TGFβ1 induces IL‐6 and inhibits IL‐8 release in human bronchial epithelial cells: The role of Smad2/3

Human bronchial epithelial (HBE) cells contribute to asthmatic airway inflammation by secreting cytokines, chemokines, and growth factors, including interleukin (IL)‐6, IL‐8 and transforming growth factor (TGF) β1, all of which are elevated in asthmatic airways. This study examines the signaling pathways leading to TGFβ1 induced IL‐6 and IL‐8 in primary HBE cells from asthmatic and non‐asthmatic volunteers. HBE cells were stimulated with TGFβ1 in the presence or absence of signaling inhibitors. IL‐6 and IL‐8 protein and mRNA were measured by ELISA and real‐time PCR respectively, and cell signaling kinases by Western blot. TGFβ1 increased IL‐6, but inhibited IL‐8 production in both asthmatic and non‐asthmatic cells; however, TGF induced significantly more IL‐6 in asthmatic cells. Inhibition of JNK MAP kinase partially reduced TGFβ1 induced IL‐6 in both cell groups. TGFβ1 induced Smad2 phosphorylation, and blockade of Smad2/3 prevented both the TGFβ1 modulated IL‐6 increase and the decrease in IL‐8 production in asthmatic and non‐asthmatic cells. Inhibition of Smad2/3 also increased basal IL‐8 release in asthmatic cells but not in non‐asthmatic cells. Using CHIP assays we demonstrated that activated Smad2 bound to the IL‐6, but not the IL‐8 promoter region. We conclude that the Smad2/3 pathway is the predominant TGFβ1 signaling pathway in HBE cells, and this is altered in asthmatic bronchial epithelial cells. Understanding the mechanism of aberrant pro‐inflammatory cytokine production in asthmatic airways will allow the development of alternative ways to control airway inflammation. J. Cell. Physiol. 225: 846–854, 2010. © 2010 Wiley‐Liss, Inc.     

Inflammatory cytokines can enhance CD44-mediated airway epithelial cell adhesion independently of CD44 expression

In airways, the cell surface molecule CD44 is upregulated on bronchial epithelial cells in areas of damage. We have shown that a blocking standard CD44 (CD44s) antibody caused a 77% (+/- 19%) inhibition of cell migration at 3 h after mechanical damage and decreased epithelial cell repair of cells grown on cell culture filter inserts. With the use of primary human bronchial epithelial cells and the bronchial epithelial cell line 16HBE 14o-, a CD44s antibody inhibited >95% (P < 0.01) of cell binding to hyaluronic acid (HA). The cytokines TNF-alpha, IFN-gamma, IL-1 beta, and IL-4 stimulated a 2- to 3.5-fold increase in CD44-dependent cell binding to HA. IFN-gamma treatment did not increase CD44 expression as assessed by flow cytometry, although phorbol myristate acetate treatment did. This indicates that IFN-gamma-induced cell binding to HA did not require increased CD44 expression. These data indicate that CD44 is important for bronchial epithelial cell binding to HA and that cytokines known to be expressed in inflammation can increase HA binding independently of the level of CD44 expression.     

Hsp72 induces inflammation and regulates cytokine production in airway epithelium through a TLR4- and NF-kappaB-dependent mechanism

