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1.  Fosfomycin Suppresses Chemokine Induction in Airway Epithelial Cells Infected with Respiratory Syncytial Virus▿  
Respiratory syncytial virus (RSV) infects airway epithelial cells, causing bronchiolitis and pneumonia. Inflammation is mediated by various cytokines secreted from RSV-infected airway epithelial cells, and it promotes the pathogenesis of RSV-related diseases. Fosfomycin (FOF) is approved as a treatment for various bacterial infectious diseases, including respiratory infectious diseases, in Japan. FOF is suggested to exhibit immunomodulatory effects on lipopolysaccharide-stimulated monocytes and T lymphocytes, in addition to its antimicrobial activity. We investigated the effect of FOF on the cytokine production of an airway epithelial cell line, A549, infected with RSV. RSV-induced cytokines, such as regulated on activation, normal T-cell expressed and secreted (RANTES), interleukin-8 (IL-8), and IL-6, in infected A549 cells. We found that FOF decreased the levels of RSV-induced RANTES and IL-8 but not the level of RSV-induced IL-6. The RANTES promoter was activated by RSV infection. Site-directed mutagenesis analysis of the RANTES promoter showed that NF-κB-binding motifs had a critical role in RSV-induced RANTES promoter activity. A luciferase reporter gene assay and a DNA-binding assay indicated that FOF suppressed the NF-κB activity induced by RSV infection. These results demonstrate that FOF treatment suppresses the RSV-induced transcription of the chemokines RANTES and IL-8 in airway epithelial cells.
PMCID: PMC2691042  PMID: 19369476
2.  Cell-Specific Expression of RANTES, MCP-1, and MIP-1α by Lower Airway Epithelial Cells and Eosinophils Infected with Respiratory Syncytial Virus 
Journal of Virology  1998;72(6):4756-4764.
Respiratory syncytial virus (RSV) is the major cause of acute bronchiolitis in infancy, a syndrome characterized by wheezing, respiratory distress, and the pathologic findings of peribronchial mononuclear cell infiltration and release of inflammatory mediators by basophil and eosinophil leukocytes. Composition and activation of this cellular response are thought to rely on the discrete target cell selectivity of C-C chemokines. We demonstrate that infection in vitro of human epithelial cells of the lower respiratory tract by RSV induced dose- and time-dependent increases in mRNA and protein secretion for RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α). Production of MCP-1 and MIP-1α was selectively localized only in epithelial cells of the small airways and lung. Exposure of epithelial cells to gamma interferon (IFN-γ), in combination with RSV infection, induced a significant increase in RANTES production that was synergistic with respect to that obtained by RSV infection or IFN-γ treatment alone. Epithelial cell-derived chemokines exhibited a strong chemotactic activity for normal human blood eosinophils. Furthermore, eosinophils were susceptible to RSV and released RANTES and MIP-1α as a result of infection. Therefore, the inflammatory process in RSV-induced bronchiolitis appears to be triggered by the infection of epithelial cells and further amplified via mechanisms driven by IFN-γ and by the secretion of eosinophil chemokines.
PMCID: PMC110009  PMID: 9573240
3.  Multiple cis Regulatory Elements Control RANTES Promoter Activity in Alveolar Epithelial Cells Infected with Respiratory Syncytial Virus 
Journal of Virology  2001;75(14):6428-6439.
Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part through its ability to induce chemokine synthesis in infected airway epithelial cells. RANTES (regulated upon activation, normally T-cell expressed and presumably secreted) is a CC chemokine which recruits and activates monocytes, lymphocytes, and eosinophils, all cell types present in the lung inflammatory infiltrate induced by RSV infection. In this study, we analyzed the mechanism of RSV-induced RANTES promoter activation in human type II alveolar epithelial cells (A549 cells). Promoter deletion and mutagenesis experiments indicate that RSV requires the presence of five different cis regulatory elements, located in the promoter fragment spanning from −220 to +55 nucleotides, corresponding to NF-κB, C/EBP, Jun/CREB/ATF, and interferon regulatory factor (IRF) binding sites. Although site mutations of the NF-κB, C/EBP, and CREB/AP-1 like sites reduce RSV-induced RANTES gene transcription to 50% or less, only mutations affecting IRF binding completely abolish RANTES inducibility. Supershift and microaffinity isolation assays were used to identify the different transcription factor family members whose DNA binding activity was RSV inducible. Expression of dominant negative mutants of these transcription factors further established their central role in virus-induced RANTES promoter activation. Our finding that the presence of multiple cis regulatory elements is required for full activation of the RANTES promoter in RSV-infected alveolar epithelial cells supports the enhanceosome model for RANTES gene transcription, which is absolutely dependent on binding of IRF transcription factors. The identification of regulatory mechanisms of RANTES gene expression is fundamental for rational design of inhibitors of RSV-induced lung inflammation.
