Swine dust induces cytokine secretion from human epithelial cells and alveolar macrophages

Exposure to swine dust causes airway inflammation with increased levels of proinflammatory cytokines, and inflammatory cells in nasal and bronchoalveolar lavage fluid (BALF) in healthy subjects. Earlier studies have suggested that lipopolysaccharides (LPS) might be an important proinflammatory factor in swine dust. Since respiratory epithelial cells and alveolar macrophages are target cells for the inhaled dust, we therefore compared the release of proinflammatory cytokines from normal human bronchial epithelial cells (NHBE), an epithelial cell line (A549) and from human alveolar macrophages obtained from BALF from healthy subjects in vitro after incubation with dust collected in swine houses or LPS. Swine dust or LPS was added to the wells with A549 cells or macrophages and incubated for 8 h at concentrations of 12.5, 25, 50 and 100 μg/ml. NHBE cells were incubated with swine dust at a concentration of 25, 50 or 100 μg/ml or with LPS at a concentration of 50 or 100 μg/ml and incubated for 24 h. The supernatants were collected, centrifuged, and IL-6, IL-1β and tumour necrosis factor-alpha (TNF-α) production was measured using an ELISA method and expressed per 10⁶ cells. Swine dust and LPS caused a dose-dependant increase of IL-6 production in NHBE cells, swine dust being more potent than LPS. In A549 cells, only swine dust, but not LPS caused an increase of IL-6 production. Neither swine dust nor LPS induced IL-1β or TNF-α release from A549 cells. Both swine dust and LPS caused a dose-dependent increase of IL-1β, IL-6 and TNF-α in alveolar macrophages. Swine dust which contained 2.2 (0.2) ng endotoxin/100 μg swine dust (0.02‰) was almost as potent as LPS in inducing cytokine release from alveolar macrophages in vitro. We conclude that both epithelial cells and alveolar macrophages have the capability to contribute to the release of proinflammatory cytokines following exposure to swine dust. Some agent(s) other than LPS in the dust contribute to the marked airway inflammatory reaction.     

Interleukin-8, interleukin-6, and soluble tumour necrosis factor receptor type I release from a human pulmonary epithelial cell line (A549) exposed to respiratory syncytial virus.

The release of interleukin-8 (IL-8), interleukin-6 (IL-6) and the soluble forms of the tumour necrosis factor receptor (sTNF-R) from human pulmonary type II-like epithelial cells (A549) after respiratory syncytial virus (RSV) infection was analysed. RSV infection alone induced a time- and RSV dose-dependent IL-8 and IL-6 release from A549 cells. Furthermore, the soluble form of the TNF-RI was also secreted in a time- and RSV dose-dependent fashion. The soluble TNF-RII was not detected in the cell supernatant of infected epithelial cells. The effect of various cytokines [IL-1 alpha/beta, TNF-alpha/beta, IL-3, IL-6, interferon-gamma (IFN-gamma), transforming growth factor-beta 2 (TGF-beta 2)] and colony-stimulating factors [granulocyte (G)-CSF; granulocyte-macrophage (GM)-CSF] on the IL-8 release from A549 cells was also studied. Our data show that the proinflammatory cytokines IL-1 alpha/beta and TNF-alpha/beta induced an IL-8 release in non-infected A549 cells, and increased the IL-8 release of RSV-infected A549 cells synergistically. In addition, IL-3, G-CSF, IFN-gamma and TGF-beta 2, albeit at high concentrations, induced a low IL-8 release from non-infected A549 cells. The enhanced IL-8 secretion rates were accompanied with elevated cytoplasmic IL-8 mRNA steady state levels, as was shown by Northern blot analysis. Cellular co-culture experiments performed with A549 cells and polymorphonuclear granulocytes or peripheral blood mononuclear cells revealed that increased IL-8 amounts were secreted in the co-culture of non-infected as well as RSV-infected cells. The present study suggests a central role for the airway epithelium during RSV infection with regard to cytokine and cytokine receptor release, resulting in a recruitment and activation of inflammatory and immune effector cells. Our data also suggest that paracrine cytokine networks and cell-cell contact are involved in the regulation of IL-8 secretion within the microenvironment of the bronchial epithelium.     