Heat shock proteins are generally regarded as intracellular proteins acting as molecular chaperones; however, Hsp72 is also detected in the extracellular compartment. Hsp72 has been identified in the bronchoalveolar lavage fluid (BALF) of patients with acute lung injury. To address whether Hsp72 directly activated airway epithelium, human bronchial epithelial cells (16HBE14o-) were treated with recombinant Hsp72. Hsp72 induced a dose-dependent increase in IL-8 expression, which was inhibited by the NF-kappaB inhibitor parthenolide. Hsp72 induced activation of NF-kappaB, as evidenced by NF-kappaB trans-activation and by p65 RelA and p50 NF-kappaB1 binding to DNA. Endotoxin contamination of the Hsp72 preparation was not responsible for these effects. Next, BALB/c mice were challenged with a single intratracheal inhalation of Hsp72 and killed 4 h later. Hsp72 induced significant up-regulation of KC, TNF-alpha, neutrophil recruitment, and myeloperoxidase in the BALF. A similar challenge with Hsp72 in TLR4 mutant mice did not stimulate the inflammatory response, stressing the importance of TLR4 in Hsp72-mediated lung inflammation. Last, cultured mouse tracheal epithelial cells (MTEC) from BALB/c and TLR4 mutant and wild-type mice were treated ex vivo with Hsp72. Hsp72 induced a significant increase in KC expression from BALB/c and wild-type MTEC in an NF-kappaB-dependent manner; however, TLR4 mutant MTEC had minimal cytokine release. Taken together, these data suggest that Hsp72 is released and biologically active in the BALF and can regulate airway epithelial cell cytokine expression in a TLR4 and NF-kappaB-dependent mechanism.     

Cigarette Smoke Modulates Repair and Innate Immunity following Injury to Airway Epithelial Cells

Cigarette smoking is the main risk factor associated with chronic obstructive pulmonary disease (COPD), and contributes to COPD development and progression by causing epithelial injury and inflammation. Whereas it is known that cigarette smoke (CS) may affect the innate immune function of airway epithelial cells and epithelial repair, this has so far not been explored in an integrated design using mucociliary differentiated airway epithelial cells. In this study, we examined the effect of whole CS exposure on wound repair and the innate immune activity of mucociliary differentiated primary bronchial epithelial cells, upon injury induced by disruption of epithelial barrier integrity or by mechanical wounding. Upon mechanical injury CS caused a delayed recovery in the epithelial barrier integrity and wound closure. Furthermore CS enhanced innate immune responses, as demonstrated by increased expression of the antimicrobial protein RNase 7. These differential effects on epithelial repair and innate immunity were both mediated by CS-induced oxidative stress. Overall, our findings demonstrate modulation of wound repair and innate immune responses of injured airway epithelial cells that may contribute to COPD development and progression.     

Peroxisome proliferator-activated receptor-gamma in cystic fibrosis lung epithelium

The pathophysiology of cystic fibrosis (CF) inflammatory lung disease is not well understood. CF airway epithelial cells respond to inflammatory stimuli with increased production of proinflammatory cytokines as a result of increased NF-kappaB activation. Peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibits NF-kappaB activity and is reported to be reduced in CF. If PPARgamma participates in regulatory dysfunction in the CF lung, perhaps PPARgamma ligands might be useful therapeutically. Cell models of CF airway epithelium were used to evaluate PPARgamma expression and binding to NF-kappaB at basal and under conditions of inflammatory stimulation by Pseudomonas aeruginosa or TNFalpha/IL-1beta. An animal model of CF was used to evaluate the potential of PPARgamma agonists as therapeutic agents in vivo. In vitro, PPARgamma agonists reduced IL-8 and MMP-9 release from airway epithelial cells in response to PAO1 or TNFalpha/IL-1beta stimulation. Less NF-kappaB bound to PPARgamma in CF than normal cells, in two different assays; PPARgamma agonists abrogated this reduction. PPARgamma bound less to its target DNA sequence in CF cells. To test the importance of the reported PPARgamma inactivation by phosphorylation, we observed that inhibitors of ERK, but not JNK, were synergistic with PPARgamma agonists in reducing IL-8 secretion. In vivo, administration of PPARgamma agonists reduced airway inflammation in response to acute infection with P. aeruginosa in CF, but not wild-type, mice. In summary, PPARgamma inhibits the inflammatory response in CF, at least in part by interaction with NF-kappaB in airway epithelial cells. PPARgamma agonists may be therapeutic in CF.     