PMCID: PMC114366  PMID: 11413310
4.  Rhinovirus-Infected Epithelial Cells Produce More IL-8 and RANTES Compared With Other Respiratory Viruses 
The environmental factors human rhinoviruses (HRVs) and house dust mites (HDMs) are the most common causes of acute exacerbations of asthma. The aim of this study was to compare the chemokine production induced by HRVs in airway epithelial cells with that induced by other respiratory viruses, and to investigate synergistic interactions between HRVs and HDMs on the induction of inflammatory chemokines in vitro.
A549 human airway epithelial cells were infected with either rhinovirus serotype 7, respiratory syncytial virus (RSV)-A2 strain, or adenovirus serotype 3 and analyzed for interleukin (IL)-8 and regulated on activation, normal T-cell expressed and secreted (RANTES) release and mRNA expression. Additionally, activation of nuclear factor (NF)-κB and activator protein (AP)-1 were evaluated. The release of IL-8 and RANTES was also measured in cells stimulated simultaneously with a virus and the HDM allergen, Der f1.
HRV caused greater IL-8 and RANTES release and mRNA expression compared with either RSV or adenovirus. NF-κB and AP-1 were activated in these processes. Cells incubated with a virus and Der f1 showed an increased IL-8 release. However, compared with cells incubated with virus alone as the stimulator, only HRV with Der f1 showed a statistically significant increase.
IL-8 and RANTES were induced to a greater extent by HRV compared with other viruses, and only HRV with Der f1 acted synergistically to induce bronchial epithelial IL-8 release. These findings may correspond with the fact that rhinoviruses are identified more frequently than other viruses in cases of acute exacerbation of asthma.
PMCID: PMC3695236  PMID: 23814675
Rhinovirus; asthma; regulated on activation, normal T-cell expressed and secreted; IL-8; Der f1
5.  Respiratory Syncytial Virus (RSV) F and G Proteins Induce IL-1α, CC and CXC Chemokine Responses by Normal Human Bronchoepithelial Cells 
The Journal of infectious diseases  2010;201(8):1201-1207.
Human respiratory syncytial virus (RSV) is a ubiquitous respiratory virus causing serious lower respiratory tract disease in infants and young children worldwide. Studies have shown that RSV infection modulates chemokine expression patterns suggesting that particular cytokine expression profiles may be indicators of disease severity. In this study, we show that RSV F or G protein treatment of fully differentiated primary human bronchial epithelial (NHBE) cells induces apical and basolateral secretion of IL-8, IP-10, MCP-1, and RANTES. Purified RSV G (attachment) protein was shown to stimulate the secretion of IL-1α, and RANTES, while purified F (fusion) protein elicited the production of IL-8, IP-10, and RANTES. Studies with UV-inactivated RSV showed that treatment of NHBE cells induces apical IL-8, IP-10, and MCP-1 secretion independent of infection suggesting that RSV proteins alone modify the chemokine response pattern which may affect the early immune response prior to infection.
PMCID: PMC2839062  PMID: 20205592
RSV; chemokine; NHBE cells; human; bronchoepithelial
6.  Inhibition of viral replication reverses respiratory syncytial virus-induced NF-kappaB activation and interleukin-8 gene expression in A549 cells. 
Journal of Virology  1996;70(12):9079-9082.
Previous studies have demonstrated that respiratory syncytial virus (RSV) infection of airway epithelial cells results in the expression of a number of cytokines, such as interleukin-8 (IL-8), that are transcriptionally regulated by nuclear factor kappaB (NF-kappaB). In the studies reported here, we demonstrate that treatment of RSV-infected A549 cells with 100 microg of ribavirin (a viral replication inhibitor) per ml results in reversal of RSV-induced NF-kappaB activation, IL-8 mRNA expression, and IL-8 protein production in A549 cells. These data confirm that viral replication is a key step in RSV-induced NF-kappaB activation and IL-8 production.
PMCID: PMC191016  PMID: 8971048
7.  Clarithromycin Suppresses Human Respiratory Syncytial Virus Infection-Induced Streptococcus pneumoniae Adhesion and Cytokine Production in a Pulmonary Epithelial Cell Line 
Mediators of Inflammation  2012;2012:528568.
Human respiratory syncytial virus (RSV) sometimes causes acute and severe lower respiratory tract illness in infants and young children. RSV strongly upregulates proinflammatory cytokines and the platelet-activating factor (PAF) receptor, which is a receptor for Streptococcus pneumoniae, in the pulmonary epithelial cell line A549. Clarithromycin (CAM), which is an antimicrobial agent and is also known as an immunomodulator, significantly suppressed RSV-induced production of interleukin-6, interleukin-8, and regulated on activation, normal T-cell expressed and secreted (RANTES). CAM also suppressed RSV-induced PAF receptor expression and adhesion of fluorescein-labeled S. pneumoniae cells to A549 cells. The RSV-induced S. pneumoniae adhesion was thought to be mediated by the host cell's PAF receptor. CAM, which exhibits antimicrobial and immunomodulatory activities, was found in this study to suppress the RSV-induced adhesion of respiratory disease-causing bacteria, S. pneumoniae, to host cells. Thus, CAM might suppress immunological disorders and prevent secondary bacterial infections during RSV infection.