A critical role for interleukin-1 receptor accessory protein in interleukin-1 signaling

Interleukin-1 (IL-1) is a central molecule in inflammation and immune responses whose pleiotropic activities are mediated by the type I IL-1 receptor (IL-1RI). The IL-1RI alone on the cell surface is silent after binding of the ligand. We show that the recently identified IL-1RI accessory protein (IL-1RAcP) converts the silent into a fully functional IL-1RI complex. Although transfection of IL-1RAcP into IL-1RAcP-deficient EL4D6/76 cells did not alter the binding kinetics or dissociation constants of the ¹²⁵I-labeled IL-1α/IL-1RI complex, a very early event, internalization of the activated receptor complex, and a late event, IL-1-stimulated IL-2 production, were successfully restored. Therefore, recruitment of IL-1RAcP is a critical early step in the signaling cascade mediated by the IL-1RI activation complex.     

Immunocytochemical localization of cyclo-oxygenase isoforms in cultured human airway structural cells

BACKGROUND: Cyclo-oxygenase (COX) exists as two isoforms, COX-1, the constitutive isoform, and COX-2, which is inducible by cytokines or inflammatory stimuli and may participate in airway inflammation. OBJECTIVE: To determine the basal distribution of COX isoforms, and their regulation by interleukin-1 beta (IL-1beta), bradykinin (BK) and dexamethasone (Dex) in cultured airway structural cells. METHODS: We measured COX-1 and COX-2 in cultured human airway smooth muscle (HASM) cells, MRC5 fibroblasts and normal human epithelial cells (NHBE) using immunocytochemical analysis. RESULTS: The majority of all types of untreated cultured cells expressed COX-1 (75% of HASM, 75% of MRC5 fibroblasts and 72% of NHBE cells). Fibroblasts and smooth muscle cells showed low constitutive COX-2 expression (2 and 8%, respectively) but this was higher in NHBE cells (28%). IL-1beta (24 h incubation) or BK (4 h incubation) had no effect on COX-1 expression in any of the cells studied. In contrast, there was a two- and 1.5-fold rise in the percentage of NHBE cells expressing COX-2; a 7.5- and sixfold rise in the percentage of HASM cells expressing COX-2 and a 33.5- and 20.5-fold increase in the percentage of fibroblasts expressing COX-2 after IL-1beta or BK treatment, respectively. Pretreatment with dexamethasone abolished IL-1beta- and BK-stimulated COX-2 induction in all cells studied. CONCLUSION: COX-1 is expressed constitutively in human airway fibroblasts, smooth muscle and epithelial cells but epithelial cells also show constitutive expression of COX-2. Both IL-1beta and BK induced COX-2 expression in all cells studied and this induction was blocked by dexamethasone. Immunocytochemical techniques can be successfully used to detect the distribution of COX isoforms in cell cultures.     

Cytokine-stimulated human lung alveolar epithelial cells release eotaxin-2 (CCL24) and eotaxin-3 (CCL26).

Asthma is a complex inflammatory disease characterized by a prolonged underlying airway inflammation resulting from cytokine-orchestrated signaling between many types of cells, including airway epithelial cells. Trafficking, recruitment, and activation of cells in airway disease are, in part, modulated by the newly discovered CC subfamily of chemokines, eotaxin (CCL11), eotaxin-2 (CCL24) and eotaxin-3 (CCL26), which transduce signals by acting as agonists for the CCR3 receptor. The specific cytokine stimuli that modulate CCL24 and CCL26 release in airway epithelial cells remain poorly defined. Thus, human 549 alveolar type II epithelium-like cells were stimulated singly and with combinations of 1-100 ng/ml tumor necrosis-factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-4, cytokines known to be elevated in the airways of asthmatics. Release of CCL11, CCL24, and CCL26 was quantified by ELISA, and CCR3 receptors monitored by immunocytochemistry and FACS analysis. Results suggest that epithelial cells release CCL11 during the first 24 h of stimulation, in contrast to a significant increase in CCL24 and CCL26 release after 24-48 h of stimulation. Differential release of the eotaxins in response to cytokine combinations was noted. The alveolar type II epithelial cells were found to possess constitutive CCR3 receptors, which increased after proinflammatory cytokine stimulation. The airway epithelium CCR3 receptor/eotaxin ligand signal transduction system may be an important target for development of novel mechanism-based adjunctive therapies designed to interrupt the underlying chronic inflammation in allergic and inflammatory disorders.     

