Korean J Asthma Allergy Clin Immunol.  2006 Mar;26(1):19-26.

The Mechanism of RANTES Production by Rhinovirus in Alveolar Epithelial Cells: Evidence for NF-kappaB-dependent Transcriptional Activation

Abstract

BACKGROUND
Rhinovirus (RV) is a well-known cause of upper respiratory tract infections and an important trigger of asthmatic exacerbation. RV can augment tissue eosinophilia when combined with antigen in appropriately sensitized patients. RANTES, a subfamily of chemokines with double cysteine motif, is a potent eosinophil chemoattractant.
OBJECTIVE
This study was designed to investigate RV stimulation of RANTES and NF-kappaB-dependent transcriptional mechanism in alveolar epithelial cells in vitro. METHOD: After RV14 infecion on A549 cells, RANTES protein and mRNA were measured using ELISA and RT- PCR. To further understand the transcriptional mechanisms of RANTES production, NF-kappaB activity was checked with gel shift assay including supershift and competition assay. The mechanism of RANTES promoter activation was analyzed by cis-element assay with mutagenesis experiment. NF-kappaB activity was inhibited with antioxidant, TLCK, TPCK, and mutant IkappaBalpha. RESULT: RANTES and mRNA levels were increased and peaked at 12 hours after infection with RV14. RV also stimulated NF-kappaB-DNA binding activity. Supershift assays revealed that this binding was due to p65 and, to a lesser extent, p50 NF-kappaB subunits. Cis-element analysis of RANTES promoter showed that NF-kappaB binding site between -50 and -40 was the most important region. A dominant negative mutant of IkappaBalpha abrogated RV-induced RANTES promoter activity. Selective antioxidants, N-acetylcysteine, and NF-kappaB blockers (TLCK, TPCK) inhibited RV-stimulated RANTES production and promoter activity in alveolar epithelial cells.
CONCLUSION
RV is a potent stimulator of RANTES production in A549 cells in vitro. This inductive effect is, to a great extent, transcriptionally mediated and dependent on NF-kappaB activation. The most important subuinits of NF-kappaB are p65/p50, which are interacted with RNATES promoter site between -50 and -40. RANTES production, are inhibited by antioxidant and NF-kappaB blockers.

MeSH Terms

Acetylcysteine
Antioxidants
Binding Sites
Chemokine CCL5*
Chemokines
Cysteine
Enzyme-Linked Immunosorbent Assay
Eosinophilia
Eosinophils
Epithelial Cells*
Humans
Mutagenesis
NF-kappa B
Polymerase Chain Reaction
Respiratory Tract Infections
Rhinovirus*
RNA, Messenger
Tosyllysine Chloromethyl Ketone
Tosylphenylalanyl Chloromethyl Ketone
Transcriptional Activation*
Acetylcysteine
Antioxidants
Chemokine CCL5
Chemokines
Cysteine
NF-kappa B
RNA, Messenger
Tosyllysine Chloromethyl Ketone
Tosylphenylalanyl Chloromethyl Ketone