Transcriptional Regulation of IL-8 by Staphylococcus aureus in Human Conjunctival Cells Involves Activation of AP-1

Abstract

purpose. To identify signal transduction pathways involved in interleukin (IL)-8 expression by human conjunctival cells challenged with Staphylococcus aureus.

methods. Conjunctival cells were cultured in the presence of live or heat-killed S. aureus. IL-8 protein and mRNA were determined by ELISA and RT-PCR, respectively. Activation of mitogen-activated protein kinases (MAPKs) and NF-κB was analyzed by Western blot analysis with phosphospecific antibodies. Conjunctival cells were transfected with wild-type (wt) or mutated IL-8 promoters (IL-8-97, lacking the AP-1 site; IL-8-97 mutant C/EBP; IL-8-97 mutant NF-κB; IL-8/AP-1 double mutant for C/EBP and NF-κB) or c-Jun-NH₂-terminal kinase (JNK)–responsive GAL-c-Jun. In further experiments, cells were cotransfected with wt IL-8 promoter and expression plasmids for p38MAPK-responsive C/EBP homologous protein (CHOP) or wt or dominant negative transactivation domain mutant (TAM-67) c-Jun. A protein–DNA binding study was performed by electrophoretic mobility shift assay (EMSA), to identify the transcription factors bound to the IL-8 promoter.

results. S. aureus induced significant IL-8 expression and synthesis in human conjunctival epithelial cells by activating c-Jun phosphorylation and transactivation potential via JNK. The IL-8 promoter activation was NF-κB- and p38MAPK-independent. Transfection and EMSA experiments suggested that only AP-1 transcription factors were necessary for optimal IL-8 expression.

conclusions. Human conjunctival epithelial cells possess the ability to respond to Gram-positive S. aureus and to activate the innate immune response by the IL-8 gene expression. These results are the first to delineate the transcription factors involved in S. aureus–induced IL-8 release by conjunctival epithelium.

Bacterial ocular infections are one of the major groups of eye diseases. Although many cases show a benign course, some can be associated with sight-threatening complications despite appropriate antibiotic therapy. ^{1 2 3} Moreover, these infections are frequently caused by antibiotic-resistant bacterial strains. ^{4 5 6 7} Staphylococcus aureus is the most common agent of bacterial conjunctivitis and keratitis and is responsible for up to 34% of bacterial infections. ^{8 9} In addition, the ability of S. aureus to reside intracellularly enables long-term colonization of the host and the maintenance of a chronic infectious state. ^{5 10} Given the limitations of traditional treatment, there is a need to explore alternative therapeutic strategies, including drugs that interfere with proinflammatory signal transduction pathways. To this end, it is of crucial importance to understand the mechanisms by which pathogens induce inflammation. However, the inflammatory response of conjunctival cells to S. aureus is poorly understood. Recent evidence suggests that conjunctival epithelial cells, in addition to their functions as a barrier and stabilizer of the tear film, ^{11 12} may play an active role in ocular surface defense via the expression and secretion of proinflammatory mediators. ^{13 14} Among these, IL-8/CXCL8 is an important chemotactic cytokine and a key mediator in neutrophil recruitment in inflammatory tissue. ¹⁵ Neutrophils increase in conjunctival epithelium of patients with vernal keratoconjunctivitis ¹⁰ and the number of them correlates with IL-8 levels. ¹⁶ In this study, we investigated the mechanisms underlying human conjunctival cell responses to S. aureus. We demonstrated that this pathogen induces IL-8 promoter activation via c-Jun NH₂-terminal kinase (JNK) activation and c-Jun phosphorylation, in a NF-κB- and p38MAPK-independent manner. 

Methods

Bacterial Strain

S. aureus, strain 347, a recent clinical isolate, was grown to the late log phase in Todd-Hewitt broth (Oxoid, Milan, Italy) at 37°C and harvested by centrifugation. Killed bacteria were prepared by heat treatment (80°C for 45 minutes), which was followed by extensive washing with distilled water and lyophilization. One microgram of killed lyophilized bacteria corresponded to approximately 1.5 × 10⁶ colony forming units (CFU). The effects of live bacteria were also studied and compared to that obtained with killed bacteria. To this end, bacteria were grown to late log phase and washed four times in phosphate-buffered saline (PBS) before addition to cell cultures. 