Interleukin-1β regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages

The success of Streptococcus pneumoniae (the pneumococcus) as a pulmonary pathogen is related to its restriction of innate immune responses by respiratory epithelial cells. The mechanisms used to overcome this restriction are incompletely elucidated. Pulmonary chemokine expression involves complex cellular and molecular networks, involving the pulmonary epithelium, but the specific cellular interactions and the cytokines that control them are incompletely defined. We show that serotype 2 or 4 pneumococci induce only modest levels of CXCL8 expression from epithelial cell lines, even in the absence of a polysaccharide capsule. In contrast, coculture of A549 cells with the macrophage-like THP-1 cell line, differentiated with vitamin D, or monocyte-derived macrophages enhanced CXCL8 release. Supernatants from the THP-1 cell line prime A549 cells to release CXCL8 at levels similar to cocultures. Interleukin-1Ra (IL-1Ra) inhibits CXCL8 release from cocultures and reduces the activity of macrophage-conditioned media, but inhibition of tumor necrosis factor alpha (TNF-α) had only a minimal effect on CXCL8 release. Release of IL-1β but not TNF-α was upregulated in cocultures. IL-1 type 1 receptor knockout C57BL/6 and BALB/c mice confirmed the importance of IL-1 signaling in CXC chemokine expression and neutrophil recruitment in vivo. In fulminant disease, increased IL-1 signaling resulted in increased neutrophils in the airway and more invasive disease. These results demonstrate that IL-1 is an important component of the cellular network involving macrophages and epithelial cells, which facilitates CXC chemokine expression and aids neutrophil recruitment during pneumococcal pneumonia. They also highlight a potential clinical role for anti-IL-1 treatment to limit excessive neutrophilic inflammation in the lung.     

Interleukin-1beta responses to Mycoplasma pneumoniae infection are cell-type specific

Interleukin-1beta (IL-1beta) is a major proinflammatory cytokine that is involved in many important cellular functions such as proliferation, differentiation, and activation of different cell types. Its mature form is released from the cells in response to various bacterial and viral infections, and it plays a significant role in host defense. Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans following attachment to respiratory epithelium, as well as extrapulmonary infections. Very little is known about the role of cytokines in pathogenesis or the response of target cells to M.pneumoniae attachment. The purpose of this study was to investigate the ability of M. pneumoniae to induce IL-1beta in human lung epithelial carcinoma A549 and in human monocytic U937 cell lines. Following M. pneumoniae infection, both IL-1beta mRNA and protein were induced in A549 cells vs. no induction in uninfected cells; however, the protein remained inside the A549 cells. Similarly, M. pneumoniae infection strongly increased mRNA and extracellular protein levels in U937 cells, which unlike A549 cells did exhibit baseline constitutive levels. De novo IL-1beta protein expression was verified by cycloheximide studies. M. pneumoniae infection did not affect constitutive caspase-1 mRNA or protein levels in either cell line. Reduced caspase-1 activity in A549 cell lysates suggests the presence of an endogenous caspase-1 inhibitory component in the A549 cells. These collective data confirm previous studies that show that M. pneumoniae is a potent inducer of cytokines following adherence to host target cells, and establish that IL-1beta release in response to M. pneumoniae infection is cell-type specific, thus emphasizing the importance of carefully considering multiple cell types in M. pneumoniae pathogenesis studies involving both immune cells and cytokine release patterns.     

