-Akt signaling to induce IL-1α mRNA and protein expression. Furthermore, we demonstrate that in normal cervical tissue explants the induction of IL-1α by SP is via the activation of EP2/EGFR/PI3 kinase-Akt signaling.

Conclusion

SP-mediated induction of IL-1α in normal and neoplastic cervical epithelial cells suggests that SP may promote cervical inflammation as well as progression of cervical cancer in sexually active women.