PMCID: PMC3384978  PMID: 22761540
8.  Synergistic Upregulation of Interleukin-8 Secretion from Pulmonary Epithelial Cells by Direct and Monocyte-Dependent Effects of Respiratory Syncytial Virus Infection 
Journal of Virology  2000;74(18):8425-8433.
Respiratory syncytial virus (RSV) infection is the major cause of severe bronchiolitis in infants. Pathology of this infection is partly due to excessive proinflammatory leukocyte influx mediated by chemokines. Although direct infection of the respiratory epithelium by RSV may induce chemokine secretion, little is known about the role of cytokine networks. We investigated the effects of conditioned medium (CM) from RSV-infected monocytes (RSV-CM) on respiratory epithelial (A549) cell chemokine release. RSV-CM, but not control CM (both at a 1:5 dilution), stimulated interleukin-8 (IL-8) secretion from A549 cells within 2 h, and secretion increased over 72 h to 11,360 ± 1,090 pg/ml without affecting cell viability. In contrast, RSV-CM had only a small effect on RANTES secretion. RSV-CM interacted with direct RSV infection to synergistically amplify IL-8 secretion from respiratory epithelial cells (levels of secretion at 48 h were as follows: RSV-CM alone, 8,140 ± 2,160 pg/ml; RSV alone, 12,170 ± 300 pg/ml; RSV-CM plus RSV, 27,040 ± 5,260 pg/ml; P < 0.05). RSV-CM induced degradation of IκBα within 5 min but did not affect IκBβ. RSV-CM activated transient nuclear binding of NF-κB within 1 h, while activation of NF-IL6 was delayed until 8 h and was still detectable at 24 h. Promoter-reporter analysis demonstrated that NF-κB binding was essential and that NF-IL6 was important for IL-8 promoter activity in RSV-CM-activated cells. Blocking experiments revealed that the effects of RSV-CM depended on monocyte-derived IL-1 but that tumor necrosis factor alpha was not involved in this network. In summary, RSV infection of monocytes results in and amplifies direct RSV-mediated IL-8 secretion from respiratory epithelial cells by an NF-κB-dependent, NF-IL6-requiring mechanism.
PMCID: PMC116353  PMID: 10954542
9.  Effect of respiratory syncytial virus infection on regulated on activation, normal T-cells expressed and secreted production in a murine model of asthma 
Korean Journal of Pediatrics  2011;54(11):456-462.
Synthesis of regulated on activation, normal T-cells expressed and secreted (RANTES) in the airway has previously been shown to be elevated after respiratory syncytial virus (RSV) infection. However, since few studies have examined whether RSV-infected asthma patients express a higher level of RANTES than do normal individuals, we used a murine model of asthma to address this question.
We prepared Dermatophagoides farinae-sensitized mice as an asthma model, and then infected them with RSV and analyzed the changes in airway responsiveness and the cell populations and cytokine levels of bronchoalveolar lavage fluid.
RANTES synthesis increased in response to RSV infection in both control mice and in asthma model (D. farinae) mice. However, there was no significant difference in the amount of RANTES produced following RSV infection between control and D. farinae mice. RSV infection affected neither interferon-γsynthesis nor airway responsiveness in either control or D. farinae mice.
RSV infection did not induce more RANTES in a murine model of asthma than in control mice.
PMCID: PMC3254891  PMID: 22253642
Respiratory syncytial virus; RANTES; Animal models; Asthma
10.  Cigarette Smoke Condensate Enhances Respiratory Syncytial Virus–Induced Chemokine Release by Modulating NF-kappa B and Interferon Regulatory Factor Activation 
Toxicological Sciences  2008;106(2):509-518.
Exposure to cigarette smoke is a risk factor contributing to the severity of respiratory tract infections associated with respiratory syncytial virus (RSV). Stimulation of airway epithelial cells by either RSV or cigarette smoke condensate (CSC) has been shown to induce secretion of the proinflammatory chemokines. However, the effect of coexposure of airway epithelial cells to CSC and RSV on inducible chemokine production has not been previously investigated. The results of this study indicate that CSC costimulation significantly increased RSV-induced interleukin-8 (IL-8) and monocyte chemoattactant protein-1 gene and protein expression when compared with each stimulus alone. Promoter deletion studies identified the interferon stimulatory response element (ISRE) of the IL-8 promoter as a critical region responsible for the synergistic increase of IL-8 gene transcription during mixed exposure. CSC costimulation enhanced RSV-induced activation of interferon regulatory factor (IRF)-1 and IRF-7, which bind to the ISRE site. CSC also furthered RSV-induced activation of the transcription factor nuclear factor kappa B (NF-κB), as shown by increased NF-κB DNA binding to its specific site of the IL-8 promoter and increased NF-κB–driven gene transcription. Therefore, our data demonstrate that a combined exposure to CSC and RSV synergistically increases chemokine expression in airway epithelial cells, suggesting that CSC contributes to an exuberant immune response to RSV by stimulating overlapping signal transduction pathways.
