MultiplecisRegulatory Elements Control RANTES Promoter Activity in Alveolar Epithelial Cells Infected with Respiratory Syncytial Virus

Abstract

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-B, C/EBP, Jun/CREB/ATF, and interferon regulatory factor Respiratory syncytial virus (RSV) is an enveloped, negativesense single-stranded RNA virus (18). Since its isolation, RSV has been identified as a leading cause of epidemic respiratory tract illness in children in the United States and worldwide. In fact, RSV is so ubiquitous that it will infect 100% of children before the age of 3 (15). In infants and young children, RSV is the most common etiologic agent of bronchiolitis and is also responsible for 50% of pneumonia cases in children up to 2 years of age (38). Each year approximately 100,000 children are hospitalized with RSV disease, with an estimated annual cost close to $300 million in the United States alone (15, 17).The main targets of RSV infection are respiratory epithelial cells. In bronchiolitis and pneumonia, RSV antigen can be identified in epithelial cells from throughout the lower respiratory tract, with less virus found in lungs of children with bronchiolitis than in lungs of children with pneumonia, where large amounts of viral antigen are detected. Necrosis of the airway epithelium is associated with mononuclear cell infiltration, mainly peribronchial and perivascular in bronchiolitis, and between the interalveolar walls, leading to alveolar filling, in pneumonia (reviewed in reference 38). Moreover, the presence of cell-specific inflammatory mediators in nasopharyngeal secretions and in tracheobronchial aspirates of children with bronchiolitis suggests that RSV infection triggers the migration to the airways and local activation of eosinophil and basophil leukocytes (11,13).Much of the cellular response at sites of tissue inflammation is controlled by gradients of chemotactic factors that direct leukocyte transendothelial migration and movement through the extracellular matrix. The composition of this cellular response is dependent on the discrete target cell selectivity of these chemotactic molecules. Chemokines, a family of small chemotactic cytokines, regulate the migration and activation of leukocytes and therefore play a key role in inflammatory and infectious processes of the lung (29). RANTES (regulated upon activation, normal T-cell expressed and presumably secreted) is a member of the CC branch of the che...

Transcriptional Mechanisms Regulating Alveolar Epithelial Cell-specific CCL5 Secretion in Pulmonary Tuberculosis

“…For example, in IL-1␤-stimulated astrocytoma cells (56) and T cells (26), deletions to 400 base pairs have little effect on CCL5 promoter activity. This was also the situation in alveolar epithelial cells stimulated by either RSV or TNF␣, where a Ϫ220 deletion of the CCL5 promoter retained near wild type activity (54,55). …”

Activation of Monocytic Cells Through Fcγ Receptors Induces the Expression of Macrophage-Inflammatory Protein (MIP)-1α, MIP-1β, and RANTES

“…Many different monocytic cell lines, including THP-1 cells, show constitutive nuclear B activity (31), and unlike MIP-1␣, RANTES has two B sites near the TATA signal, thus explaining possible interactions with components of the general transcription machinery that could account for the expression of RANTES detected in resting cells. In contrast, the delayed pattern of RANTES mRNA induction by IC agrees with the characterization of RANTES as an unusual gene induced late after T lymphocyte activation, the expression of which has been related to a novel transcription factor termed RANTES factor of late activated T lymphocytes, which belongs to the Kruppel-like family of transcription factors (32), although other factors have been associated with the cell-specific pattern of transcriptional regulation of RANTES, among them NF-B (33), C/EBP␤ (34), and IFN regulatory factor (35).…”

Induction of RANTES/CCL5 by herpes simplex virus is regulated by nuclear factor κB and interferon regulatory factor 3

“…We and others have previously found that these two transcription factors become activated during HSV infection (Preston et al, 2001;Paludan, 2001). NF-kB and IRF-3 have also been shown to be essential for RANTES expression in response to some virus infections (Lin et al, 1999;Genin et al, 2000;Casola et al, 2001), whereas alternative mechanisms, notably MAP kinase-dependent pathways, play major roles in RANTES expression during other virus infections (Kujime et al, 2000). Our results did not support the idea that p38 is involved in RANTES transcription, but the data do not exclude a minor role for p38 at the posttranscriptional level.…”

“…Various sets of transcription factors are responsible for expression of RANTES depending on the cellular context and inducing agent. Several studies have shown that nuclear factor kB (NF-kB), the mitogen-activated protein (MAP) kinase system and the interferon regulatory factor (IRF) family are involved in virus-induced expression of RANTES (Lin et al, 1999;Genin et al, 2000;Kujime et al, 2000;Casola et al, 2001;Pazdrak et al, 2002).…”

“…Selection was performed using G418 at a concentration of 300 mg ml 21 . Transient transfections were carried out with a luciferase-linked construct of the wild-type RANTES promoter (Casola et al, 2001). Transfection efficiency and variation within plates was investigated using a GFP-vector (pEGFP-N1; Clontech), counting transfected cells by fluorescence microscopy.…”