Glycosylation profiles of airway epithelium after repair of mechanical injury in guinea pigs

Glycosylated structures on the cell surface have a role in cell adhesion, migration, and proliferation. Repair of the airway epithelium after injury requires each of these processes, but the expression of cell surface glycosylation of airway epithelial cells after injury is not known. We examined cell surface glycosylation using lectin-binding profiles of normal and repairing epithelia in Hartley guinea pigs from 0 to 14 days after mechanical injury. The epithelium regenerated completely over 7 days. In normal trachea, galactose- or galactosamine-specific lectins (14 of 20 tested) labelled epithelial cells, but fucose, mannose, and other sugar-specific lectins (15 tested) did not. GSA-2, a glucosamine-specific lectin, labelled epithelial cells weakly in uninjured tracheas, but intense labelling was noted in basal and non-ciliated columnar cells adjacent to the injury site over 3h to 14 days after injury. Labelling of these cells peaked at 12h and 5 days after injury respectively. Similar patterns were seen with lectins AlloA and HAA but not with CPA during repair. The binding of the lectin DSA to proteins collected from primary cultures of airway epithelial cells decreased substantially after treatment for 24h with either transforming growth factor- or interleukin-1, but that of the CPA lectin did not. We demonstrate changes in glycosylation profiles of airway epithelial cells coordinate with repair after mechanical injury. These changes may be useful to study mechanisms by which repair is regulated.     

Hepatocyte growth factor and other fibroblast secretions modulate the phenotype of human bronchial epithelial cells

The luminal airway surface is lined with epithelial cells that provide a protective barrier from the external environment and clear inhaled pathogens from the lung. To accomplish this important function, human bronchial epithelial (HBE) cells must be able to rapidly regenerate a mucociliary layer of cells following epithelial injury. Whereas epithelial-fibroblast interactions are known to modulate the airway architecture during lung development and repair, little is known about how these two cells interact. Using a primary HBE and lung fibroblast coculture system, we demonstrate that 1) subepithelial fibroblasts provide a suitable environment for differentiation of HBE cells into a polarized ciliated phenotype despite being cultured in media that induces terminal squamous differentiation and growth arrest in the absence of fibroblasts, 2) HBE cells cocultured with subepithelial fibroblasts exhibit augmented ciliogenesis, accelerated wound repair, and diminished polarized ion transport compared with cells grown in control conditions, and 3) hepatocyte growth factor (HGF) is important for subepithelial fibroblast modulation of HBE cell differentiation. These results provide a model to study fibroblast modulation of epithelial phenotype and indicate that HGF secreted by subepithelial fibroblasts contributes to HBE cell differentiation.     

Barrier Disrupting Effects of Alternaria Alternata Extract on Bronchial Epithelium from Asthmatic Donors

Sensitization and exposure to the allergenic fungus Alternaria alternata has been associated with increased risk of asthma and asthma exacerbations. The first cells to encounter inhaled allergens are epithelial cells at the airway mucosal surface. Epithelial barrier function has previously been reported to be defective in asthma. This study investigated the contribution of proteases from Alternaria alternata on epithelial barrier function and inflammatory responses and compared responses of in vitro cultures of differentiated bronchial epithelial cells derived from severely asthmatic donors with those from non-asthmatic controls. Polarised 16HBE cells or air-liquid interface (ALI) bronchial epithelial cultures from non-asthmatic or severe asthmatic donors were challenged apically with extracts of Alternaria and changes in inflammatory cytokine release and transepithelial electrical resistance (TER) were measured. Protease activity in Alternaria extracts was characterised and the effect of selectively inhibiting protease activity on epithelial responses was examined using protease inhibitors and heat-treatment. In 16HBE cells, Alternaria extracts stimulated release of IL-8 and TNFα, with concomitant reduction in TER; these effects were prevented by heat-treatment of the extracts. Examination of the effects of protease inhibitors suggested that serine proteases were the predominant class of proteases mediating these effects. ALI cultures from asthmatic donors exhibited a reduced IL-8 response to Alternaria relative to those from healthy controls, while neither responded with increased thymic stromal lymphopoietin (TSLP) release. Only cultures from asthmatic donors were susceptible to the barrier-weakening effects of Alternaria. Therefore, the bronchial epithelium of severely asthmatic individuals may be more susceptible to the deleterious effects of Alternaria.     

