Induction of proinflammatory cytokines in human lung epithelial cells during Chlamydia pneumoniae infection

Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute respiratory diseases such as pneumonia and bronchitis. Previous studies have established that C. pneumoniae can induce cytokines in mouse and/or human cells, but little information is available on the cytokine response of respiratory epithelial cells, a first line of infection. In this study, heparin treatment of C. pneumoniae significantly reduced its ability to induce interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) mRNA in human lung carcinoma cells, indicating that cytadherence is an important early stimulus for induction of proinflammatory mediators. Although the IL-8, gamma interferon, and TNF-alpha message was consistently induced by infection of A549 cells not treated with heparin, only an elevation of IL-8 protein was detected in A549 supernatants. A549 IL-beta and IL-6 mRNA and supernatant protein profiles were not significantly changed by infection. Heat or UV inactivation of C. pneumoniae only partially reduced the cytokine response, and inhibition of C. pneumoniae protein or DNA synthesis did not affect its ability to induce cytokine gene expression. To prevent stress-induced cytokine release by the A549 cells, centrifugation was not utilized for infection experiments. These experiments establish the importance of cytadherence in cytokine release by cells of respiratory epithelial origin and suggest that further work in the area of cytokine mediators is warranted to gain valuable pathogenic and therapeutic insights.     

Mycoplasma pneumoniae subtype-independent induction of proinflammatory cytokines in THP-1 cells

Mycoplasma pneumoniae can be divided into two main subtypes depending on the amino acid sequences of the P1 adhesin and the P65 protein, both located in the attachment organelle. Differences between these subtypes in infectivity, virulence and interaction with host cells have not been extensively studied. Using ELISA to measure released protein and real-time PCR to quantify mRNA, we have demonstrated that both M. pneumoniae subtypes significantly increased tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8) at comparable levels in THP-1 cells over a 72 h period of time. However, subtype 2 induced a statistically significant increase (P<0.001) in the release of interleukin-1beta at 24 h post-infection compared to subtype 1. These data provide evidence that the induction of proinflammatory cytokine gene and protein expression by M. pneumoniae is not dependent on the infecting subtype.     

Regulation of alpha1-proteinase inhibitor release by proinflammatory cytokines in human intestinal epithelial cells

alpha1-Proteinase inhibitor (alpha1-PI) is the main serine proteinase inhibitor in human plasma. Apart from its synthesis in the liver, this anti-inflammatory protein is also synthesized by and excreted from human intestinal epithelial cells. Antiinflammatory actions of alpha1-PI are thought to be of relevance in the pathogenesis of inflammatory bowel disease. To investigate the role of macrophage-derived cytokines on alpha1-PI secretion from intestinal epithelial cells, we cultured Caco-2 cells until differentiation (14 days in culture) on permeable filter supports. Monolayers of differentiated Caco-2 cells were then co-cultured with human peritoneal macrophages, grown on plastic in the basolateral chamber. Under these conditions, alpha1-PI secretion from Caco-2 cells was enhanced by 45%, probably by a direct action of macrophage-derived cytokines on Caco-2 cells. To extend this observation further, we treated differentiated Caco-2 cells with macrophage-derived proinflammatory cytokines (IL-1beta, IL-8, TNF-alpha), as well as with lymphocyte-derived cytokines IL-2, IL-6 and IFN-gamma. As early as after 24h treatment, IL-2 and IL-8 induced a significant and dose-dependent increase of alpha-1-PI secretion into cell culture medium; this effect was completely reversed after immunoneutralization by the antibodies against IL-2 and IL-8 alpha1-PI secretion was only slightly decreased after treatment with IFN-gamma, while IL-1beta, IL-6 and TNF-alpha had no effect. alpha1-PI secretion correlated well with the expression of this protein in differentiated Caco-2 cells after cytokine treatment, as confirmed by Western blot. Our data imply that, in vitro, alpha1-PI secretion in enterocyte-like Caco-2 cells is up-regulated by IL-2 and IL-8. Our results suggest that both lymphocyte- and macrophage-derived cytokines regulate secretion of the anti-inflammatory protein alpha1-PI in intestinal epithelial cells.     

Alterations in nucleotide content of human lung fibroblasts infected with Mycoplasma pneumoniae.