Interleukin-1 receptor antagonist attenuates airway hyperresponsiveness following exposure to ozone

The role of an interleukin (IL)-1 receptor antagonist (IL-1Ra) on the development of airway hyperresponsiveness (AHR) and airway inflammation following acute O(3) exposure in mice was investigated. Exposure of C57/BL6 mice to O(3) at a concentration of 2.0 ppm or filtered air for 3 h resulted in increases in airway responsiveness to inhaled methacholine (MCh) 8 and 16 h after the exposure, and an increase in neutrophils in the bronchoalveolar lavage (BAL) fluid. IL-1beta expression, assessed by gene microarray, was increased 2-fold 4 h after O(3) exposure, and returned to baseline levels by 24 h. Levels of IL-1beta in lung homogenates were also increased 8 h after O(3) exposure. Administration of (human) IL-1Ra before and after O(3) exposure prevented development of AHR and decreased BAL fluid neutrophilia. Increases in chemokine levels in lung homogenates, tumor necrosis factor-alpha, MIP-2, and keratinocyte chemoattractant following O(3) exposure were prevented by IL-1Ra. Inhalation of dexamethasone, an inhibitor of IL-1 production, blocked the development of AHR, BAL fluid neutrophilia, and decreased levels of IL-1 following O(3) exposure. In summary, acute exposure to O(3) induces AHR, neutrophilic inflammation, epithelial damage, and IL-1. An IL-1Ra effectively prevents the development of altered airway function, inflammation, and structural damage.     

Regulation of NF-kappaB and ICAM-1 expression in human airway epithelial cells.

The aim of this study was to elucidate the redox regulation of cytokine-induced NF-kappaB activation and NF-kappaB mediated gene induction in A549 cells and primary cultures of human airway epithelial cells. In A549 cells, Western blot analysis showed transient depletion of IkappaBalpha after 15 min IL-1beta treatment followed by its reappearance after 60 min, indicating efficient NF-kappaB-driven gene induction. A similar pattern was observed in primary epithelial cells however, the kinetics were slower and depletion was less. In primary airway epithelial cells IkappaBalpha levels were 59.8+/-8.5% of control following 30 min treatment with IL-1beta and in A549 cells 29.1+/-8.5% of control following 15 min IL-1beta treatment. Cytokine-induced IkappaBalpha depletion was associated with NF-kappaB nuclear accumulation and subsequent resynthesis of IkappaBalpha and upregulation of ICAM-1 in both cell types. The antioxidant, NAC (20 mM) had no effect on the kinetics of cytokine-induced IkappaBalpha depletion or NF-kappaB p65 nuclear translocation in either cell type and failed to influence kappaB dependent IkappaBalpha resynthesis. H2O2 treatment alone or in combination with cytokines had no significant effects on IkappaBalpha depletion, NF-kappaB p65 nuclear translocation or ICAM-1 expression in either cell type but did cause significant activation of p38 MAPK. These results suggest that cytokine-induced NF-kappaB activation in cultured human airway epithelial cells does not involve an NAC-sensitive oxidant stress and that H2O2-induced oxidant stress does not result in effective NF-kappaB activation and NF-kappaB mediated gene induction.     

Peroxisome proliferator-activated receptor-gamma agonists inhibit respiratory syncytial virus-induced expression of intercellular adhesion molecule-1 in human lung epithelial cells

Respiratory syncytial virus (RSV) is the major causative agent of severe lower respiratory tract disease and death in infants worldwide. The epithelial cells of the airways are the target cells for RSV infection and the site of the majority of the inflammation associated with the disease. However, despite five decades of intensive RSV research there exist neither an effective active vaccine nor a promising antiviral and anti-inflammatory therapy. Recently, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily, has been shown to possess anti-inflammatory properties. Therefore, we hypothesized whether the detrimental increase of intercellular adhesion molecule-1 (ICAM-1) on RSV-infected lung epithelial cells (A549 and primary normal human bronchial epithelial cells (NHBE)) might be modulated by natural and synthetic PPAR-gamma agonists (15d-PGJ2, ciglitazone, troglitazone, Fmoc-Leu). Our data show that all PPAR-gamma agonists under study significantly down-regulated the RSV-induced expression of ICAM-1 on A549- and NHBE cells in a dose-dependent manner resulting in a reduced beta2 integrin-mediated adhesion of monocytic effector cells (U937) to RSV-infected A549 cell monolayers. In contrast, the PPAR-alpha agonist bezafibrate had no impact on the RSV-induced ICAM-1 expression. The reduced ICAM-1 expression was associated with a diminished ICAM-1 mRNA level and binding activity of nuclear factor-kappaB (p65/p50) in A549 cells. These findings suggest that PPARgamma agonists have beneficial effects in the suppression of the inflammatory response during RSV infection and therefore might have clinical efficacy in the course of severe RSV-infection.     

