Induction of interleukin 8 (IL-8) production by Pseudomonas nitrite reductase in human alveolar macrophages and epithelial cells.

Interleukin-8 (IL-8) participates in the generation of dense neutrophil accumulations in bronchopulmonary infections caused by Pseudomonas aeruginosa (P. aeruginosa). We have recently reported that nitrite reductase, a bifunctional enzyme located in the periplasmic space of P. aeruginosa, induces IL-8 generation in bronchial epithelial cells (K. Oishi et al. Infect. Immun. 65: 2648-2655, 1997). We examined whether or not Pseudomonas nitrite reductase (PNR) could also stimulate human alveolar macrophages (AM) and pulmonary type II epithelial-like cells (A549) to induce IL-8 production and mRNA expression as well as the production of TNF alpha and IL-1beta. We demonstrated a time- and dose-dependent IL-8 protein synthesis and IL-8 mRNA expression, but no TNF alpha or IL-1beta production, by A549 cells in response to PNR. New protein translation was not required for PNR-mediated IL-8 mRNA expression in the same cells. Furthermore, simultaneous stimulation of PNR with serial doses of TNF alpha or IL-1beta resulted in additive IL-8 production in A549 cells. In adherent AM, PNR enhanced IL-8 protein synthesis and IL-8 mRNA expression in a time-dependent fashion. PNR similarly induced a time-dependent production of TNF alpha and IL-1beta by human adherent AM. Neutralization of TNF alpha or IL-1beta did not influence the levels of IL-8 production in adherent AM culture. We also evaluated whether the culture supernatants of the A549 cells or AM stimulated with PNR could similarly mediate neutrophil migration in vitro. When anti-human IL-8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in the culture supernatants of these cells stimulated with 5 microg/ml of PNR, the mean percent reduction of NCF activities were 49-59% in A549 cells and 24-34% in AM. Our present data support that PNR directly stimulates AM and pulmonary epithelial cells to produce IL-8. PNR also mediates neutrophil migration, in part, through IL-8 production from AM and pulmonary epithelial cells. These data suggest the contribution of PNR to the pathogenesis of bronchopulmonary infections due to P. aeruginosa.     

Contribution of IL-1 beta and TNF-alpha to the initiation of the peripheral lung response to atmospheric particulates (PM10)

Alveolar macrophages (AM) play a key role in clearing atmospheric particulates from the lung surface and stimulating epithelial cells to produce proinflammatory mediators. The present study examines the role of "acute response" cytokines TNF-alpha and IL-1 beta released by AM exposed to ambient particulate matter with a diameter of <10 microm (PM(10)) in amplifying the proinflammatory mediator expression by A549 cells and human bronchial epithelial cells (HBEC). The results showed that supernatants from human AM incubated 24 h with PM(10) (100 microg/ml) contained more TNF-alpha, IL-1 beta, granulocyte-macrophage colony stimulating factor, IL-6, and IL-8 than nonexposed AM supernatants. The 3-h treatment of A549 cells with PM(10)-exposed AM supernatants increased TNF-alpha, IL-1 beta, IL-8, regulated on activation normal T-cells expressed and secreted (RANTES), and leukemia inhibitory factor mRNA compared with the treatment with nonexposed AM supernatants and, compared with untreated A549 cells, additionally increased ICAM-1 and monocyte chemotactic protein-1 mRNA. Preincubating PM(10)-exposed AM supernatants with anti-IL-1 beta antibodies reduced all the above mediators as well as VEGF mRNA expression (P < 0.05), while anti-TNF-alpha antibodies were less effective (P > 0.05), and the combination of the two antibodies most effective. When HBEC were treated similarly, anti-TNF-alpha antibodies had the greatest effect. In A549 cells PM(10)-exposed AM supernatants increased NF-kappa B, activator protein (AP)-1 and specificity protein 1 binding, while anti-TNF-alpha and anti-IL-1 beta antibodies reduced NF-kappa B and AP-1 binding. We conclude that AM-derived TNF-alpha and IL-1 beta provide a major stimulus for the production of proinflammatory mediators by lung epithelial cells and that their relative importance may depend on the type of epithelial cell target.     

Rhinovirus-16 Induced Release of IP-10 and IL-8 Is Augmented by Th2 Cytokines in a Pediatric Bronchial Epithelial Cell Model

Background

In response to viral infection, bronchial epithelial cells increase inflammatory cytokine release to activate the immune response and curtail viral replication. In atopic asthma, enhanced expression of Th2 cytokines is observed and we postulated that Th2 cytokines may augment the effects of rhinovirus-induced inflammation.