PMCID: PMC2721674  PMID: 18723827
RSV; cigarette smoke condensate; chemokines; IRF; NF-κB
11.  Expression of Respiratory Syncytial Virus-Induced Chemokine Gene Networks in Lower Airway Epithelial Cells Revealed by cDNA Microarrays 
Journal of Virology  2001;75(19):9044-9058.
The Paramyxovirus respiratory syncytial virus (RSV) is the primary etiologic agent of serious epidemic lower respiratory tract disease in infants, immunosuppressed patients, and the elderly. Lower tract infection with RSV is characterized by a pronounced peribronchial mononuclear infiltrate, with eosinophilic and basophilic degranulation. Because RSV replication is restricted to airway epithelial cells, where RSV replication induces potent expression of chemokines, the epithelium is postulated to be a primary initiator of pulmonary inflammation in RSV infection. The spectrum of RSV-induced chemokines expressed by alveolar epithelial cells has not been fully investigated. In this report, we profile the kinetics and patterns of chemokine expression in RSV-infected lower airway epithelial cells (A549 and SAE). In A549 cells, membrane-based cDNA macroarrays and high-density oligonucleotide probe-based microarrays identified inducible expression of CC (I-309, Exodus-1, TARC, RANTES, MCP-1, MDC, and MIP-1α and -1β), CXC (GRO-α, -β, and -γ, ENA-78, interleukin-8 [IL-8], and I-TAC), and CX3C (Fractalkine) chemokines. Chemokines not previously known to be expressed by RSV-infected cells were independently confirmed by multiprobe RNase protection assay, Northern blotting, and reverse transcription-PCR. High-density microarrays performed on SAE cells confirmed a similar pattern of RSV-inducible expression of CC chemokines (Exodus-1, RANTES, and MIP-1α and -1β), CXC chemokines (I-TAC, GRO-α, -β, and -γ, and IL-8), and Fractalkine. In contrast, TARC, MCP-1, and MDC were not induced, suggesting the existence of distinct genetic responses for different types of airway-derived epithelial cells. Hierarchical clustering by agglomerative nesting and principal-component analyses were performed on A549-expressed chemokines; these analyses indicated that RSV-inducible chemokines are ordered into three related expression groups. These data profile the temporal changes in expression by RSV-infected lower airway epithelial cells of chemokines, chemotactic proteins which may be responsible for the complex cellular infiltrate in virus-induced respiratory inflammation.
PMCID: PMC114473  PMID: 11533168
12.  Helicobacter pylori Induces RANTES through Activation of NF-κB  
Infection and Immunity  2003;71(7):3748-3756.
Helicobacter pylori-infected gastric mucosa displays a conspicuous infiltration of mononuclear cells and neutrophils. RANTES (short for “regulated upon activation, normal T cell expressed and secreted”) is a chemoattractant cytokine (chemokine) important in the infiltration of T lymphocytes and monocytes. RANTES may therefore contribute to the cellular infiltrate in the H. pylori-infected gastric mucosa. The aim of this study was to analyze the molecular mechanism responsible for H. pylori-mediated RANTES expression. We observed that gastric epithelial cells produced RANTES upon coculture with H. pylori. In addition, H. pylori induced RANTES mRNA expression and an increase in luciferase activity in cells which were transfected with a luciferase reporter construct derived from the RANTES promoter, in gastric epithelial cells, indicating that the induction of RANTES production occurred at the transcriptional level. Induction of RANTES was dependent on an intact cag pathogenicity island. Activation of the RANTES promoter by H. pylori occurred through the action of NF-κB. Transfection of kinase-deficient mutants of IκB kinase (IKK) and NF-κB-inducing kinase (NIK) inhibited H. pylori-mediated RANTES activation. In contrast, tumor necrosis factor alpha- or interleukin-1/Toll-like receptor signaling molecules—such as mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1, MyD88, and interleukin-1 receptor-associated kinase—did not play a role in RANTES activation by H. pylori. Collectively, H. pylori induced NF-κB activation through an intracellular signaling pathway that involved IKK and NIK, leading to RANTES gene transcription. RANTES induction by H. pylori may play an important role in gastric inflammation.
PMCID: PMC162018  PMID: 12819056
13.  Interaction of Neisseria meningitidis with Human Meningeal Cells Induces the Secretion of a Distinct Group of Chemotactic, Proinflammatory, and Growth-Factor Cytokines  
Infection and Immunity  2002;70(8):4035-4044.
