NR.It is possible that PNR suppressed the transcription of the IκBα gene. To examine this possibility, we carried out Northern blot analysis of IκBα mRNA expression in A549 cells stimulated with PNR. A549 cells were cultured with PNR or medium, and total RNA was extracted at 1 h of culture. Induction of IκBα mRNA by PNR was observed (Fig.8). It was reported previously that transcription of the IκBα gene is regulated by the NF-κB binding site in the promoter and is activated by NF-κB p65 or c-Rel (12). These results clearly indicate that the decrease of IκBα protein induced by PNR did not result from suppression of transcription but from modulation of posttranscriptional events, probably at the protein level. Cumulatively, these results indicate that PNR activates A549 cells, as reflected by a loss of IκBα protein and up-regulation of its mRNA.
Induction of IκBα mRNA in A549 cells by PNR. A549 cells were cultured with PNR (5 μg/ml, lane 3) or medium alone (lane 2) for 1 h. Total RNA was assessed for IκBα expression. Equal loading was assessed by hybridization of a stripped blot with a probe for GAPDH.
P. aeruginosa affects neutrophil recruitment into the airways by direct and indirect mechanisms. Among the chemotactic factors for neutrophils produced by the airways, IL-8 is one of the most potent. Because epithelial cells are sources of IL-8 in the airways, we and others hypothesized that P. aeruginosa may affect neutrophil migration by stimulating the production of IL-8 in airway epithelial cells. This hypothesis provides an important model to elucidate the mechanism by which P. aeruginosa infection increases IL-8 levels and causes dense neutrophil infiltration in the airways of patients with CAD. Previous studies have shown that a nonprotein factor of less than 1 kDa in culture supernatant of P. aeruginosa stimulates IL-8 expression and production by bronchial epithelial cells (16). Pilin-mediated adherence of P. aeruginosa as well as Pseudomonas autoinducer was reported to be a potent stimulus for IL-8 production by bronchial epithelial cells (5). Through the analysis of an inducer among Pseudomonas products for IL-8 production in bronchial epithelial cells, we have recently reported the nitrite reductase fromP. aeruginosa to be a potent IL-8 inducer in respiratory cells (27). The PNR, with a molecular mass of 60,204 Da, is recognized as a periplasmic component active in energy generation (35). This enzyme oxidizes reduced Pseudomonascytochrome c551 under aerobic conditions and also reduces nitrite to nitric oxide by using reducedPseudomonas cytochrome c551 under anaerobic conditions (38, 39). It seems that the purified PNR did not stimulate epithelial cells to produce IL-8 by released nitric oxide through its enzymatic reaction, because in vitro cultures were under aerobic conditions without electron donors such asPseudomonas cytochrome c551(38). Our previous studies with reducedPseudomonas cytochrome c551 under aerobic conditions showed loss of potent cytochrome oxidase activity in the purified PNR (27). Indeed, the purified PNR did not stimulate nitric oxide production under the culture conditions (data not shown). Therefore, the enzymatic activity of PNR is not essential for the IL-8-inducing activity of PNR, and a direct stimulation of bronchial epithelial cells by the PNR is a possible mechanism for IL-8 gene induction (27).
We have recently reported that PNR could be released from P. aeruginosa after exposure to antimicrobial agents or complement and that released PNR is active in inducing IL-8 production by bronchial epithelial cells (33). Moreover, we found increased levels of immunoglobulin G and immunoglobulin A antibodies against PNR in sera from patients with CAD and P. aeruginosainfections. However, hyperimmune sera from these patients did not neutralize PNR-mediated IL-8 induction in bronchial epithelial cells (26).
This study was done to define the sites in the IL-8 promoter that are required for PNR induction of IL-8 gene expression in airway epithelium. Optimal PNR-induced IL-8 gene activation required an NF-κB binding site in the 5′-flanking region of the gene. In addition, PNR incubation directly results in activation of the transcription factor that binds to this site, NF-κB.
We initially demonstrated, by use of luciferase constructs with serial deletions in the IL-8 promoter, that the region between −1481 and −98 bp in the 5′-flanking region of the IL-8 gene is not essential for PNR-induced IL-8 gene expression. Further deletions up to −50 bp completely abolished the response of the promoter to PNR. This suggests that the region between −98 and −50 bp contains elements that are essential for PNR induction of IL-8 gene activation. Within this segment of the 5′-flanking region of the IL-8 gene, there are several potential binding sites for transcription factors, including NF-κB and NF-IL-6 (Fig. 2A).
Several prior studies, using various stimuli and cell lines, demonstrated that NF-κB and NF-IL-6 play a vital role in the regulation of IL-8 gene expression. The studies also suggested that each factor may possess a variable degree of importance, depending on the cell line and the stimulus used (22), and that induction of the IL-8 gene by TNF-α, IL-1, phorbol myristate acetate, and hepatitis B virus protein X requires the cooperative effect of NF-κB and NF-IL-6 (15, 23). On the other hand, IL-8 gene activation by TNF-α, gamma interferon, cytomegalovirus, and paclitaxel appears to be mediated by the AP-1 site in conjunction with the NF-κB site in a human gastric cancer cell line (40), a monocytic cell line (24), and a human ovarian carcinoma cell line (13). In contrast to previous reports, we now report that PNR induction of IL-8 is independent of intact NF-IL-6 and AP-1 sites. These results suggest that different sets of nuclear transcription factors may be responsible for the regulation of IL-8 gene transcription in a cell type- and stimulus-specific manner. Interestingly, AP-1 was the preferred transcription factor (over NF-IL-6) for cooperative interaction with NF-κB for IL-8 gene expression in respiratory syncytial virus-infected A549 cells (17). However, our present results demonstrate that the activation of NF-κB, but not that of NF-IL-6 and AP-1, was indispensable for PNR-induced IL-8 gene transcription, suggesting that the molecular mechanism involved in IL-8 gene transcription differed, even within the same cell, depending on the employed stimuli.
To complement these observations, we also determined if PNR could induce the degradation of IκBα in A549 epithelial cells. Similar to other transient stimuli, PNR induced the degradation of IκBα in A549 cells. Degradation of IκBα in PNR-induced A549 cells was associated with translocation of NF-κB and increased steady-state transcription of IκBα mRNA. Further, the levels of IκBα protein in PNR-induced A549 cells were restored to a level similar to that of unstimulated A549 cells after 3 h of incubation. Restoration of IκBα in PNR-stimulated A549 cells was sensitive to the effect of cycloheximide, indicating a dependency on new protein synthesis.
Infections with P. aeruginosa trigger dense neutrophil infiltrations in the airways of patients with CAD (28). PNR-induced NF-κB activation and the subsequent up-regulation of IL-8 could contribute to inflammatory cell recruitment. Because of its pivotal role in inflammation, NF-κB could be an obvious target for new types of anti-inflammatory treatments for CAD. Blocking the activation of NF-κB may prevent the early events in the inflammatory cascade, decreasing P. aeruginosa-induced inflammation.
In summary, we have shown that PNR regulates IL-8 gene expression at the transcriptional level, acting through the 5′-flanking region of the IL-8 gene. The transcriptional factor NF-κB is necessary for PNR-induced IL-8 up-regulation, indicating one molecular mechanism ofP. aeruginosa-induced airway inflammation.
We thank Dean W. Ballard for providing the cDNA probe encoding human IκBα. We gratefully acknowledge Mika Yamamoto and Masako Sasaki for excellent technical assistance and Bent W. Nielsen for his helpful comments during the preparation of the manuscript.
Oishi, K. Unpublished data.
Carbone P. P.
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