Zinc-dependent cytoadherence of Legionella pneumophila to human alveolar epithelial cells in vitro

Microbial adherence to host cells is an early key step in the establishment of infection. During the course of Legionnaire's disease, Legionella interactions with host cells are best documented for resident macrophages. However, L. pneumophila can also replicate within type I and type II pneumocytes, which cover almost the entire alveolar surface. In the presence of zinc, we observed a significant and concentration-dependent increase in L. pneumophila adherence to and invasion of type II pneumocytes. The zinc-dependent adherence mechanism seemed to be host-cell-independent, as a similar increase in cytoadherence was observed with macrophages. We also found that zinc-dependent adherence of L. pneumophila appears to involve recognition of zinc-binding pneumocyte receptors by a bacterial adhesin, and heparan-sulfated host cell receptors, but not type IV pili.     

Effects of topical corticosteroids on inflammatory mediator–induced eicosanoid release by human airway epithelial cells

Background: Airway epithelial cells are among the first cells to come in contact with aerosolized corticosteroids. However, the relative potencies and time course of action of the several commonly used aerosolized corticosteroids on eicosanoid production by airway epithelial cells are unknown. Objectives: This study compared the effects of fluticasone, budesonide, and triamcinolone on eicosanoid output by human airway epithelial cells in vitro. We also determined the spectrum of eicosanoids affected and the mechanism for corticosteroid action. Methods: Cultured BEAS-2B airway epithelial cells (a transformed cell line) were exposed to corticosteroids (1 nmol/L to 1 μmol/L) for 2 to 48 hours and then assayed for basal- and bradykinin (BK)-stimulated eicosanoid output. The eicosanoid profile was identified by HPLC in tritiated arachidonic acid prelabelled cells, and PGE₂, the major eicosanoid product, was quantitated by RIA. The effect of corticosteroids on the immunoreactivity of key proteins involved in eicosanoid metabolism (ie, cyclooxygenase [COX], phospholipase A2 [PLA₂], and Clara cell protein, a PLA₂ inhibitor) was determined by Western blotting. Results: Eicosanoid output was largely confined to prostaglandins with values of 5 ± 2 and 82 ± 35 ng PGE₂/10⁶ cells for basal- and BK stimulation, respectively (n = 8). All 3 corticosteroids inhibited basal- and BK-induced PGE₂ output in a dose- and time-dependent manner. Fluticasone and budesonide completely eliminated PGE₂ output in nanomolar concentrations in contrast to triamcinolone, which required micromolar concentration. The rank order of potency was: fluticasone = budesonide > triamcinolone. The time course of action for PGE₂ inhibition also differed, with budesonide acting more slowly than the other 2 corticosteroids (P = .04). All 3 corticosteroids markedly reduced COX2 with little effect on COX1, cPLA₂ (Type IV), or iPLA₂ (Type VI) immunoreactivity or their relative distribution in cytosol versus membrane fractions. Clara cell protein immunoreactivity was undetectable in control and corticosteroid-treated cell lysates. Conclusion: These results show that in a human airway epithelial cell line, the 3 inhaled corticosteroids commonly used to treat asthma differ in onsets of action as inhibitors of prostaglandin synthesis and vary considerably in potency. All 3 corticosteroids act mechanistically in similar fashion by inhibiting COX2 synthesis. (J Allergy Clin Immunol 1999;103:1081-91.)     

Identification of Legionella species by ribotyping and other molecular methods

There are currently more than 40 species of Legionella and the identification of most of them by standard methods is often technically difficult. The aim of this study was to use a ribotyping method with endonuclease HindIII and a probe consisting of a set of five oligonucleotides (referred to as OligoMix5). A total of 123 strains, including 78 type or reference strains corresponding to 44 species, eight clinical and 37 environmental isolates were tested. The usefulness of the method was demonstrated for the identification at the species level of all of the 123 Legionella isolates tested, with each species showing a specific profile. Among the 15 serogroups of Legionella pneumophila, eight patterns were obtained. For the 45 field strains, the randomly amplified polymorphic DNA (RAPD) technique and intergenic 16S-23S ribosomal spacer PCR analysis (ITS 16-23S) were also used. Altogether, these three methods allowed the identification of all of strains tested. However, ribotyping has proven to be more effective than the other methods.     