The interactions of Neisseria meningitidis with cells of the leptomeninges are pivotal events in the progression of bacterial leptomeningitis. An in vitro model based on the culture of human meningioma cells was used to investigate the role of the leptomeninges in the inflammatory response. Following challenge with meningococci, meningioma cells secreted specifically the proinflammatory cytokine interleukin-6 (IL-6), the CXC chemokine IL-8, the CC chemokines monocyte chemoattractant protein 1 (MCP-1) and regulated-upon-activation, normal-T-cell expressed and secreted protein (RANTES), and the cytokine growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). A temporal pattern of cytokine production was observed, with early secretion of IL-6, IL-8, and MCP-1 followed by later increases in RANTES and GM-CSF levels. IL-6 was induced equally by the interactions of piliated and nonpiliated meningococci, whereas lipopolysaccharide (LPS) had a minimal effect, suggesting that other, possibly secreted, bacterial components were responsible. Induction of IL-8 and MCP-1 also did not require adherence of bacteria to meningeal cells, but LPS was implicated. In contrast, efficient stimulation of RANTES by intact meningococci required pilus-mediated adherence, which served to deliver increased local concentrations of LPS onto the surface of meningeal cells. Secretion of GM-CSF was induced by pilus-mediated interactions but did not involve LPS. In addition, capsule expression had a specific inhibitory effect on GM-CSF secretion, which was not observed with IL-6, IL-8, MCP-1, or RANTES. Thus, the data demonstrate that cells of the leptomeninges are not inert but are active participants in the innate host response during leptomeningitis and that there is a complex relationship between expression of meningococcal components and cytokine induction.
PMCID: PMC128145  PMID: 12117909
14.  RANTES and Monocyte Chemoattractant Protein–1 (MCP-1) Play an Important Role in the Inflammatory Phase of Crescentic Nephritis, but Only MCP-1 Is Involved in Crescent Formation and Interstitial Fibrosis 
The Journal of Experimental Medicine  1997;185(7):1371-1380.
The involvement of chemokines in inflammation is well established, but their functional role in disease progression, and particularly in the development of fibrosis, is not yet understood. To investigate the functional role that the chemokines monocyte chemoattractant protein–1 (MCP-1) and RANTES play in inflammation and the progression to fibrosis during crescentic nephritis we have developed and characterized a murine model for this syndrome. Significant increases in T-lymphocytes and macrophages were observed within glomeruli and interstitium, paralleled by an induction of mRNA expression of MCP-1 and RANTES, early after disease initiation. Blocking the function of MCP-1 or RANTES resulted in significant decreases in proteinuria as well as in numbers of infiltrating leukocytes, indicating that both MCP-1 and RANTES (regulated upon activation in normal T cells expressed and secreted) play an important role in the inflammatory phase of crescentic nephritis. In addition, neutralization of MCP-1 resulted in a dramatic decrease in both glomerular crescent formation and deposition of type I collagen. These results highlight a novel role for MCP-1 in crescent formation and development of interstitial fibrosis, and indicate that in addition to recruiting inflammatory cells this chemokine is critically involved in irreversible tissue damage.
PMCID: PMC2196251  PMID: 9104823
15.  Transcriptional activation of the interleukin-8 gene by respiratory syncytial virus infection in alveolar epithelial cells: nuclear translocation of the RelA transcription factor as a mechanism producing airway mucosal inflammation. 
Journal of Virology  1996;70(12):8773-8781.
The most common cause of epidemic pediatric respiratory disease, respiratory syncytial virus (RSV), stimulates interleukin-8 (IL-8) synthesis upon infecting airway epithelium, an event necessary for the development of mucosal inflammation. We investigated the mechanism for enhanced IL-8 production in human A549 type II pulmonary epithelial cells. Infection with sucrose-purified RSV (pRSV) produced a time-dependent increase in the transcriptional initiation rate of the IL-8 gene. Transient transfection of the human IL-8 promoter mutated in the binding site for nuclear factor-kappaB (NF-kappaB) demonstrated that this sequence was essential for pRSV-activated transcription. Gel mobility shift assays demonstrated pRSV induction of sequence-specific binding complexes; these complexes were supershifted only by antibodies directed to the potent NF-kappaB transactivating subunit RelA. Both Western immunoblot and indirect immunofluorescence assays showed that cytoplasmic RelA in uninfected cells became localized to the nucleus after pRSV infection. RelA activation requires replicating RSV, because neither conditioned medium nor UV-inactivated pRSV was able to stimulate its translocation. We conclude that RelA undergoes changes in subcellular distribution in airway epithelial cells upon pRSV infection. The ability of replicating RSV to activate RelA translocation may play an important role in activating IL-8 and other inflammatory gene products necessary for airway mucosal inflammation seen in RSV disease.
PMCID: PMC190974  PMID: 8971006
16.  Selective induction of monocyte and not neutrophil-attracting chemokines after influenza A virus infection 
The Journal of Experimental Medicine  1996;184(3):1191-1196.
