To demonstrate binding specificity, 100-fold molar excess (10 ng) of a specific or non-specific oligonucleotide (5′-ATTCGATCGGGGCGGGGCGAGC-3′) was included in the binding reaction
To demonstrate binding specificity, 100-fold molar excess (10 ng) of a specific or non-specific oligonucleotide (5′-ATTCGATCGGGGCGGGGCGAGC-3′) was included in the binding reaction. TNF and RSV strongly induced Rel A the activation subunit of NF-B, whereas only TNF was able to substantially induce the p50 subunit. Consistent with the expression studies, RSV but not TNF induction of Rel A and p50 were markedly inhibited by SMAD9 NAC, providing a mechanism by which TNF and RSV can differentially activate chemokine gene expression via NF-B. Conclusions These data suggest that RSV induction of chemokine gene expression, in contrast to TNF, involves redox-sensitive NF-B complexes containing predominantly Rel A. Background Respiratory syncytial virus (RSV) belongs to the Pneumovirinae subfamily of the Paramyxovirodae family of enveloped single-stranded negative sense RNA viruses. RSV infection of the lower respiratory tract cells results in cell death and sloughing into the lumen of the respiratory tree. Worldwide, RSV is the leading cause of infant mortality from respiratory infections and is so highly contagious that by age two nearly all children have been infected. RSV infection in infancy cause severe bronchiolitis and pneumonia and may predispose children to the subsequent development of asthma, the most common chronic illness of childhood . Many studies have indicated that chemokines can play an important role in the onset and severity of asthma and it has been demonstrated that RSV illness of lung epithelial cells raises chemokine production, even though mechanisms involved are mainly unfamiliar [2-5]. The chemotactic cytokines, or chemokines, compose a large superfamily of small structurally related polypeptides that perform important tasks in host defense by recruiting specific subsets of leukocytes to sites of swelling and injury . Chemokines have been connected with a number of inflammatory diseases and conditions, including asthma, sepsis, inflammatory bowel disease, and adult respiratory stress syndrome [7-9]. The chemokine superfamily can be divided into two major groups based on MIF Antagonist the position of the 1st two of four-conserved cysteine residues in the amino terminus, which are either adjacent (CC subfamily) or separated by one amino acid (CXC subfamily). The CXC chemokines such as IL-8 were originally identified as potent activators and chemoattractants for neutrophils, whereas the CC chemokines such as MCP-1 and RANTES mostly entice monocytes and eosinophils respectively . Chemokines are secreted inside a stimulus-and cell type-specific manner [11-17] and are regulated primarily at the level of gene transcription [18-24]. The transcriptional promoters of IL-8, RANTES and MCP-1 consist of binding sites for the redox-responsive transcription element NF-B, which has been shown to be important for his or her rules by viral infections and cytokines [18,20,23,25-34]. We previously shown the chemokines IL-8, MCP-1 and RANTES are differentially controlled in A549 airway epithelial cells [35-38]. To further elucidate the mechanisms of chemokine manifestation in A549, we have compared the induction of IL-8, MCP-1 and RANTES by RSV illness with that of TNF. Our findings suggest that RSV induction of chemokine gene manifestation involves a redox-sensitive NF-B signaling mechanism that differs from that mediated by TNF and including mainly the Rel A subunit of NF-B. Materials and methods Materials Dulbecco’s Modified Eagle Medium (DMEM), fetal MIF Antagonist bovine serum (FBS), Dulbecco’s phosphate buffered saline (DPBS), antibiotic/antimycotic, 1% trypsin/EDTA, Hanks Balanced Salt Remedy (HBSS) and TRIZOL were purchased from Invitrogen Gibco Cell Tradition (Carlsbad, CA). N-acetyl-L-cysteine, dexamethasone, glycerol and MTT tetrazolium salt were from Sigma (St. Louis, MO). TNF was from R&D systems (Minneapolis, MN). ELISA kits were purchased from Pierce Endogen (Rockford, IL). Human being CK5 RiboQuant ribonuclease safety MIF Antagonist assay kit was purchased from BD Pharmingen (San Diego, CA). [-32P]UTP (250 Ci) was from Perkin Elmer Existence Sciences (Boston, MA). Gel shift assay system was purchased from Promega (Madison, WI). [-32P]ATP (500 Ci) was from ICN (Costa Mesa, CA). Antibodies were purchased from Santa Cruz Biotechnology (Santa MIF Antagonist Cruz, CA). The A549 cell collection and RSV Very long strain.