细胞外调节蛋白激酶信号通路参与机械牵张诱导肺泡上皮细胞高迁移率族蛋白B1的表达调控

目的 探讨细胞外调节蛋白激酶（ERK）信号通路在机械牵张诱导肺泡上皮细胞（A549）表达高迁移率族蛋白B1（HMGB1）中的作用。 方法 肺泡上皮细胞A549分为A、B、C 3组,A组为对照组;B组A549细胞施加14%牵张应变,牵张时间为4 h;C组细胞的牵张模式与B组相同,只是于施加牵张前用ERK的特异性抑制剂PD98059预处理A549细胞2h。分别用免疫细胞化学染色和RT-PCR检测细胞HMGB1蛋白和mRNA的表达,用Western blotting检测ERK激酶的活性。 结果 A549细胞施加14 %牵张应变后,HMGB1蛋白和mRNA表达明显增加,ERK激酶活性明显增高（P＜0.05）;该诱导激活作用可被PD98059阻断。 结论 机械牵张通过ERK信号通路,调节A549细胞的HMGB1基因和蛋白表达。     

A method for estabilishing primary cultures of human gastric epithelial cells

Long-term culture of human gastric epithelial cells has been difficult, and at present no normal human gastric epithelial cell lines are readily available. As part of our experiments to study pathogenesis of H. pylori, a bacterium that infects the stomach, we developed methods to culture normal human gastric epithelial cells. Primary cultures of human gastric epithelial cells can be established from gastric biopsies taken at upper G.I. endoscopy. Enzymatically isolated gastric epithelial-like cells are present in tight colonies on culture dishes within 24 hours of placing the cells in culture. Cells isolated stain positively for cytokeratin and produce neutral mucins, indicating that they are mucin secreting epithelial cells, consistent with gastric epithelial cells. Epithelial cells can be maintained up to 4 weeks in culture with evidence of DNA synthesis up through the first week of culture.     

IL-1beta secretion induced by Aggregatibacter (Actinobacillus) actinomycetemcomitans is mainly caused by the leukotoxin

Aggregatibacter (Actinobacillus) actinomycetemcomitans forms a leukotoxin that selectively lyses primate neutrophils, monocytes and triggers apoptosis in promyeloic cells and degranulation of human neutrophils. Recently, we showed that the leukotoxin causes activation of caspase-1 and abundant secretion of bio-active IL-1β from human macrophages. In this study, we show that high levels of IL-β correlated with a high proportion of A. actinomycetemcomitans in clinical samples from a patient with aggressive periodontitis. To determine the relative contribution of leukotoxin to the overall bacteria-induced IL-1β secretion, macrophages were isolated from peripheral blood and exposed to different concentrations of live A. actinomycetemcomitans strains with either no, low or high production of leukotoxin. Cell lysis and levels of IL-1β, IL-6, TNF-α and caspase-1 were measured by ELISA and flow cytometry. Leukotoxin was the predominant cause of IL-1β secretion from macrophages, even in the A. actinomycetemcomitans strain with low leukotoxin production. Macrophages exposed to non-leukotoxic bacteria accumulated cytosolic pro-IL-1β, which was secreted by a secondary exposure to leukotoxic bacteria. In conclusion, the present study shows for the first time that A. actinomycetemcomitans-induced IL-1β secretion from human macrophages in vitro is mainly caused by leukotoxin.     

RNA干扰对人肺泡上皮A549细胞巨噬细胞移动抑制因子基因表达抑制作用

目的探讨巨噬细胞移动抑制因子(MIF)小干扰RNA(siRNA)干扰对人肺泡上皮A549细胞株MIF基因表达的影响.方法将MIF siRNA用转染剂Interferin介导转染体外培养的A549细胞,通过RT-PCR方法观察不同浓度MIF siRNA转染后,对MIF mRNA表达的影响;采用细胞免疫荧光方法分析MIF蛋白表达的强弱;应用MTT方法测定MIF siRNA转染对细胞的毒性,及脂多糖(LPS)刺激对A549细胞生存率的影响.结果 (1)MIF siRNA转染人肺泡上皮A549细胞株后,可下调细胞中MIF mRNA的表达,只有当MIF siRNA转染浓度大于50 nmol/L时,才能明显下调MIF mRNA的表达;(2)MIF siRNA转染人肺泡上皮A549细胞株后,A549细胞MIF的荧光蛋白表达也明显降低,在此基础上再加入MIF刺激,MIF的蛋白表达又明显增加;(3)MIF siRNA转染对A549细胞几乎没有毒性,且对LPS刺激A549细胞导致细胞死亡及细胞的生存率下降有一定的保护作用.结论 MIF siRNA能在A549细胞特异沉默目的基因MIF和抑制MIF蛋白的表达,且对LPS刺激导致细胞死亡有一定的保护作用.     