The nucleotide content of normal MRC-5 human lung fibroblasts and fibroblasts infected with Mycoplasma pneumoniae PI 1428 was determined. Nucleotides from control and infected fibroblasts were extracted with 5% trichloracetic acid. After neutralization of the extracts, the nucleotides in the extracts were separated by anion-exchange chromatography. Significant differences were found between the nucleotide content of the control and infected cells. Nucleotide triphosphate levels were twofold higher in the control fibroblasts than in the infected fibroblasts 4 h after the initiation of infection. At the same time, nucleotide diphosphate and monophosphate levels were higher in the infected fibroblasts than in the control fibroblasts. Determination of the energy charge ratio for each set of nucleotides (adenosine, guanosine, cytidine, and uridine) demonstrated a shift of nucleotide content in the infected fibroblasts. Immediately after infection, the energy charge for each set of nucleotides was higher for the control fibroblasts than it was for the infected fibroblasts. This pattern continued throughout the infection period with only minor exceptions. The work presented here indicates a loss of energy charge in fibroblasts infected with M. pneumoniae and may help to explain some of the metabolic changes and cell damage which accompany infection.     

Immunohistochemical labelling of cytokines in lung lesions of pigs naturally infected with Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae (Mh) is the primary agent of porcine enzootic pneumonia (PEN), a chronic respiratory disease endemic to pig farms, and characterized histologically by infiltration of mononuclear cells in airways and prominent hyperplasia of the bronchus-associated lymphoid tissue (BALT). To gain further insight into the pathogenesis of PEN, cytokine expression in the lung, with particular attention to the BALT, was examined immunohistochemically in pigs naturally infected with Mh. An increase (P < 0.05) in proinflammatory and immunoregulatory cytokines (especially interleukin [IL]-2, IL-4 and tumour necrosis factor [TNF]-alpha, and to a lesser extent IL-1 [alpha and beta] and IL-6) was detected in the BALT, which showed intense lymphoid hyperplasia. IL-1beta and TNF-alpha were also detected in the bronchoalveolar exudate of infected pigs, and IL-6 and IL-8 were demonstrated in mononuclear cells of the alveolar septa. The results showed that in Mh infection, macrophage and lymphocyte activation results in the expression of a number of cytokines capable of inducing lung lesions and lymphoreticular hyperplasia of the BALT.     

Mycoplasma pneumoniae host-pathogen studies in an air-liquid culture of differentiated human airway epithelial cells

The interaction between Mycoplasma pneumonaie and the airway epithelium in vivo is complex and multifaceted. While multiple in vitro studies have been conducted studying this interaction with cell lines and animal cell and organ culture models, the interactions between M. pneumoniae and fully differentiated human airway epithelium in air-liquid interface culture remains unexplored. In the present study we investigated M. pneumoniae interactions with airway epithelium utilizing an air-liquid interface culture of differentiated normal human bronchial epithelial (NHBE) cells. Utilizing confocal microscopy we found that M. pneumoniae cells bound initially to ciliated epithelial cells, but colonization became more evenly distributed over the entire surface with time. M. pneumoniae infection resulted in stimulation of intercellular adhesion molecule 1 (ICAM-1) gene expression and soluble ICAM-1 production in this culture system.     

Induction of proinflammatory cytokines from human respiratory epithelial cells after stimulation by nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) causes repeated respiratory infections in patients with chronic lung diseases. These infections are characterized by a brisk inflammatory response which results in the accumulation of polymorphonucleated cells in the lungs and is dependent on the expression and secretion of proinflammatory cytokines. We hypothesize that multiple NTHi molecules, including lipooligosaccharide (LOS), mediate cellular interactions with respiratory epithelial cells, leading to the production of proinflammatory cytokines. To address this hypothesis, we exposed 9HTEo- human tracheal epithelial cells to NTHi and compared the resulting profiles of cytokine gene expression and secretion using multiprobe RNase protection assays and enzyme-linked immunosorbent assays (ELISA), respectively. Dose-response experiments demonstrated a maximum stimulation of most cytokines tested, using a ratio of 100 NTHi bacterial cells to 1 9HTEo- tracheal epithelial cell. Compared with purified LOS, NTHi bacterial cells stimulated 3.6- and 4.5-fold increases in epithelial cell expression of interleukin-8 (IL-8) and IL-6 genes, respectively. Similar results were seen with epithelial cell macrophage chemotactic protein 1, IL-1alpha, IL-1beta, and tumor necrosis factor alpha expression. Polymyxin B completely inhibited LOS stimulation but only partially reduced NTHi whole cell stimulation. Taken together, these results suggest that multiple bacterial molecules including LOS contribute to the NTHi stimulation of respiratory epithelial cell cytokine production. Moreover, no correlation was seen between NTHi adherence to epithelial cells mediated by hemagglutinating pili, Hia, HMW1, HMW2, and Hap and epithelial cytokine secretion. These data suggest that bacterial molecules beyond previously described NTHi cell surface adhesins and LOS play a role in the induction of proinflammatory cytokines from respiratory epithelial cells.     