探讨肺纤维化时表达单核细胞趋化蛋白-1的主要效应细胞

目的研究凝血酶对人肺上皮细胞及正常人肺成纤维细胞释放单核细胞趋化蛋白-1（MCP-1）的调节作用。方法人肺支气管上皮细胞（normal human bronchial epithelial cells,NHBE）、人肺癌上皮细胞系A549及正常人肺成纤维细胞（normal human lung fibroblasts,NH-LF）分别培养于96孔或6孔板培养板内,凝血酶诱导4h和24h后,收集上清液及细胞裂解液。ELISA方法检测MCP-1蛋白水平;实时荧光定量RT-PCR技术检测MCP-1mRNA水平。结果凝血酶可诱导NHBE、A549及NH-LF细胞释放MCP-1,但NH-LF细胞的MCP-1释放量远远高于NHBE及A549细胞;凝血酶还可诱导NH-LF细胞mRNA表达。结论人肺成纤维细胞可能是肺内表达MCP-1的主要来源细胞,因此在肺纤维化时可能起重要作用。     

IL-1 receptor-type expression in relation to atopy.

BACKGROUND: IL-1 has 2 receptors, type I (IL-1RI) and type II (IL-1RII), which have 2 forms each, membrane (m) and soluble (s). When IL-1 binds to mIL-1RI, the active receptor, an inflammatory response is initiated, which does not occur when IL-1 binds to mIL-1RII, the decoy receptor. Both sIL-1RI and sIL-1RII function as IL-1-mopping mechanisms. We hypothesized that the ratio of active (mIL-1RI) to inactive (mIL-1RII, sIL-1RI, and sIL-1RII) receptors is important in determining the amount of inflammation produced in allergic reactions. OBJECTIVE: Our aim was to compare the concentrations of mIL-1RI and mIL-1RII on cultured PBLs and sIL-1RI, sIL-1RII, and IL-1beta in sera and supernatants of cultured PBMCs from atopic and nonatopic subjects. METHODS: The membrane receptors, soluble receptors, and IL-1beta concentrations were measured by ELISA with specific mAbs. RESULTS: Although there was no difference in the level of serum IL-1beta between the 2 groups, PBMCs from atopic persons spontaneously secreted higher levels of IL-1beta than those from nonatopic donors (P < .05). PBLs from atopic subjects compared with those from nonatopic individuals expressed higher mIL-1RI (P < .0001) and mIL-1RII (P < .05). Levels of both the soluble receptors from both serum (P < .0001) and PBMCs (P < .05) of nonatopic donors were higher than those found in atopic donors. CONCLUSION: This augmentation of mIL-1RI concomitant with a reduction in soluble receptors may be an important contributory factor to the inflammation that occurs with allergen exposure.     

IL-1, IL-18, and IL-33 families of cytokines

Summary: The interleukin-1 (IL-1), IL-18, and IL-33 families of cytokines are related by mechanism of origin, receptor structure, and signal transduction pathways utilized. All three cytokines are synthesized as precursor molecules and cleaved by the enzyme caspase-1 before or during release from the cell. The NALP-3 inflammasome is of crucial importance in generating active caspase-1. The IL-1 family contains two agonists, IL-1α and IL-1β, a specific inhibitor, IL-1 receptor antagonist (IL-1Ra), and two receptors, the biologically active type IL-1R and inactive type II IL-1R. Both IL-1RI and IL-33R utilize the same interacting accessory protein (IL-1RAcP). The balance between IL-1 and IL-1Ra is important in preventing disease in various organs, and excess production of IL-1 has been implicated in many human diseases. The IL-18 family also contains a specific inhibitor, the IL-18-binding protein (IL-18BP), which binds IL-18 in the fluid phase. The IL-18 receptor is similar to the IL-1 receptor complex, including a single ligand-binding chain and a different interacting accessory protein. IL-18 provides an important link between the innate and adaptive immune responses. Newly described IL-33 binds to the orphan IL-1 family receptor T1/ST2 and stimulates T-helper 2 responses as well as mast cells.     