Methods

Primary bronchial epithelial cell cultures from pediatric subjects were treated with Th2 cytokines for 24 h before infection with RV16. Release of IL-8, IP-10 and GM-CSF was measured by ELISA. Infection was quantified using RTqPCR and TCID₅₀. Phosphatidyl inositol 3-kinase (PI3K) and P38 mitogen activated protein kinase (MAPK) inhibitors and dexamethasone were used to investigate differences in signaling pathways.

Results

The presence of Th2 cytokines did not affect RV replication or viral titre, yet there was a synergistic increase in IP-10 release from virally infected cells in the presence of Th2 cytokines. Release of IL-8 and GM-CSF was also augmented. IP-10 release was blocked by a PI3K inhibitor and IL-8 by dexamethasone.

Conclusion

Th2 cytokines increase release of inflammatory cytokines in the presence of rhinovirus infection. This increase is independent of effects of virus replication. Inhibition of the PI3K pathway inhibits IP-10 expression.     

Activation of lymphocytes induced by bronchial epithelial cells with prolonged RSV infection

Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells,which might be responsible for susceptibility to asthma; however, the underlying mechanism is not clear. This study determined the activation of lymphocytes and drift of helper T (Th) subsets induced by RSV-infected human bronchial epithelial cells (HBECs) in vitro. HBECs had prolonged infection with RSV, and lymphocytes isolated from human peripheral blood were co-cultured with RSV-infected HBECs. Four groups were established, as follows: lymphocytes (group L); lymphocytes infected with RSV (group RL); co-culture of lymphocytes with non-infected HBECs (group HL); and co-culture of lymphocytes with infected HBECs (group HRL). After co-culture with HBECs for 24 hours, lymphocytes were collected and the following were determined in the 4 groups: cell cycle status; apoptosis rate; and concentrations of IL-4, IFN-γ, and IL-17 in the supernatants. Cell cycle analysis for lymphocytes showed a significant increase in S phase cells, a decrease in G1 phase cells, and a higher apoptosis rate in group HRL compared with the other three groups. In group HRL, the levels of IL-4, IFN-γ, and IL-17 in supernatants were also higher than the other three groups. For further study, lymphocytes were individually treated with supernatants from non-infected and RSV-infected HBECs for 24 h. We showed that supernatants from RSV-infected HBECs induced the differentiation of Th2 and Th17 subsets, and suppressed the differentiation of Treg subsets. Our results showed that HBECs with prolonged RSV infection can induce lymphocyte proliferation and apoptosis, and enhance the release of cytokines by lymphocytes. Moreover, subset drift might be caused by RSV-infected HBECs.     

Respiratory synctial virus infection in BALB/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant Th2-like cytokine pattern.

Vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) caused excessive disease in infants upon subsequent natural infection with RSV. Recent studies with BALB/c mice have suggested that T cells are important contributors to lung immunopathology during RSV infection. In this study, we investigated vaccine-induced enhanced disease by immunizing BALB/c mice with live RSV intranasally or with FI-RSV intramuscularly. The mice were challenged with RSV 6 weeks later, and the pulmonary inflammatory response was studied by analyzing cells obtained by bronchoalveolar lavage 4 and 8 days after challenge. FI-RSV-immunized mice had an increased number of total cells, granulocytes, eosinophils, and CD4+ cells but a decreased number of CD8+ cells. The immunized mice also had a marked increase in the expression of mRNA for the Th2-type cytokines interleukin-5 (IL-5) and IL-13 as well as some increase in the expression of IL-10 (a Th2-type cytokine) mRNA and some decrease in the expression of IL-12 (a Th1-type cytokine) mRNA. The clear difference in the pulmonary inflammatory response to RSV between FI-RSV- and live-RSV-immunized mice suggests that this model can be used to evaluate the disease-enhancing potential of candidate RSV vaccines and better understand enhanced disease.     