Regulation of autoimmunity by proinflammatory cytokines

Studies extending over a decade have provided compelling evidence to suggest that chronic expression of proinflammatory cytokines in vivo leads to unique regulatory properties that target the cognate immune response in a way that appears to be beneficial to the host. This review focuses on the prototypic proinflammatory cytokine tumour necrosis factor α, because recent studies of autoimmune disease in mice and man have unraveled a novel and unexpected immunosuppressive role for this inflammatory mediator during the effector phase of the autoimmune process. So far, T lymphocytes would appear to be important cellular targets of this immunoregulatory effect.     

Parasitism of hamster trachea epithelial cells by Mycoplasma pneumoniae.

Hamster trachea epithelial (HTE) cells were employed as a model system for Mycoplasma pneumoniae pathogenesis. To more closely mimic natural infection, M. pneumoniae was forced to rely upon host cells (as opposed to the growth medium) for nutrients, and infections were initiated with relatively low mycoplasma doses and monitored for extended time periods. A time- and dose-dependent decline in the viability of infected cells was observed; however, viability never declined below 50% of that in uninfected controls. Protein and RNA synthesis actually increased above control levels in infected cells, despite a concomitant decrease in viability. This response was pronounced at higher multiplicities of infection but was only transient at lower doses. In parallel studies in which a culture medium capable of supporting M. pneumoniae growth was used, loss of viability was accelerated. With a low-dose infection a transient increase followed by a precipitous decline in macromolecular synthesis was observed, relative to that in uninfected controls. At higher doses, however, macromolecular synthesis decreased dramatically and in proportion to the loss of viability. The requirement for HTE cells for mycoplasma growth under the experimental culture conditions was demonstrated by quantitating viable mycoplasmas in the culture medium in the presence or absence of HTE cells over 4 days. The increase in mycoplasma number was negligible in the absence of HTE cells, while a 30-fold increase was observed in the presence of HTE cells. These findings demonstrate the feasibility of long-term, low-dose studies of M. pneumoniae pathogenesis with trachea epithelial cells and a nonpermissive culture medium. This experimental system should facilitate the elucidation of the mechanism(s) responsible for host cell injury, and perhaps reveal how host cells respond to infection.     

人偏肺病毒感染人肺上皮细胞后Toll样受体的表达及其功能

目的 观察人偏肺病毒(human metapneumoviras)感染人肺上皮细胞后Toll样受体(TLR)表达变化及其信号通路的功能,探讨hMPV诱导气道炎症的部分机制.方法 hMPV感染体外培养的人肺上皮细胞株A549,检测病毒在A549细胞中的生长曲线,并通过RT-PCR,real-timeRT-PCR方法检测细胞TLR mRNA的表达,ELISA检测细胞培养上清IFN-α及TNF-α的表达.结果 (1)hMPV可在A549细胞中复制,感染后3 d达高峰10~(5.2)TCID_(50)/ml.(2)RT-PCR结果提示:hMPV感染A549细胞6 h后大部分TLR的表达均上调.(3)定量PCR结果提示:hMPV感染A549细胞后TLR3-4、TLR7-9的表达增高,且有时间依赖性,而紫外灭活的hMPV刺激细胞后TLR表达无明显变化.(4)ELISA结果提示hMPV感染后24 h,IFN-α及TNF-α的表达均明显升高.结论 人偏肺病毒感染A549细胞后可上调TLR表达,其诱导的炎性反应与部分TLR介导的信号转导途径有关.     

