Respiratory syncytial virus and TNFalpha induction of chemokine gene expression involves differential activation of Rel A and NF-kappaB1

Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), Dulbecco's phosphate buffered saline (DPBS), antibiotic/antimycotic, 1% trypsin/EDTA, Hanks Balanced Salt Solution (HBSS) and TRIZOL were purchased from Invitrogen Gibco Cell Culture (Carlsbad, CA). N-acetyl-L-cysteine, dexamethasone, glycerol and MTT tetrazolium salt were obtained from Sigma (St. Louis, MO). TNFα was obtained from R&D systems (Minneapolis, MN). ELISA kits were purchased from Pierce Endogen (Rockford, IL). Human CK5 RiboQuant ribonuclease protection assay kit was purchased from BD Pharmingen (San Diego, CA). [α-³²P]UTP (250 μCi) was obtained from Perkin Elmer Life Sciences (Boston, MA). Gel shift assay system was purchased from Promega (Madison, WI). [γ-³²P]ATP (500 μCi) was obtained from ICN (Costa Mesa, CA). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The A549 cell line and RSV Long strain were obtained from the American Type Culture Collection (Rockville, MD).

RSV, Long strain, was grown on HEp-2, a human tracheal epithelial cell line. Cells were grown to 50% confluence in DMEM containing 7% FBS and 1% antibiotic/antimycotic. After two washes with 1X DPBS, a minimal volume of RSV, at a multiplicity of infection (MOI) of 1 or greater, containing less than 1% FBS was added. The virus and cells were incubated for 2 hours, after which DMEM was added to bring culture to normal growth volume and 7% FBS. Cultures were then incubated for 48 hours. All plates were scraped and contents were transferred to 50 ml conical tubes. The virus/cell cocktail was vortexed briefly and large debris was removed in a tabletop centrifuge. RSV was purified by ultracentrifugation through 30% glycerol in HBSS. RSV stocks were resuspended in HBSS containing 0.5% bovine serum albumin and 100 mM magnesium sulfate and frozen at -70° C.

The A549 human type II lung carcinoma cell line was grown and maintained in DMEM containing 7% FBS and 1% antibiotic/antimycotic. For treatment, cells were grown to approximately 75% confluence, washed once with 1X DPBS and incubated overnight with DMEM containing 1% FBS. Cultures were then washed twice with 1X DPBS and pretreated with inhibitors DEX or NAC in serum-free DMEM to final concentrations as indicated in the figures. RSV and TNFα were added to the DMEM/inhibitor mix to final concentrations as indicated in the figures. One hour after agonist addition, FBS was added to 1% concentration for the duration of the experiment.

After addition of RSV and TNFα, cells were cultured for 24 h. Supernatants were collected and spun at high speed to remove cellular debris. The resulting suspension was frozen at -70°C until assayed following manufacturer's instructions by chemokine-specific ELISA (Endogen).

RNase protection assays (RPA) were performed using an RPA kit purchased from BD Pharmingen. In brief, total RNA was isolated from stimulated A549 cells with TRIZOL. The hCK-5 multiprobe template set was used to synthesize RNA probes for the chemokines lymphotactin, RANTES, IP-10, MIP-1α, MIP-1β, IL-8, MCP-1, and I-309 as well as the house-keeping genes L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) labeled with [α-³²P]UTP using T7 RNA polymerase. 3 × 10⁵ cpm of labeled probe were hybridized with 10 μg of total RNA for 16 h at 56°C. MRNA-probe hybrids were treated with RNase cocktail and phenol-chloroform extracted. Protected hybrids were resolved on a 6% denaturing polyacrylamide sequencing gels and exposed to a Molecular Dynamics detection screen overnight. Laser densitometry was performed using a Molecular Dynamics Storm scanner system (Molecular Dynamics, Inc., Sunny vale, CA).

Electrophoretic mobility shift assays (EMSA) were performed essentially as described previously [39]. Briefly, nuclear protein extracts (7 μg protein) prepared form A549 cells by the method of Osborn et al[40] were incubated with 5 × 10⁵ cpm (~0.1 ng) of ³²P end-labeled consensus NF-κB oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3') probe contained in the Promega gel shift assay system for 20–30 min at room temperature in 10 μl reaction volume using the kit provided reaction buffer. 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. Protein-DNA and protein-DNA-antibody complexes were resolved in 5% polyacrylamide gels preelectrophoresed for 30 min at room temperature in 0.25X TBE buffer (22.5 mM Tris-borate and 0.5 mM EDTA, pH 8.3). Gels were dried and exposed to radiographic film with an intensifying screen at -70°C.

NF-κB subunits Rel A (p65) and NF-κB1 (p50) were quantified using Trans-AM™ transcription factor assay kit from Active Motif (Carlsbad, CA). The assay is essentially an ELISA in which the consensus NF-κB binding site (5'-GGGACTTTCC-3') is immobilized onto the 96 well plate. Nuclear cell extracts (3 μg) were added to the wells and assayed for either Rel A or p50 binding as per the manufacturer's instructions. Optical density was determined on a spectophotometer at 450 nm.