呼吸道合胞病毒感染诱导BALB/c鼠固有免疫细胞分泌IL-33

IL-33是新发现的细胞因子,在病毒感染所诱发的气道炎症反应中发挥重要作用。研究发现感染呼吸道合胞病毒的BALB/c鼠肺组织内IL-33水平明显升高。但RSV感染后分泌IL-33的免疫细胞类型,尤其是感染早期分泌IL-33的固有免疫细胞类型目前尚不清楚。通过分离培养BALB/c鼠肺泡巨噬细胞及树突状细胞,采用ELISA及实时荧光定量PCR法检测上述固有免疫细胞RSV感染后IL-33分泌水平的变化。结果显示,RSV感染72 h后BALB/c鼠肺泡巨噬细胞IL-33mRNA水平明显升高,而树突状细胞RSV感染24 h后IL-33mRNA水平开始上升,48 h达峰值。研究证实RSV感染诱导BALB/c鼠固有免疫细胞分泌IL-33,进而介导感染后炎症的发生发展。     

Different respiratory syncytial virus and Quillaja saponin formulations induce murine peritoneal cells to express different proinflammatory cytokine profiles

The recognition of a pathogen or a vaccine antigen formulation by cells in the innate immune system leads to production of proinflammatory cytokines, which will determine the ensuing acquired immune response quantitatively and qualitatively. Tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 and IL-6 are the first set of cytokines produced upon such an encounter, which have roles both in protective immunity and immunopathogenesis evident with respiratory syncytial virus (RSV). RSV antigens in different physical adjuvant-vaccine formulations were analysed for their capacity to provoke cultured murine peritoneal cells to produce these three proinflammatory cytokines. RSV immunostimulating complex (ISCOM), i.e. both antigen and adjuvant are incorporated in the same particle, induced high levels of IL-1alpha being of the same magnitude or higher than those of live RSV and lipopolysaccharide (LPS). Live virus and LPS induced higher levels of IL-6 and TNF-alpha than ISCOM and so did non-adjuvanted UV-inactivated RSV but only at high doses. ISCOM-Matrix, i.e. ISCOM without antigens, admixed as a separate entity to inactivated RSV, downregulated or blocked the cytokine response to the inactivated RSV in contrast to ISCOM. Kinetic studies showed that ISCOM induced cytokine production first detected at hours 1, 2, 4 for TNF-alpha, IL-6 and IL-1alpha respectively, which was earlier than for the other antigen formulations containing corresponding doses of antigen and/or Quillaja adjuvant. Peak values for production of TNF-alpha and IL-6 were at 8 h and for IL-1alpha at 72 h following stimulation with ISCOM. The delayed appearance of IL-1alpha may reflect the cell-bound nature of this cytokine.     

Cytokine regulation of IL-1 beta gene expression in the human polymorphonuclear leukocyte

Although recently polymorphonuclear leukocytes (PMN) have been identified as producers of IL-1 beta in response to LPS and granulocyte/monocyte colony stimulating factor, little is known regarding the ability of other cytokines to induce the production of IL-1 beta in the PMN. Inasmuch as IL-1 and TNF have been shown to be important priming agents, as well as agents that induce migration of PMN, we investigated their effect on IL-1 beta gene expression in human peripheral blood PMN. In the present study, we demonstrate that human peripheral blood PMN produce IL-1 beta in response to IL-1 alpha, IL-1 beta, and TNF-alpha. Control (unstimulated) human PMN had virtually undetectable levels of IL-1 beta mRNA. Either IL-1 beta or TNF, induced PMN to transiently express IL-1 beta mRNA with peak expression at 1 h, returning to untreated levels by 2 h. A dose response indicated that as little as 0.05 ng/ml of IL-1 beta or TNF resulted in IL-1 beta induction, with maximal effects at 1 ng/ml of IL-1 beta and 5 ng/ml of TNF. IL-1 alpha or IL-1 beta exhibited similar dose responses in IL-1 beta mRNA induction. Inasmuch as cytokines have been shown to have synergistic effects in cell function studies, we induced PMN with a combination of maximally effective doses of TNF plus IL-1 beta. They demonstrated a cooperative effect on IL-1 beta gene expression, in that mRNA levels were sustained for three hours. IL-1 beta Ag expression, as measured by ELISA, paralleled IL-1 beta mRNA expression with cell associated peak levels at 2 to 4 h. IL-1 beta Ag levels in PMN lysates and supernatants correlated with IL-1 beta mRNA levels, i.e., TNF + IL-1 greater than TNF greater than IL-1. Thus, these studies represent the first demonstration of IL-1 and TNF induction of IL-1 beta gene expression in the PMN. Furthermore, the time course of induction is unique to the PMN, with peak induction of mRNA at 1 h, which is consistent with the short lived nature of these cells in inflammatory lesions.     