Streptococcus uberis internalizes and persists in bovine mammary epithelial cells

Streptococcus uberis is one of the most important emerging bovine mastitis pathogens and chronic persistent intramammary infections (IMI) are often described. To define the ability of S. uberis to persist intracellularly, studies on time-dependent internalization and survival of S. uberis strains in bovine mammary epithelial cells were conducted. Two S. uberis strains (UT366 and UT888) and a Staphylococcus aureus strain used as positive control, all isolated from cows with clinical mastitis were cocultured with bovine mammary epithelial cells (MAC-T) and persistent survival in host epithelial cells for extended periods (120 h) studied. Of S. uberis strains tested, UT366 showed highest internalization values at 60 min of incubation whereas at 8 h of incubation the corresponding values for UT888 were the highest. Of both strains of S. uberis tested, UT366 seems to internalize bovine mammary cells more efficiently initially, however, during the first 8 h, UT888 seems to survive intracellularly better than UT366. Results showed that both S. uberis strains could survive intracellularly up to 120 h without apparent loss of host cells viability. S. aureus internalized more efficiently than all strains tested and host cell death was observed after 72 h of incubation. These results indicate that S. uberis can survive within mammary epithelial cells for extended time without apparent loss of host cells viability. Intracellular persistence of S. uberis may be associated with the spread of the infection to deeper tissues and development of persistent IMI.     

NOX1在TNF-α诱导的肺泡上皮细胞氧化损伤和炎症中的作用研究

目的:探索NADPH氧化酶1(NOX1)在肿瘤坏死因子α(TNF-α)诱导的肺泡上皮细胞A549氧化损伤和炎症中的作用。方法:以real-time PCR和Western blot法测定TNF-α处理后肺泡上皮细胞中NOX1的m RNA和蛋白表达水平。在肺泡上皮细胞中转染NOX1 si RNA及其阴性对照,以TNF-α诱导以后,用real-time PCR和Western blot测定细胞中NOX1水平。硫代巴比妥酸法检测细胞中丙二醛(MDA)含量,黄嘌呤氧化法检测细胞中超氧化物歧化酶(SOD)活性,ELISA法检测细胞培养液中的白细胞介素4(IL-4)、白细胞介素6(IL-6)和白细胞介素1β(IL-1β)含量,流式细胞术检测细胞凋亡率,Western blot检测凋亡蛋白cleaved caspase-3的水平。结果:TNF-α诱导处理后A549细胞中NOX1 m RNA和蛋白水平均升高(P 0. 05),NOX1 si RNA转染可以下调细胞中NOX1的m RNA和蛋白表达(P 0. 05),转染si RNA阴性对照对细胞中NOX1的m RNA和蛋白表达没有影响。TNF-α处理后细胞中MDA含量升高,SOD活性降低,细胞分泌的IL-4、IL-6和IL-1β增多,细胞凋亡率和凋亡蛋白cleaved caspase-3水平均升高,与没有经TNF-α处理的细胞比较,差异有统计学意义(P 0. 05)。下调NOX1的细胞经TNF-α诱导后,细胞中MDA含量降低,SOD活性升高,细胞分泌的IL-4、IL-6和IL-1β减少,凋亡率及凋亡蛋白cleaved caspase-3水平降低,与只经过TNF-α诱导处理的细胞比较,差异有统计学意义(P 0. 05)。结论:TNF-α诱导肺泡上皮细胞表达NOX1。下调NOX1表达可减少细胞氧化损伤和炎症因子分泌,减少细胞凋亡。     

Polymorphism of Candida albicans is a major factor in the interaction with human dendritic cells

Morphological plasticity of Candida albicans is a major virulence factor. Using pH-dependent dimorphism we show, that human dendritic cells (DC) recognize filamentous forms and blastoconidia of a virulent C. albicans isolate (strain SC5314). Heat inactivated and viable blastoconidia are rapidly phagocytosed by human DC. However, viable yeast cells start to filament inside the DC at later stages of infection, leading to penetration and loss of cellular integrity. The cytokine burst of human DC induced upon contact with Candida is dominated by the granulocyte-activating, chemotactic factor IL-8 and the proinflammatory mediator TNF-alpha. Blastoconidia induce markedly lower cytokine levels than filamentous forms. Whereas IL-8 secretion is mainly cell mass dependent, release of TNF-alpha, a major proinflammatory cytokine, is clearly dependent on the morphology of Candida.     