Interleukin-1beta modulates proinflammatory cytokine production in human epithelial cells

Periodontitis is a chronic human inflammatory disease initiated and sustained by dental plaque microorganisms. A major contributing pathogen is Porphyromonas gingivalis, a gram-negative bacterium recognized by Toll-like receptor 2 (TLR2) and TLR4, which are expressed by human gingival epithelial cells (HGECs). However, it is still unclear how these cells respond to P. gingivalis and initiate inflammatory and immune responses. We have reported previously that HGECs produce a wide range of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha (TNF-alpha), and IL-1beta. In this study, we show that IL-1beta has a special role in the modulation of other inflammatory cytokines in HGECs challenged with P. gingivalis. Our results show that the increased production of IL-1beta correlates with the cell surface expression of TLR4, and more specifically, TLR4-normal HGECs produce fourfold more IL-1beta than do TLR4-deficient HGECs after challenge. Moreover, blocking the IL-1beta receptor greatly reduces the production of "secondary" proinflammatory cytokines such as IL-8 or IL-6. Our data indicate that the induction of IL-1beta plays an important role in mediating the release of other proinflammatory cytokines from primary human epithelial cells following challenge with P. gingivalis, and this process may be an inflammatory enhancement mechanism adopted by epithelial cells.     

IL-1beta, IL-1Ra and sIL-1RII in the culture supernatants of PMN and PBMC and the serum levels in patients with inflammation and patients with cancer disease of the same location.

An inflammation or an other malignant process may create a microenvironment that modulates the production and activity of cytokines and their regulators. In the present study we compared the secretion of IL-1beta and its regulatory proteins: IL-IRa and sIL-1RII by PMN and PBMC derived from patients with inflammation and patients with cancer disease of the same location. We also examined the serum levels of these mediators in groups of patients. The results obtained revealed changes in the secretion of IL-1beta and IL-1Ra which are more characteristic of PMN and PBMC from cancer patients than of the cells from patients with inflammation. In contrast, the secretion of sIL-1RII is more characteristic of PMN and PBMC derived from patients with inflammation. Furthermore, PMN appear to play more significant role in the secretion of IL-1Ra into the circulation of cancer patients than PBMC. In contrast, PBMC affect to a large extent the secretion of IL-1beta and sIL-1RII into the circulation of patients with inflammation than PMN. Concluding, the secretion of IL-1beta and its regulatory proteins may depend on the type of immune cells and the type of the disease.     

Involvement of epidermal growth factor receptor-linked signaling responses in Pseudomonas fluorescens-infected alveolar epithelial cells

Pseudomonas fluorescens is an opportunistic indoor pathogen that can cause severe airway proinflammatory responses. Pulmonary epithelium, like other mucosal epithelial linings of the body, constitutes the first line of defense against airway microbial pathogens. Mucosal epithelial cells can be a sentinel of pathogenic bacteria via stimulation of specific cell surface receptors, including the epidermal growth factor receptor (EGFR) and Toll-like receptor (TLR). This study addressed the involvement of EGFR in airway epithelial pathogenesis by P. fluorescens. Human A549 pneumocytes showed prolonged production of proinflammatory interleukin-8 (IL-8) in response to infection with P. fluorescens, which was via the nuclear factor-kappa B (NF-κB) signaling pathway. Production of proinflammatory cytokine IL-8 was not mediated by P. fluorescens lipopolysaccharide, a representative TLR4 agonist, but was mediated through EGFR-linked signals activated by the opportunistic bacteria. Moreover, EGFR signals were involved in NF-κB signal-mediated production of proinflammatory cytokines. Along with persistent NF-κB activation, P. fluorescens enhanced the EGFR phosphorylation and subsequent activation of downstream mediators, including protein kinase B or extracellular-signal-regulated kinases 1/2. Blocking of EGFR-linked signals increased epithelial susceptibility to pathogen-induced epithelial cell death, suggesting protective roles of EGFR signals. Thus, airway epithelial exposure to P. fluorescens can trigger antiapoptotic responses via EGFR and proinflammatory responses via TLR4-independent NF-κB signaling pathway in human pneumocytes.