Candida albicans and Streptococcus salivarius modulate IL-6, IL-8, and TNF-alpha expression and secretion by engineered human oral mucosa cells

We investigated the involvement of oral epithelial cells via two cytokines (IL-6 and TNF-alpha) and one chemokine (IL-8) in local defences against live yeast (Candida albicans) and bacteria (Streptococcus salivarius) using an engineered human oral mucosa model. We report that the yeast changed from the blastospore to the hyphal form and induced significant tissue disorganization at later contact periods (24 and 48 h) compared to the bacteria. However, this effect did not reduce the viability or total number of epithelial cells. Gene activation analyses revealed that IL-6, IL-8 and TNF-alpha mRNA levels rose in tissues in contact with live C. albicans or S. salivarius. Gene activation was followed by an upregulation of protein secretion. IL-6 levels were higher after contact with C. albicans than with S. salivarius. IL-8 levels after contact with S. salivarius were higher than with C. albicans. Our study suggests that S. salivarius is more efficient at inducing proinflammatory mediator release than C. albicans. These results provide additional evidence for the contribution of oral epithelial cells to the inflammatory response against fungi and bacteria.     

Polarized Secretion of Interleukin (IL)-6 and IL-8 by Human Airway Epithelia 16HBE14o- Cells in Response to Cationic Polypeptide Challenge

Background

The airway epithelium participates in asthmatic inflammation in many ways. Target cells of the epithelium can respond to a variety of inflammatory mediators and cytokines. Damage to the surface epithelium occurs following the secretion of eosinophil-derived, highly toxic cationic proteins. Moreover, the surface epithelium itself is responsible for the synthesis and release of cytokines that cause the selective recruitment, retention, and accumulation of various inflammatory cells. To mimic the damage seen during asthmatic inflammation, the bronchial epithelium can be challenged with highly charged cationic polypeptides such as poly-l-arginine.

Methodology/Principal Findings

In this study, human bronchial epithelial cells, 16HBE14o- cells, were “chemically injured” by exposing them to poly-l-arginine as a surrogate of the eosinophil cationic protein. Cytokine antibody array data showed that seven inflammatory mediators were elevated out of the 40 tested, including marked elevation in interleukin (IL)-6 and IL-8 secretion. IL-6 and IL-8 mRNA expression levels were elevated as measured with real-time PCR. Cell culture supernatants from apical and basolateral compartments were collected, and the IL-6 and IL-8 production was quantified with ELISA. IL-6 and IL-8 secretion by 16HBE14o- epithelia into the apical compartment was significantly higher than that from the basolateral compartment. Using specific inhibitors, the production of IL-6 and IL-8 was found to be dependent on p38 MAPK, ERK1/2 MAPK, and NF-κB pathways.

Conclusions/Significance

The results clearly demonstrate that damage to the bronchial epithelia by poly-l-arginine stimulates polarized IL-6 and IL-8 secretion. This apically directed secretion of cytokines may play an important role in orchestrating epithelial cell responses to inflammation.     

白细胞介素23在呼吸道合胞病毒感染致Th1、Th2及Th17细胞分化中的作用及机制

本研究旨在探讨白细胞介素23(interleukin 23,IL-23)在呼吸道合胞病毒(respiratory syncytial virus,RSV)感染支气管上皮细胞BEAS-2B后对Th1、Th2和Th17细胞分化的影响及作用机制。将RSV感染BEAS-2B后的上清液与淋巴细胞共孵育,并分别阻断IL-23受体(IL-23 receptor,IL-23R)、IL-23p19亚基及p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)信号通路。应用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测上清液中细胞因子γ干扰素(interferon γ,IFN-γ)、IL-4、IL-17的浓度。同时,应用实时聚合酶链反应(polymerase chain reaction,PCR)检测相关转录因子(t-bet、gata3、rorγt)和信号转导子(stat4、stat6、stat3)的表达。结果显示,RSV感染后IFN-γ、IL-4和IL-17蛋白表达上调,转录因子及信号转导子的表达也有所增加。阻断IL-23和p38 MAPK信号通路后,Th1、Th2和Th7细胞分泌的细胞因子及转录因子表达均明显下降。结果提示,阻断IL-23后可在基因转导层面抑制RSV感染上皮细胞后诱导的Th1、Th2和Th17细胞分化,此过程可能与p38 MAPK信号通路有关。     

Enhancement of IL-6 receptor beta chain (gp130) expression by IL-6, IL-1 and TNF in human epithelial cells.