Intracellular survival of persistent group A streptococci in cultured epithelial cells

Group A streptococcus (GAS) is the principle etiologic agent of bacterial pharyngotonsillitis and a wide range of other diseases. Failure to eradicate GAS from patients has been documented in 5-30% of patients with pharyngotonsillitis, in spite of the continued sensitivity of GAS to penicillin and other beta-lactams. It was recently proposed that eradication failure might be attributed to the ability of GAS to maintain an intracellular reservoir during antibiotic treatment. We have previously shown that strains derived from patients with bacterial eradication failure, despite antibiotic treatment (persistent strains), adhered to and were internalized by cultured epithelial cells more efficiently than strains that were successfully eradicated. Since, penicillin and other beta-lactams do not penetrate well into mammalian cells, intracellular survival of GAS is crucial in order to persist during prolonged antibiotic treatment. In this study, we compared the survival of GAS strains from cases of eradication failure and eradication success, using an epithelial cell culture model. We found that persistent strains show significantly increased intracellular survival, compared to the 'eradication success' strains. This finding supports the idea that an intracellular reservoir of GAS plays a role in the etiology of antibiotic eradication failure.     

Agaphelin modulates the activation of human bronchial epithelial cells induced by lipopolysaccharide and IL-4

Sand fly saliva presents molecules with potential to development of compounds for treatment of inflammatory diseases. Agaphelin, isolated from the saliva of the mosquito Anopheles gambiae, demonstrates anti-inflammatory properties such as neutrophils chemotaxis inhibition. Here, we extend these results and evaluated the role of agaphelin (0.1−100 nM) in an in vitro model consisting in the activation of human bronchial epithelial cells (BEAS-2B) by IL-4 (50 ng/mL) or lipopolysaccharide (LPS; 10 ng/mL). Agaphelin is non-cytotoxic for BEAS-2B cells. Notably, agaphelin markedly reduces CCL2 and IL-8 production induced by IL-4 or LPS, without altering the IL-10 production. The TLR4 expression and STAT1 phosphorylation induced by LPS were inhibited by agaphlin. In addition, agaphelin decreased the phosphorylation of STAT6 induce by IL-4, whose effect was independent of IL-4-binding activity. Taken together, these findings identify agaphelin as a potential anti-inflammatory therapeutic agent for airway inflammations.     

Production of IL-12, IL-23 and IL-27p28 by bone marrow-derived conventional dendritic cells rather than macrophages after LPS/TLR4-dependent induction by Salmonella Enteritidis

Induction of the interleukin-12 (IL-12) cytokine family comprising IL-12, IL-23, IL-27, and IL-12p40 by intracellular pathogens is required for orchestration of cell-mediated immune responses. Macrophages (MΦ) have been shown to be a source of IL-12 following TLR4-dependent activation by Salmonella (S.). In this study another antigen-presenting cell type, the conventional dendritic cell (cDC), was analyzed and its cytokine responses compared with those of MΦ. We generated bone marrow-derived conventional dendritic cells (BMDC) and macrophages (BMMΦ) by incubating murine bone marrow cells with supernatants containing granulocyte/macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), respectively. Stimulation of BMDC and BMMΦ with S. enterica serovar Enteritidis (SE) or LPS resulted in the release of IL-12 and IL-23 by BMDC but not by BMMΦ. Furthermore, BMDC secreted approx. 20-fold more IL-12p40 and IL-27p28 than BMMΦ. However, BMDC and BMMΦ produced similar levels of IL-10. Using BMDC originating from wild-type (wt), TLR2^def and TLR4^def mice, we show that in BMDC the induction of IL-12, IL-23, and IL-27p28 by SE is dependent on TLR4, whereas low-level production of p40 is also mediated by pattern recognition receptors (PRR) other than TLR4. Interestingly, LPS- and SE-provoked responses of BMDC were remarkably similar indicating that LPS is the primary danger molecule of SE. Taken together, our results point to cDC rather than MΦ as the major producers of the IL-12 family members during in vitro infection with SE. The mechanisms of recognition of SE, however, appear to be the same for cDC and MΦ     