It is characteristic for virus infections that monocytes/macrophages and lymphocytes infiltrate infected tissue while neutrophils are absent. To understand the mechanisms selectively attracting mononuclear cells in viral diseases, we examined in an influenza A virus model the expression and regulation of chemokines as candidate molecules responsible for the immigration of leukocytes into inflamed tissue. After influenza A virus infection of human monocytes, a rapid expression of the mononuclear cell attracting CC-chemokine genes MIP-1, MCP-1, and RANTES occurred which was followed by the release of chemokine proteins. In striking contrast to CC-chemokines, the expression of the prototype neutrophil CXC-chemoattractants IL-8 and GRO-alpha was completely suppressed after influenza A infection. The release of other neutrophil chemotactic factors was excluded by microchemotaxis assays. These results suggest that the virus-specific induction of mononuclear cell-attracting chemokines accounts for the preferential influx of mononuclear leukocytes into virus-infected tissue.
PMCID: PMC2192790  PMID: 9064338
17.  Role of CCL5 (RANTES) in Viral Lung Disease 
Journal of Virology  2006;80(16):8151-8157.
CCL5/RANTES is a key proinflammatory chemokine produced by virus-infected epithelial cells and present in respiratory secretions of asthmatics. To examine the role of CCL5 in viral lung disease, we measured its production during primary respiratory syncytial virus (RSV) infection and during secondary infection after sensitizing vaccination that induces Th2-mediated eosinophilia. A first peak of CCL5 mRNA and protein production was seen at 18 to 24 h of RSV infection, before significant lymphocyte recruitment occurred. Treatment in vivo with Met-RANTES (a competitive chemokine receptor blocker) throughout primary infection decreased CD4+ and CD8+ cell recruitment and increased viral replication. In RSV-infected, sensitized mice with eosinophilic disease, CCL5 production was further augmented; Met-RANTES treatment again reduced inflammatory cell recruitment and local cytokine production. A second wave of CCL5 production occurred on day 7, attributable to newly recruited T cells. Paradoxically, mice treated with Met-RANTES during primary infection demonstrated increased cellular infiltration during reinfection. We therefore show that RSV induces CCL5 production in the lung and this causes the recruitment of RSV-specific cells, including those making additional CCL5. If this action is blocked with Met-RANTES, inflammation decreases and viral clearance is delayed. However, the exact effects of chemokine modulation depend critically on time of administration, a factor that may potentially complicate the use of chemokine blockers in inflammatory diseases.
PMCID: PMC1563837  PMID: 16873271
18.  Chemokines in the limbal form of vernal keratoconjunctivitis 
The British Journal of Ophthalmology  2000;84(12):1360-1366.
BACKGROUND/AIMS—Chemokines are a family of low molecular weight cytokines that attract and activate leucocytes. The CC chemokines act on eosinophils, basophils, monocytes, and lymphocytes, suggesting that they play an important part in allergic diseases. The aims of this study were to investigate the expression of the CC chemokines, RANTES, eotaxin, monocyte chemotactic protein (MCP) 1, MCP-2, and MCP-3 in the conjunctiva of patients with vernal keratoconjunctivitis (VKC) and to determine the cellular source of these chemokines.
METHODS—Conjunctival biopsy specimens from nine subjects with active VKC, and six control subjects were studied by immunohistochemical techniques using a panel of monoclonal and polyclonal antibodies directed against RANTES, eotaxin, MCP-1, MCP-2, and MCP-3. The phenotype of inflammatory cells expressing chemokines was examined by sequential double immunohistochemistry.
RESULTS—In the normal conjunctiva, superficial epithelial cells showed a constitutive, weak cytoplasmic expression of eotaxin. Few inflammatory cells in the perivascular areas expressed RANTES, MCP-1, MCP-2, and MCP-3. In VKC specimens, the epithelium showed intense cytoplasmic eotaxin staining in all cells, and cytoplasmic RANTES staining mainly in the superficial layers. Furthermore, RANTES and eotaxin were expressed on the vascular endothelium mainly in the upper substantia propria. Compared with normal controls, VKC specimens showed significantly more inflammatory cells expressing RANTES, eotaxin, MCP-1, and MCP-3 (p<0.001, 0.0028, 0.0092, and <0.001, respectively). In VKC specimens, the numbers of inflammatory cells expressing RANTES were significantly higher than the numbers of inflammatory cells expressing eotaxin, MCP-1, and MCP-2 (all p values <0.001). Colocalisation studies revealed that the majority of inflammatory cells expressing chemokines were CD68 positive monocytes/macrophages.
CONCLUSIONS—These results demonstrate an increase in the expression of RANTES, eotaxin, MCP-1, and MCP-3 in the conjunctiva of patients with VKC compared with control subjects. These data suggest a potential role for these chemokines in the pathogenesis of VKC. Antagonists of chemokine receptors may provide new therapeutic modalities in VKC.