Analysis of the IL-6 Receptor beta chain (gp130) mRNA expression on the two human epithelial cell lines UAC and Hep3B reveals that it is enhanced by IL-6, IL-1 and TNF treatment. In the case of UAC cells, TNF action might be mediated by IL-6. For Hep3B cells, TNF seems to exert a direct effect on gp130, as no IL-6 expression is detected after stimulation by this cytokine. On the same cells, increase of the binding of an anti-gp130 monoclonal antibody was observed after treatment by TNF, which denotes the effective appearance of new gp130 molecules on the cell surface. All this cytokines seem to act selectively on the beta chain of the IL-6 receptor. This probably reflects the importance for some cells to have gp130 represented on their membrane in inflammatory contexts.     

Differential regulation of epithelial-derived C-C chemokine expression by IL-4 and the glucocorticoid budesonide.

Airway epithelial cells are a rich source of eosinophil-selective C-C chemokines. We investigated whether cytokines and the topical glucocorticoid budesonide differentially regulate RANTES, monocyte chemoattractant protein-4 (MCP-4), and eotaxin mRNA and protein expression in the human bronchial epithelial cell line BEAS-2B and in primary human bronchial epithelial cells by Northern blot analysis and ELISAs. Eotaxin and MCP-4 mRNA expression induced by TNF-alpha alone or in combination with IFN-gamma was near-maximal after 1 h, peaked at 4 and 8 h, respectively, remained unchanged up to 24 h, and was protein synthesis independent. In contrast, RANTES mRNA was detectable only after 2 h and slowly increased to a peak at 24 h, and was protein synthesis dependent. Induction of eotaxin and MCP-4 mRNA showed a 10- to 100-fold greater sensitivity to TNF-alpha compared with RANTES mRNA. IL-4 and IFN-gamma had selective effects on chemokine expression; IL-4 selectively up-regulated the expression of eotaxin and MCP-4 and potentiated TNF-alpha-induced eotaxin, while IFN-gamma markedly potentiated only the TNF-alpha-induced expression of RANTES. Although budesonide inhibited the expression of chemokine mRNA to a variable extent, it effectively inhibited production of eotaxin and RANTES protein. Budesonide inhibited both RANTES- and eotaxin promoter-driven reporter gene activity. Budesonide also selectively accelerated the decay of eotaxin and MCP-4 mRNA. These results point to IL-4 as a possible mediator by which Th2 cells may induce selective production of C-C chemokines from epithelium and indicate that glucocorticoid inhibit chemokine expression through multiple mechanisms of action.     

Roflumilast Inhibits Respiratory Syncytial Virus Infection in Human Differentiated Bronchial Epithelial Cells

Respiratory syncytial virus (RSV) causes acute exacerbations in COPD and asthma. RSV infects bronchial epithelial cells (HBE) that trigger RSV associated lung pathology. This study explores whether the phosphodiesterase 4 (PDE4) inhibitor Roflumilast N-oxide (RNO), alters RSV infection of well-differentiated HBE (WD-HBE) in vitro. WD-HBE were RSV infected in the presence or absence of RNO (0.1-100 nM). Viral infection (staining of F and G proteins, nucleoprotein RNA level), mRNA of ICAM-1, ciliated cell markers (digital high speed videomicroscopy, β-tubulin immunofluorescence, Foxj1 and Dnai2 mRNA), Goblet cells (PAS), mRNA of MUC5AC and CLCA1, mRNA and protein level of IL-13, IL-6, IL-8, TNFα, formation of H₂O₂ and the anti-oxidative armamentarium (mRNA of Nrf2, HO-1, GPx; total antioxidant capacity (TAC) were measured at day 10 or 15 post infection. RNO inhibited RSV infection of WD-HBE, prevented the loss of ciliated cells and markers, reduced the increase of MUC5AC and CLCA1 and inhibited the increase of IL-13, IL-6, IL-8, TNFα and ICAM-1. Additionally RNO reversed the reduction of Nrf2, HO-1 and GPx mRNA levels and consequently restored the TAC and reduced the H₂O₂ formation. RNO inhibits RSV infection of WD-HBE cultures and mitigates the cytopathological changes associated to this virus.     