Cells and Culture Conditions

Experiments were performed in compliance with the Declaration of Helsinki. Ten healthy volunteers (20 eyes, three men, seven women; mean age, 39.5 years; range, 24–50) were enrolled. After informed consents were obtained, conjunctival epithelial samples were collected by brush cytology. ¹⁷ After topical anesthesia (0.4% oxybuprocaine hydrochloride) was applied to the upper tarsal conjunctiva, mucous discharge was carefully removed with forceps, and the conjunctiva was scraped (Cytobrush-S; Medscand, Malmo, Sweden) several times. The collected cells were suspended in 1 mL of PBS with 0.1% bovine serum albumin (BSA) and centrifuged (500g, 10 minutes). Primary human conjunctival cells were then grown for 24 hours in RPMI 1640 medium supplemented with penicillin (50 IU/mL), streptomycin (50 μg/mL), and 10% heat-inactivated fetal calf serum (FCS; Celbio, Milan, Italy) at 37°C in a 5% CO₂ humidified atmosphere. The human conjunctival cell line (Wong-Kilbourne derivative of Chang conjunctiva, clone 1-5-C-4, ATCC CCL-20.2; American Type Culture Collection, Manassas, VA) was cultured as just described. Cultures were renewed from frozen stocks every 2 months. 

Cytokine Production Assay

Primary conjunctival epithelial cells and Chang cells were cultured in flat-bottom 24-well plates (5 × 10⁵ cells/well) as just described and stimulated with different concentrations of heat-killed or live S. aureus. In the case of live bacteria, after a 2-hour exposure, the monolayers were extensively washed, and medium containing gentamicin (50 μg/mL) was added to eliminate the extracellular bacteria. At 6 hours after stimulation, supernatants were collected and analyzed for IL-8 with a commercial immunoassay (human IL-8 ELISA kit; Bio Source International, Nivelles, Belgium) with a sensitivity of 10 pg/mL, according to the manufacturer’s instructions. 

RNA Isolation and RT-PCR

Primary and Chang cells were cultured in 35-mm plates (2 × 10⁶ cells/well) and stimulated for the indicated times with heat-killed or live S. aureus. Total RNA was extracted (Trizol; Invitrogen, Milan, Italy) according to the manufacturer’s instructions, and the amount of total RNA was determined by measuring the absorbance at 260 nm. For RT-PCR, 1 μg total RNA was reverse transcribed in a total volume of 20 μL (IMProm-II Reverse Transcriptase kit; Promega, Milan, Italy) according to the manufacturer’s instructions. Twenty microliters of RT products were brought to a volume of 100 μL containing 2 mM MgCl₂, 0.2 mM PCR nucleotide mix, 1 μM concentration of both the upstream and downstream PCR primers (Sigma Genosys, Pampisford, Cambs, UK), 5 units of Taq DNA polymerase (Transgenomic, Bergamo, Italy), and 10× polymerase reaction buffer (Promega). Two pairs of primers were used. The primer sequences were: IL-8 sense, 5′-ATG ACT TCC AAG CTG GCC GTG GCT-3′; IL-8 antisense, 5′-TCT CAG CCC TCT TCA AAA ACT TCT C-3′; β-actin sense, 5′-TGA CGG GGT CTA CCC ACA CTG TGC CCC ATC TA-3′; and β-actin antisense, 5′-CTA GAA GCA TTG CGC TGG ACG ATG GAG GG-3′. Amplification was performed in a DNA thermal cycler (Applied Biosystems, Milan, Italy) after initial denaturation at 94°C for 4 minutes, followed by 37 cycles of PCR using the following temperature and time profile: denaturation at 94°C for 3 minutes; primer annealing for 50 seconds at 58°C for IL-8 and at 62°C for β-actin, respectively; primer extension at 72°C for 1 minute; and final extension of 72°C for 7 minutes. The PCR products were visualized by electrophoresis on 1% agarose gel in 1× TBE buffer (89 mM Tris borate, 2 mM EDTA, pH 8.3) after staining with 0.5 μg/mL ethidium bromide. Stained gels were captured by digital camera (Eastman-Kodak, New Haven, CT). 

Western Blot Analysis

Primary and Chang cells were cultured in 3 × 10⁶ cells/well and after stimulation with S. aureus for different times at a final concentration of 50 μg/mL were lysed at 4°C in 300 μL of lysis buffer (MPER; Pierce, Rockford, IL) and 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 1 mg/mL leupeptin. Western blot analysis was performed as previously described. ¹⁸ Phospho-specific p38, ATF-2, c-Jun and IκB-α rabbit antibodies, diluted 1:500 have been used, as described by the manufacturer (Cell Signaling Technology, Milan, Italy). A mouse monoclonal 1:1000 diluted primary anti-β-actin antibody was used to normalize protein loading to that of specific proteins in each lane (Sigma-Aldrich, Milan, Italy). After they were washed three times in PBS with 0.3% Tween 20, membranes were incubated with secondary horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG for 1 hour. Proteins were visualized with reagents from Pierce (Supersignal West Pico Chemiluminescent Substrate System). 