As shown in Fig. 1, RSV infection of A549 epithelial cells induces the production and secretion of the chemokines IL-8, MCP-1, and RANTES. In A549 epithelial cells substantial MCP-1 was constitutively expressed whereas little or no spontaneous IL-8 or RANTES was detected. However, upon RSV infection (MOI = 3) a significant increase in chemokine production and secretion was observed 24 hr post-infection for each of the chemokines.

To determine whether the chemokine induction was mediated by increased chemokine gene expression we assessed the kinetics of mRNA synthesis by chemokine specific RNase protection. A549 cells were infected with RSV (MOI = 1) or stimulated with TNFα (100 ng/ml) and total RNA isolated at various times post-treatment over a 24 hr time course. As shown in Fig. 2, RSV rapidly induced IL-8 and MCP-1 with steady-state mRNA readily detected as early as 1 hr and continued to increase over the 24 hr time course. In contrast, RANTES mRNA was not detected until 4 hrs post infection after which its expression rapidly increased also reaching a maximum at 24 hrs. The RSV induction kinetics differed dramatically from the induction kinetics for TNFα in the same experiment. Whereas RSV induction of the chemokines did not reach a maximum until 24 hrs, TNFα induction was maximal between 4 and 8 hrs and was either unchanged or lower at 24 hrs post-infection.

The magnitude of induction also differed between TNFα and RSV infection. In general TNFα was a stronger inducer of chemokine expression. This difference was also reflected in the amount of chemokine secreted from the cells (data not shown). Together, these data indicate that RSV infection of A549 epithelial cells induces chemokine expression with distinct kinetics suggesting the mechanism of RSV induction differs from that of proinflammatory cytokines.

IL-8, MCP-1 and RANTES gene expression are regulated by the redox responsive transcription factor NF-κB [18, 20, 23, 25‐33, 41‐44]. The NF-κB signaling pathways have been shown to be differentially affected by antioxidants and glucocorticoid steroids [45]. To determine whether RSV and TNFα induction of chemokine expression might be mediated by different NF-κB signaling pathways we pretreated A549 cells with the glutathione precursor N-acetyl-L-cysteine (NAC) or the synthetic glucocorticoid dexamethasone (DEX). Twenty-four hours post infection, total RNA was isolated and steady state mRNA expression analyzed by chemokine specific RNase protection. As shown in Fig. 3, the two inhibitors had very different effects on chemokine gene expression. Most strikingly, RSV induced chemokine expression was more sensitive to NAC, whereas TNFα induced chemokine expression was conversely more sensitive to DEX. Thus, NAC preferentially inhibited chemokine expression induced by RSV, while DEX predominantly inhibited chemokine expression induced by TNFα.

NF-κB binds to the chemokine promoter as either homo- or heterodimers composed of Rel A (p65) and NF-κB1 (p50). Initially we detected TNFα and RSV induction of NF-κB by EMSA (KA Roebuck and LR Carpenter unpublished data) but because multiple gel shift complexes were induced it was not possible to distinguish the individual contributions of Rel A and p50 to the formation of these complexes. Therefore to assess the induction of the Rel A and p50 subunits individually in A549 nuclear cell extracts, we used a subunit specific NF-κB binding ELISA system that utilizes anti-Rel A and anti-p50 antibodies. As shown in Fig. 4, Rel A and p50 subunits of NF-κB are differentially expressed and induced in A549 epithelial cells. In untreated cells, no Rel A (solid bars) was detectable whereas in resting cells constitutive levels of p50 were observed (open bars), suggesting that unstimulated A549 epithelial cells contain p50 homodimers, which with regard to transcriptional activity have been shown to be functionally inert or inhibitory. In contrast, cells stimulated by TNFα (Fig. 4A) or infected by RSV (Fig. 4B) markedly induced Rel A, the transcriptionally active subunit of NF-κB. In addition, TNFα was also able to induce p50 by about 5-fold whereas RSV increased the constitutive p50 level by only about 50%. The induction of Rel A and p50 were specific since competition with either wild type or mutant consensus NF-κB oligonucleotide respectively inhibited or not the RSV and TNFα induced binding activity. These data demonstrate that TNFα is a potent inducer of both Rel A and p50 whereas RSV primarily induces the Rel A subunit of NF-κB.

To determine whether RSV and TNFα activate NF-κB differently, we examined the effects of NAC and DEX on Rel A and p50 binding activity. As shown in Fig. 4, Rel A and p50 subunits are differentially inhibited by NAC and DEX which have been shown to inhibit NF-κB by distinct mechanisms. Both NAC and DEX were unable to inhibit TNFα induction of either Rel A or p50. In contrast, in RSV infected cells both DEX and NAC inhibited Rel A and p50 binding activity. However, the inhibitory effects were most striking for Rel A suggesting the inhibitors primarily target the RSV induction of Rel A.

The differential effects of NAC on TNFα and RSV induced NF-κB binding activity correlated with its effects on chemokine gene expression (Fig. 3) and protein secretion (data not shown) suggesting that RSV induces IL-8, MCP-1 and RANTES through redox-sensitive NF-κB binding complexes. DEX also showed differential effects on NF-κB binding activity with DEX preferentially inhibiting RSV induction of NF-κB. However, the effects of DEX on NF-κB binding induced by TNFα did not correlate with its effects on chemokine expression, suggesting in TNFα activation of chemokine gene expression DEX targets another mechanism of chemokine induction not involving NF-κB.