Regulation of proinflammatory cytokines in seasonal allergic rhinitis

BACKGROUND: Mediators and cytokines have been demonstrated to be released due to nasal allergen exposure in sensitized subjects, but little is known about the release of cytokines and their antagonists under natural conditions. METHODS: Mediators - histamine, eosinophilic cationic protein (ECP), leukotrienes (LT) C4/ D4/E4 - and cytokines - interleukin (IL)-1beta, IL-8, IL-1 receptor antagonist (ra) - were measured in nasal secretions throughout the grass pollen season (6 visits) and for 6 weeks thereafter (3 visits) in patients with seasonal allergic rhinitis (n = 13) and compared to controls (n = 12). A second study was performed comparing nasal secretions of 13 subjects allergic to house dust mite to 8 controls. RESULTS: Compared to controls, leukotrienes and ECP were significantly elevated at nearly all time points in and postseason in the allergic group. Whereas IL-1beta was significantly elevated throughout the study period, IL-1ra was significantly decreased from visit 1 to 3. IL-8 showed no increase compared to controls. Data from subjects with perennial allergic rhinitis supported these findings and additionally demonstrated decreased concentrations of IL-8 and myeloperoxidase in secretions compared to controls. CONCLUSION: Allergic rhinitis represents a persistent inflammation in terms of an activation of eosinophils and constant upregulation of the proinflammatory cytokine IL-1beta in the pollen season and thereafter. We additionally could demonstrate a dysfunction of the anti-inflammatory capacity, i.e. IL-1ra, a naturally occurring antagonist. Persistent inflammation may furthermore lead to the dysregulation of local cellular immunity by reducing the number and activity of neutrophils on the mucosal surface.     

Intravenous immunoglobulin prevents release of proinflammatory cytokines in human monocytic cells stimulated with procalcitonin

Objective  

The aim of this study was to investigate whether the stimulation of monocytic cells with procalcitonin (PCT) results in the release of proinflammatory cytokines. The effects of intravenous immunoglobulin (IVIG) on the production of cytokines from the cells stimulated with PCT were also studied.     

Synthesis and regulation of accessory/proinflammatory cytokines by intestinal epithelial cells.

Intestinal epithelial cells (IEC) have been shown to act as antigen-presenting cells (APC) in vitro and may have this capacity in vivo. In order to determine whether IEC, like other APC, are able to produce accessory cytokines which may play a role in T cell activation, we assessed the accessory cytokine profile of IEC constitutively or after stimulation. We measured expression, production and regulation of accessory cytokines (IL-1 beta, IL-6, tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) by the presence of mRNA as well as secreted protein. Freshly isolated IEC from surgical specimens were cultured in the presence or absence of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), IL-1 beta or TNF-alpha. mRNA was assessed by a specific RNAse protection assay which controlled for contaminating cell populations while protein secretion was measured by ELISA (IL-1) or bioassay (TNF and IL-6). Neither IL-1 beta nor TNF-alpha were detectable in cultured IEC supernatants, supporting the lack of macrophage contamination. All IEC spontaneously secreted IL-6 at levels comparable to those of macrophages. IEC IL-6 mRNA also increased approximately 200-fold during the first 24 h of culture. LPS, IFN-gamma or TNF-alpha had no effect on spontaneous IL-6 production, and neither resulted in the secretion of IL-1 beta or TNF-alpha. However, IL-1 beta up-regulated IL-6 synthesis by 6-7-fold. IEC express a profile of cytokine mRNAs distinct from conventional APC (low level constitutive IL-6 expression but no detectable IL-1 beta, TGF-beta or TNF-alpha), adding to their uniqueness as APC.     

呼吸道合胞病毒感染人肺上皮细胞活化Toll样受体3介导的抗病毒作用研究

目的 探讨呼吸道合胞病毒( RSV)感染人肺上皮A549细胞后,Toll样受体3(TLR3)的水平变化及其产生的Ⅰ型干扰素的抗病毒作用.方法 RSV感染体外培养的人肺上皮A549细胞,并给予TLR3特异性抗体处理,分别感染4、8、12、16、24h后收集各组细胞.未感染病毒的细胞作为对照组.RT-PCR法检测TLR3、IFN-α、IFN-β,RSV F蛋白的mRNA表达水平变化.结果 RSV感染A549细胞后,TLR3、IFN-α、IFN-β,RSV F蛋白的mRNA表达量均升高且有时间依赖性,TLR3 mRNA在24h表达量是基础表达量的5倍,IFN-α、IFN-β mRNA在24 h表达量是基础表达量的4倍多,RSVF蛋白的mRNA表达量近1.7倍.TLR3抗体预先处理以抑制TLR3受体,再行RSV感染,IFN-α和IFN-β mRNA表达量虽然升高,但较感染组均有所下降,mRNA表达在12 h后显著降低,且IFN-ββ的mRNA表达量下调更明显.但RSV F基因的mRNA表达在12 h、24 h升高有显著性差异.结论 RSV感染A549细胞后可上调TLR3表达,其活化细胞介导产生的Ⅰ型干扰素起抗病毒作用,在一定程度上可抑制病毒的增殖水平.