Respiratory syncytial virus infection activates STAT signaling in human epithelial cells

Acute respiratory syncytial virus (RSV) infection causes airway inflammation and exacerbates asthma, but the mechanism of inflammation is poorly understood. The role of the STAT-signaling pathway in RSV infection in epithelial cells was examined in this study. DNA microarray analyses of RSV-infected human alveolar type II (A549) epithelial cells identified several genes whose expression was altered from -5.5 to +56.4-fold. Four of the highly expressed genes contained STAT-binding elements. In A549 and normal human bronchial epithelial cells (NHBE), RSV induced phosphorylation and nuclear translocation of STAT-1alpha that was abrogated when RSV attachment was blocked. Treatment with a JAK-2 inhibitor or transfection with dominant-negative STAT-1alpha blocked STAT-1alpha activation and RSV infection. RSV also activated STAT-3 and IL-6 specific antibodies blocked this activation. Thus, activation of the STAT-1alpha and STAT-3 pathways play a role in RSV infection.     

Ionizing radiation enhances IL-6 and IL-8 production by human endothelial cells

Irradiation exposure is known to induce an inflammatory reaction. Endothelial cells play a crucial role both in the inflammatory process and in radiation damage. Therefore, supernatants and cell lysates of (60)Co-irradiated human umbilical vein endothelial cells (HUVEC) have been assessed for the presence of pro-inflammatory cytokines. After gamma irradiation, interleukin (IL)-1alpha, IL-1beta and tumor necrosis factor (TNF)-alpha remained undetectable in both cell supernatants and cell lysates. However, a dose-dependent increase in the production of IL-6 and IL-8 has been demonstrated up to 6 days after exposure. These data indicate that the pro-inflammatory cytokines IL-6 and IL-8 may be involved in the inflammatory response of vascular endothelium induced by exposure to ionizing radiation.     

Induction of IL-6 and CCL5 (RANTES) in human respiratory epithelial (A549) cells by clinical isolates of respiratory syncytial virus is strain specific

ABSTRACT: BACKGROUND: Respiratory syncytial virus (RSV) is the major respiratory pathogen of infants and young children. During each seasonal epidemic, multiple strains of both subgroup A and B viruses circulate in the community. Like other RNA viruses, RSV genome replication is prone to errors that results in a heterogeneous population of viral strains some of which may possess differences in virulence. We sought to determine whether clinical isolates of RSV differ in their capacity to induce inflammatory cytokines IL-6 and CCL5 (previously known as RANTES [regulated upon activation, normal T-cell expressed and secreted protein]), which are known to be induced in vitro and in vivo in response to RSV, during infection of A549 cells. RESULTS: Screening of subgroup A and B isolates revealed heterogeneity among strains to induce IL-6 and CCL5. We chose two subgroup B strains, New Haven (NH)1067 and NH1125, for further analysis because of their marked differences in cytokine inducing properties and because subgroup B strains, in general, are less genetically heterogeneous as compared to subgroup A strains. At 12 and 24 hours post infection RSV strains, NH1067 and NH1125 differed in their capacity to induce IL-6 by an order of magnitude or more. The concentrations of IL-6 and CCL5 were dependent on the dose of infectious virus and the concentration of these cytokines induced by NH1125 was greater than that of those induced by NH1067 when the multiplicity of infection of NH1067 used was as much as 10-fold higher than that of NH1125. The induction of IL-6 was dependent on viable virus as infection with UV-inactivated virus did not induce IL-6. The difference in IL-6 induction most likely could not be explained by differences in viral replication kinetics. The intracellular level of RSV RNA, as determined by quantitative RT-PCR, was indistinguishable between the 2 strains though the titer of progeny virus produced by NH1125 was greater than that produced by NH1067 at 16, 24 and 36 hours but essentially equal at 48 and 72 hours. Full genome sequencing of the 2 strains revealed 193 polymorphisms and 4 insertions in NH1067when compared to NH1125 (2 single base insertions in non-coding regions and 2 duplications of 3 and 60 bases in the RSV G gene). Of the polymorphisms, 147 occurred in coding regions and only 30 resulted in amino acid changes in 7 of the RSV genes. CONCLUSIONS: These data suggest that RSV strains may not be homogeneous with regard to pathogenesis or virulence. Identification of the genetic polymorphisms associated with variations in cytokine induction may lead to insights into RSV disease and to the development of effective antiviral agents and vaccines.