Wound repair and anti-oxidative capacity is regulated by ITGB4 in airway epithelial cells

Integrin beta 4 (ITGB4) is a structural adhesion molecule which engages in maintaining the integrity of airway epithelial cells. Its specific cytomembrane structural feature strongly indicates that ITGB4 may engage in many signaling pathways and physiologic processes. However, in addition to adhesion, the specific biologic significance of ITGB4 in airway epithelial cells is almost unknown. In this article, we investigated the expression and functional properties of ITGB4 in airway epithelial cells in vivo and in vitro. Human bronchial epithelial cell line (16HBE14O-cells) and primary rat tracheal epithelial cells (RTE cells) were used to determine ITGB4 expression under ozone tress or mechanical damage, respectively. An ovalbumin (OVA)-challenged asthma model was used to investigate ITGB4 expression after antigen exposure in vivo. In addition, an ITGB4 overexpression vector and ITGB4 silence virus vector were constructed and transfected into RTE cells. Then, wound repair ability and anti-oxidation capacity was evaluated. Our results demonstrated that, on the edge of mechanically wounded cell areas, ITGB4 expression was increased after mechanical injury. After ozone stress, upregulation expression of ITGB4 was also detected. In the OVA-challenged asthma model, ITGB4 expression was decreased on airway epithelial cells accompanying with structural disruption and damage of anti-oxidation capacity. Besides, our study revealed that upregulation of ITGB4 promotes wound repair ability and anti-oxidative ability, while such abilities were blocked when ITGB4 was silenced. Taken together, these results showed that ITGB4 was a new interesting molecule involved in the regulation of wound repair and anti-oxidation processes for airway epithelial cells.     

Lipopolysaccharide-induced cytokine expression in alveolar epithelial cells: role of PKCζ-mediated p47phox phosphorylation

Chronic inflammation incited by bacteria in the saccular lung of premature infants contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). LPS-mediated type II alveolar epithelial cell (AEC) injury induces the expression of pro-inflammatory cytokines that trigger pulmonary neutrophil influx, alveolar matrix degradation and lung remodeling. We hypothesized that NADPH oxidase (Nox)-dependent mechanisms mediate LPS-induced cytokine expression in AEC. We examined the role of p47phox in mediating LPS-dependent inflammatory cytokine expression in A549 cells (which exhibit phenotypic features characteristic of type II AEC) and elucidated the proximal signaling events by which Nox is activated by LPS. LPS-induced ICAM-1 and IL-8 expression was associated with increased superoxide formation in AEC. LPS-mediated oxidative stress and cytokine expression was inhibited by apocynin and augmented by PMA demonstrating that Nox-dependent redox signaling regulates LPS-dependent pro-inflammatory signaling in AEC. In LPS-treated cells, p47phox translocated from the cytoplasm to the perinuclear region and co-localized with gp91phox. LPS also induced a temporal increase in p47phox serine304 phosphorylation in AEC. While inhibition of classical PKC and novel PKC with calphostin and rottlerin did not inhibit ICAM-1 or IL-8 expression, the myristolyated PKCζ pseudosubstrate peptide (a specific inhibitor of PKCζ) inhibited LPS-induced cytokine expression in AEC. Inhibition of PKCζ also attenuated LPS-mediated p47phox phosphorylation and perinuclear translocation in AEC. Consistent with these data, LPS activated PKCζ in AEC as evidenced by increased threonine410 phophorylation. We conclude that PKCζ-mediated p47phox activation regulates LPS-dependent cytokine expression in AEC. Selective inhibition of PKCζ or p47phox might attenuate LPS-mediated inflammation and alveolar remodeling in BPD.     