PMCID: PMC1723358  PMID: 11090473
19.  Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils  
The Journal of Experimental Medicine  1996;183(6):2437-2448.
The chemokine eotaxin is unusual in that it appears to be a highly specific chemoattractant for eosinophils. Ligand-binding studies with radiolabeled eotaxin demonstrated a receptor on eosinophils distinct from the known chemokine receptors CKR-1 and -2. The distinct eotaxin binding site on human eosinophils also bound RANTES (regulated on activation T expressed and secreted) and monocyte chemotactic protein (MCP)3. We have now isolated a cDNA from eosinophils, termed CKR-3, with significant sequence similarity to other well characterized chemokine receptors. Cells transfected with CKR-3 cDNA bound radiolabeled eotaxin specifically and with high affinity, comparable to the binding affinity observed with eosinophils. This receptor also bound RANTES and MCP-3 with high affinity, but not other CC or CXC chemokines. Furthermore, receptor transfectants generated in a murine B cell lymphoma cell line migrated in transwell chemotaxis assays to eotaxin, RANTES, and MCP-3, but not to any other chemokines. A monoclonal antibody recognizing CKR-3 was used to show that eosinophils, but not other leukocyte types, expressed this receptor. This pattern of expression was confirmed by Northern blot with RNA from highly purified leukocyte subsets. The restricted expression of CKR-3 on eosinophils and the fidelity of eotaxin binding to CKR-3, provides a potential mechanism for the selective recruitment and migration of eosinophils within tissues.
PMCID: PMC2192612  PMID: 8676064
20.  Association of TGFβ1, TNFα, CCR2 and CCR5 gene polymorphisms in type-2 diabetes and renal insufficiency among Asian Indians 
BMC Medical Genetics  2007;8:20.
Cytokines play an important role in the development of diabetic chronic renal insufficiency (CRI). Transforming growth factor β1 (TGF β1) induces renal hypertrophy and fibrosis, and cytokines like tumor necrosis factor-alpha (TNFα), chemoattractant protein-1 (MCP-1), and regulated upon activation and normal T cell expressed and secreted (RANTES) mediate macrophage infiltration into kidney. Over expression of these chemokines leads to glomerulosclerosis and interstitial fibrosis. The effect of MCP-1 and RANTES on kidney is conferred by their receptors i.e., chemokine receptor (CCR)-2 and CCR-5 respectively. We tested association of nine single nucleotide polymorphisms (SNPs) from TGFβ1, TNFα, CCR2 and CCR5 genes among individuals with type-2 diabetes with and without renal insufficiency.
Type-2 diabetes subjects with chronic renal insufficiency (serum creatinine ≥ 3.0 mg/dl) constituted the cases, and matched individuals with diabetes of duration ≥ 10 years and normoalbuminuria were evaluated as controls from four centres in India. Allelic and genotypic contributions of nine SNPs from TGFβ1, TNFα, CCR2 and CCR5 genes to diabetic CRI were tested by computing odds ratio (OR) and 95% confidence intervals (CI). Sub-analysis of CRI cases diabetic retinopathy status as dependent variable and SNP genotypes as independent variable in a univariate logistic regression was also performed.
SNPs Tyr81His and Thr263Ile in TGF β1 gene were monomorphic, and Arg25Pro in TGF β1 gene and Δ32 polymorphism in CCR5 gene were minor variants (minor allele frequency <0.05) and therefore were not considered for case-control analysis. A significant allelic association of 59029G>A SNP of CCR5 gene has been observed and the allele 59029A seems to confer predisposition to development of diabetic CRI (OR 1.39; CI 1.04–1.84). In CRI subjects a compound group of genotypes "GA and AA" of SNP G>A -800 was found to confer predisposition for proliferative retinopathy (OR 3.03; CI 1.08–8.50, p = 0.035).
Of the various cytokine gene polymorphisms tested, allele 59029A of CCR5 gene is significantly associated with diabetic renal insufficiency among Asian Indians. Result obtained for 59029G>A SNP of CCR5 gene is in conformity with reports from a Japanese population but due to sub-optimal power of the sample, replication in larger sample set is warranted.
PMCID: PMC1853079  PMID: 17428349
21.  Inducible Expression of Inflammatory Chemokines in Respiratory Syncytial Virus-Infected Mice: Role of MIP-1α in Lung Pathology 
Journal of Virology  2001;75(2):878-890.