Internalization of a non-pathogenic mycobacteria by macropinocytosis in human alveolar epithelial A549 cells

Mycobacterium smegmatis (MSM) a non-pathogenic mycobacterium is often employed as a tool to understand many aspects of the mycobacterial infections. However, its own biology and particularly its mechanism of entry into non-phagocytic cells are not well known. Previously, we demonstrated that Mycobacterium tuberculosis (MTB) invades epithelial cells by macropinocytosis. In the present study, we investigated whether MSM also invades human epithelial type II pneumocytes (A549) by macropinocytosis. Infection of A549 cells with MSM elicited actin filaments redistribution, lamellipodia formation and increased fluid phase uptake, suggesting macropinocytosis. Furthermore, macropinocytosis inhibitors like cytochalasin D and amiloride caused inhibition of fluid phase and bacterial uptake. We can conclude that MSM, like MTB, takes advantage of macropinocytosis for entry into epithelial cells, however, unlike MTB, internalized MSM are killed by host cells. These findings suggest that induction of macropinocytosis and cell invasion is not an exclusive feature of pathogenic organisms.     

Interferon-gamma stimulates the secretion of IL-1, but not of IL-6, by glomerular mesangial cells.

IL-1 activity in culture supernatant and cell lysate from rat mesangial cells stimulated with interferon-gamma (IFN-gamma) was measured by a thymocyte proliferation assay. While IFN-gamma alone had no effect on the secretion or the intracellular pool of IL-1, the enhancement by IFN-gamma of IL-1 secretion in response to lipopolysaccharide (LPS) was observed. The stimulatory effect of culture supernatant on thymocyte proliferation was abrogated by preincubation with the anti-IL-1 antibody. At least 4-h incubation with IFN-gamma and LPS was required to detect enhancing effect of IFN-gamma. The addition of as little as 1 U/ml IFN-gamma significantly increased IL-1 secretion in the presence of 10 micrograms/ml LPS. The IL-6 activity in culture supernatants was determined by measurement of thymidine uptake in mouse IL-6-dependent cell line (MH60.BSF2). Mesangial cells secreted IL-6 in culture supernatant without additional stimuli and LPS distinctly increased it as described previously. However, in contrast to IL-1 production, no effect of IFN-gamma on IL-6 secretion was observed in the presence or absence of LPS. Moreover, we determined whether enhanced IL-1 release is associated with Ia expression on mesangial cells. IFN-gamma alone and the combination with LPS induced marked expression of Ia antigen, whereas LPS alone did not. We conclude that IFN-gamma stimulates the production of IL-1, but not IL-6, by mesangial cells and suggest an important role of IFN-gamma in the pathogenesis of glomerulonephritis by regulating the mesangial production of IL-1 and the accessory cell function of mesangial cells.     

The ICOS-ligand B7-H2, expressed on human type II alveolar epithelial cells, plays a role in the pulmonary host defense system

The mechanism of immune defense against pathogens in the lung, has so far been poorly understood. Here, we show that human type II alveolar epithelial cells play a key role in defense via interactions between B7 homolog (B7h), also known as ICOS ligand, and its receptor ICOS expressed on activated T cells. The A549 alveolar type II cell line abundantly expresses B7-H2, CD40 and B7-1, but not B7-2 or hGL50. TNF-alpha significantly induced B7-H2 and CD40 expression by A549 cells, but had no effect on B7-1 or B7-2 expression. TNF-alpha-deficient mice exhibited low B7-H2 expression on alveolar epithelial cells in comparison with wild-type mice. Co-culture of TNF-alpha pre-stimulated A549 cells with CD4+ T cells promoted CD154 expression, CD4+ T cell proliferation and cytokine production, especially IFN-gamma. Monocyte-derived TNF-alpha in combination with IFN-gamma and LPS markedly induced B7-H2 expression in A549 cells. This study thus identifies a unique costimulatory pathway via alveolar epithelial type II cells that preferentially affects T helper cell function, implying that alveolar epithelial type II cells play a crucial role in innate immunity in the lung by regulating IFN-gamma-synthesis via B7-H2/ICOS interactions.