Effect of the alpha3beta1 integrin on the IL-1 stimulated activation of c-Jun N-terminal kinase (JNK) in CACO-2 cells

Intestinal epithelial cells (IEC) are capable of responding to IL-1 stimulation by producing a variety of pro-inflammatory cytokines. Recently, we have found that binding of the alpha3beta1 integrin may have a regulatory effect on IL-1 responses and intracellular signaling by suppressing cytokine secretion, mRNA expression and the downstream intracellular signaling events from IKK to NF-kappaB activation. In this study, we extend these findings by showing that treatment of the Caco-2 epithelial cells with a cross-linking anti-alpha3 integrin antibody resulted in a suppression in the levels of IL-1 induced AP-1 binding activity in nuclear extracts. Furthermore, suppressed levels of IL-1 induced c-Jun N-terminal kinase (JNK) phosphorylation and kinase activity were seen with the antibody treated cells. Cells cultured on purified laminin-5, the ligand for the alpha3beta1 integrin, did not show significantly elevated levels of JNK phosphorylation after IL-1 stimulation while cells cultured on fibronectin yielded significantly elevated levels of IL-1 induced JNK phosphorylation. These results indicate that binding of the alpha3beta1 integrin results in a suppression in the activation of the IL-1 induced intracellular signaling pathway from JNK to AP-1. This novel regulatory effect may be a potentially important mechanism to regulate IL-1 mediated responses by IEC.     

Cystic fibrosis airway epithelial cell polarity and bacterial flagellin determine host response to Pseudomonas aeruginosa

The role of epithelial polarity and bacterial factors in the control of the innate immune response of airway epithelial cells to Pseudomonas aeruginosa PAK was investigated using a human, nasal cystic fibrosis (DeltaF508/DeltaF508) epithelial cell line CF15 grown as confluent layers on permeable supports. Addition of PAK to the basal surface of CF15 layers caused significant expression changes in 1525 different genes (out of 12 625 examined), including the cytokines IL-6, IL-8, IL-1beta and TNF-alpha, as well as genes associated with leucocyte adhesion, antibacterial factors, and NF-kappaB signalling. Confocal microscopy showed that nuclear migration of NF-kappaB in all CF15 cells was preceded by PAK binding to the basal and lateral surfaces of some cells. Addition of PAK to the apical surface of CF15 monolayers elicited changes in expression of only 602 genes, including 256 not affected during basolateral PAK exposure. Over time, cytokine expression during apical PAK was similar to that exhibited by basal PAK, but the magnitudes during apical treatment were much smaller with little/no nuclear migration of NF-kappaB in CF15 cells. Furthermore, these responses depended on the presence of flagellin, but not pili on the bacteria. Thus, P. aeruginosa triggered a strong innate immune response that depended on the apical versus basolateral polarity of CF15 cells and the presence of flagellin on the bacteria.     

糖原合酶激酶3β和腺瘤性结肠息肉病蛋白在气道上皮细胞机械损伤修复过程的时空分布

为了探讨糖原合酶激酶3β（glycogen synthase kinase 3β,GSK3β）和腺瘤性结肠息肉病（adenomatous polyposis coli, APC）蛋白在气道上皮细胞（airway epithelial cells,AECs）损伤和修复中的作用,我们采用机械划线损伤的方法建立体外气道上皮损伤修复模型,采用Western blot、免疫荧光双标共聚焦成像和免疫沉淀的方法观察损伤修复过程中APC蛋白和GSK3β在AECs中表达及分布的动态变化。结果显示：（1）用Western blot方法观察到划线损伤0．5 h后即有GSK3β磷酸化增强（P〈 0．05）,6 h达到高峰（P〈0．05）,持续到12 h（P〈0．05）,24 h开始下降,而GSK3β总量大致保持一致。（2）在免疫荧光双标共聚焦成像实验中,划线损伤0 h组APC蛋白主要表达于胞浆,而划线损伤6 h后APC蛋白主要聚集于损伤前沿区的迁移活跃细胞。（3）免疫共沉淀的实验结果显示,划线损伤0 h时GSK3B和APC蛋白能共同沉淀,但在划线损伤6 h之后,两者发生了分离。以上结果表明：划线损伤后AECs立即启动修复过程,此时GSK3B的活性被抑制,促使APC蛋白游离出来;游离出来的APC蛋白则与微管正极结合,增加了微管的稳定性,从而调节细胞骨架运动,促进气道上皮的损伤修复。     