Lower respiratory tract disease caused by respiratory syncytial virus (RSV) is characterized by profound airway mucosa inflammation, both in infants with naturally acquired infection and in experimentally inoculated animal models. Chemokines are central regulatory molecules in inflammatory, immune, and infectious processes of the lung. In this study, we demonstrate that intranasal infection of BALB/c mice with RSV A results in inducible expression of lung chemokines belonging to the CXC (MIP-2 and IP-10), CC (RANTES, eotaxin, MIP-1β, MIP-1α, MCP-1, TCA-3) and C (lymphotactin) families. Chemokine mRNA expression occurred as early as 24 h following inoculation and persisted for at least 5 days in mice inoculated with the highest dose of virus (107 PFU). In general, levels of chemokine mRNA and protein were dependent on the dose of RSV inoculum and paralleled the intensity of lung cellular inflammation. Immunohisthochemical studies indicated that RSV-induced expression of MIP-1α, one of the most abundantly expressed chemokines, was primarily localized in epithelial cells of the alveoli and bronchioles, as well as in adjoining capillary endothelium. Genetically altered mice with a selective deletion of the MIP-1α gene (−/− mice) demonstrated a significant reduction in lung inflammation following RSV infection, compared to control littermates (+/+ mice). Despite the paucity of infiltrating cells, the peak RSV titer in the lung of −/− mice was not significantly different from that observed in +/+ mice. These results provide the first direct evidence that RSV infection may induce lung inflammation via the early production of inflammatory chemokines.
PMCID: PMC113984  PMID: 11134301
Virology  2007;368(1):91-101.
Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and type I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-κB, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immuno-modulatory mediators.
PMCID: PMC2266690  PMID: 17655903
hMPV; airway epithelial cells; chemokines; inflammation; transcription factors
23.  Inducible translational regulation of the NF-IL6 transcription factor by respiratory syncytial virus infection in pulmonary epithelial cells. 
Journal of Virology  1996;70(3):1554-1563.
Respiratory syncytial virus (RSV), the most common etiologic agent of epidemic pediatric respiratory disease, infects and replicates in the human airway epithelium, resulting in the induction of cellular gene products essential for immune and inflammatory responses. We describe the effect of RSV infection on nuclear factor-IL6 (NF-IL6) expression, a human basic domain-leucine zipper-containing transcription factor that alone in combination with other inducible transcription factors regulates the expression of cytokine and adhesion molecule genes. RSV-infected human type II pulmonary alveolar epithelial cells (A549) synthesize a single 45.7-kDa isoform of NF-IL6 rapidly and in a time-dependent manner. NF-IL6 is first detectable after 3 h of infection and continues to accumulate until 48 h (until the cells lose viability). NF-IL6 production could not be induced by UV-inactivated virus, demonstrating the requirement of viral replication for NF-IL6 synthesis. Immunoprecipitation after [35S]methionine metabolic labeling was done to investigate the mechanism for NF-IL6 production. There was robust NF-IL6 protein synthesis within RSV-infected (24 h) cells. Protein synthesis occurred without detectable changes in the abundance or size of the single 1.8-kb NF-IL6 mRNA. RNase protection assay of transfected chloramphenicol acetyltransferase reporter genes driven by either wild-type or mutated NF-IL6 binding sites show a virus-induced increase in NF-IL6-dependent transcription. These studies have demonstrated a novel inducible mechanism for translational control of NF-IL6 synthesis and identify this transcription factor as a potential effector of the host response to RSV infection.
PMCID: PMC189977  PMID: 8627674
24.  Induction of IL-6 and CCL5 (RANTES) in human respiratory epithelial (A549) cells by clinical isolates of respiratory syncytial virus is strain specific 
Virology Journal  2012;9:190.
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.
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.
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.
PMCID: PMC3463437  PMID: 22962966
Respiratory syncytial virus; Clinical isolates; IL-6; RANTES; CCL5; Respiratory syncytial virus genome
25.  Prostate secretions from men with CP/CPPS inhibit proinflammatory mediators 
The Journal of urology  2010;184(4):1536-1542.
In the past, numerous chemokines have been shown to be present in the expressed prostatic secretions (EPS) of patients with CP/CPPS. This study examined the functional effects of chemokines in the EPS of patients with CPPS.
Materials and Methods
The functional effects of EPS on human monocytes were studied by examining monocyte chemotaxis in response to MCP-1, a major chemoattractant previously identified in CP/CPPS. Effects on cellular signaling were determined by quantifying intracellular calcium elevation in monocytes and activation of NF-κB in benign prostate epithelial cells.
Our results show that the MCP-1 present in EPS is non-functional, with an inability to mediate chemotaxis of human monocytes or mediate signaling in either monocytes or prostate epithelial cells. Moreover, this absence of functionality could be extended to other proinflammatory cytokines such as IL-1β and TNFα, when incubated with the EPS from CPPS patients. The mechanism underlying this apparent ability to modulate pro-inflammatory cytokines involves heat labile extracellular proteases that mediate inhibition of both immune and prostate epithelial cell function.
These results may have implications for the design of specific diagnostics and therapeutics targeted at complete resolution of prostate inflammatory insults.
PMCID: PMC3660015  PMID: 20723928
Chronic pelvic pain syndrome; prostatitis; chemokines; inflammation; pelvic pain

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