SHP-1 as a critical regulator of Mycoplasma pneumoniae-induced inflammation in human asthmatic airway epithelial cells

Asthma is a chronic inflammatory disease in which airway epithelial cells are the first line of defense against exposure of the airway to infectious agents. Src homology protein (SHP)-1, a protein tyrosine phosphatase, is a negative regulator of signaling pathways that are critical to the development of asthma and host defense. We hypothesize that SHP-1 function is defective in asthma, contributing to the increased inflammatory response induced by Mycoplasma pneumoniae, a pathogen known to exacerbate asthma. M. pneumoniae significantly activated SHP-1 in airway epithelial cells collected from nonasthmatic subjects by bronchoscopy with airway brushing but not in cells from asthmatic subjects. In asthmatic airway epithelial cells, M. pneumoniae induced significant PI3K/Akt phosphorylation, NF-κB activation, and IL-8 production compared with nonasthmatic cells, which were reversed by SHP-1 overexpression. Conversely, SHP-1 knockdown significantly increased IL-8 production and PI3K/Akt and NF-κB activation in the setting of M. pneumoniae infection in nonasthmatic cells, but it did not exacerbate these three parameters already activated in asthmatic cells. Thus, SHP-1 plays a critical role in abrogating M. pneumoniae-induced IL-8 production in nonasthmatic airway epithelial cells through inhibition of PI3K/Akt and NF-κB activity, but it is defective in asthma, resulting in an enhanced inflammatory response to infection.     

Interleukin-1beta augments in vitro alveolar epithelial repair

Biologically active interleukin (IL)-1beta is present in the pulmonary edema fluid obtained from patients with acute lung injury and has been implicated as an important early mediator of nonpulmonary epithelial wound repair. Therefore, we tested the hypothesis that IL-1beta would enhance wound repair in cultured monolayers from rat alveolar epithelial type II cells. IL-1beta (20 ng/ml) increased the rate of in vitro alveolar epithelial repair by 118 +/- 11% compared with that in serum-free medium control cells (P < 0.01). IL-1beta induced cell spreading and migration at the edge of the wound but not proliferation. Neutralizing antibodies to epidermal growth factor (EGF) and transforming growth factor-alpha or inhibition of the EGF receptor by tyrphostin AG-1478 or genistein inhibited IL-1beta-induced alveolar epithelial repair, indicating that IL-1beta enhances in vitro alveolar epithelial repair by an EGF- or transforming growth factor-alpha-dependent mechanism. Moreover, the mitogen-activated protein kinase pathway is involved in IL-1beta-induced alveolar epithelial repair because inhibition of extracellular signal-regulated kinase activation by PD-98059 inhibited IL-1beta-induced alveolar epithelial repair. In conclusion, IL-1beta augments in vitro alveolar epithelial repair, indicating a possible novel role for IL-1beta in the early repair process of the alveolar epithelium in acute lung injury.     

Phosphatidylinositol 3-kinase is required for rhinovirus-induced airway epithelial cell interleukin-8 expression

Rhinovirus (RV) is a common cause of asthma exacerbations. The signaling mechanisms regulating RV-induced airway epithelial cell responses have not been well studied. We examined the role of phosphatidylinositol (PI) 3-kinase in RV-induced interleukin (IL)-8 expression. Infection of 16HBE14o- human bronchial epithelial cells with RV39 induced rapid activation of PI 3-kinase and phosphorylation of Akt, a downstream effector of PI 3-kinase. RV39 also colocalized with cit-Akt-PH, a citrogen-tagged fluorescent fusion protein encoding the pleckstrin homology domain of Akt, indicating that 3-phosphorylated PI accumulates at the site of RV infection. Inhibition of PI 3-kinase and Akt attenuated RV39-induced NF-kappaB transactivation and IL-8 expression. Inhibition of PI 3-kinase also blocked internalization of labeled RV39 into 16HBE14o- cells, suggesting that the requirement of PI 3-kinase for RV39-induced IL-8 expression, at least in part, relates to its role in viral endocytosis.