Transient Transfections

Liposome-mediated transient gene transfer was performed (FuGene 6 TR; Roche Diagnostic, Milan, Italy) as recommended by the manufacturer. Briefly, Chang cells were seeded at 3 × 10⁶ cells/well and transiently transfected with 1 μg of wild-type IL-8 (wtIL-8), or IL-8 lacking the AP-1 site (IL-8-97), or IL-8-97 mutant C/EBP (IL-8-97/mC/EBP), or IL-8-97 mutant NF-κB (IL-8-97/mNF-κB), or the IL-8 double mutant for C/EBP and NF-κB (IL-8/AP-1) promoters developed in collaboration with Hector R. Wong, (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH); pSV-nlsLacZ DNA, a β-galactosidase expression vector (0.5 μg) and empty plasmid DNA (pBSM), at a final concentration of 1.75 μg/plate. Expression plasmids for C/EBP homologous protein (CHOP), kindly provided by Hidetoshi Hayashi (Nagoya City University, Nagoya, Japan), wt c-Jun, and dominant negative transactivation domain mutant (TAM-67) c-Jun, kindly provided by Lucia Altucci, (Department of General Pathology, Second University of Naples, Naples, Italy) were transfected (0.2 μg) where indicated. Chang cells were also transiently transfected with 1 μg pFR-luc, a luciferase reporter gene including multimerized GAL4 UASs upstream of the minimal promoter, the transactivator GAL-cJun (50 ng; supplied by Stratagene, La Jolla, CA), and pSV-nlsLacZ DNA. Five hours after addition of the liposome-DNA mixture in serum-free medium, the media were changed and the cells further stabilized in RPMI 1640 containing 10% FCS. Preliminary tests were performed to define the optimal transfection conditions through the determination of β-galactosidase levels. Twenty-four hours after transfection, the cells were treated with 50 μg/mL or 3 × 10⁶ CFU of killed or live bacteria, respectively. Where indicated, 20 μM SB203580, SP600125, CAPE (Calbiochem, La Jolla, CA) were added to the culture medium 1 hour before the beginning of the additional stimulation. Cells were then harvested by washing and scraping in lysis buffer (Promega) and lysed with three cycles of rapid freeze-thawing. After the cell lysates were cleared by 15 minutes of centrifugation at 4°C, protein concentration in the crude extracts was determined with a colorimetric assay (Bio-Rad, Hercules, CA). Luciferase activity was assayed with luciferase assay reagent (Promega) according to the manufacturer’s instructions. Results were normalized for β-galactosidase activity produced by the cotransfected plasmid pnlsLAC. 

Electrophoretic Mobility Shift Assay

Nuclear fractions for the electrophoretic mobility shift assay (EMSA) were prepared (Nu-Clear extraction kit; Sigma-Aldrich) according to the manufacturer’s protocol. Protein concentrations of nuclear fractions were determined with a protein assay (Bio-Rad). Oligonucleotides containing the NF-κB/IL-8 (TCG TGG AAT TTC CTC TG), AP-1/IL-8 (GTG TGA TGA CTC AGG TTT G), and C/EBP/IL-8 (GCC ATC AGT TGC AAA TCG T) sequences were labeled using [γ-³²P]ATP (GE Healthcare, Piscataway, NJ) and T4 polynucleotide kinase (Promega). Fifteen micrograms of nuclear fractions, 40,000 cpm labeled double-stranded probe, and 2 μL of 5× binding buffer (20% glycerol, 5 mM MgCl₂, 2.5 mM EDTA, 2.5 mM dithiothreitol, 250 mM NaCl, 50 mM Tris-HCl [pH 7.5], 0.25 mg/mL poly(dI-dC)·poly(dI-dC)) were mixed in a total volume of 10 μL. In competition assays, 100× unlabeled competitors were added at the same time of probe addition. 

Densitometry and Statistical Analysis

The relative intensities of protein and nucleic acid bands were analyzed (Digital Sciences 1D program; Scientific Imaging Systems Division; Eastman-Kodak). Standard curves were run, and the data that were obtained were in the linear range of the curve. In addition, all values were normalized to their respective lane-loading controls. 

Data are expressed as the mean ± SD of n determinations. Results were analyzed by two-tailed Student’s t-test. P < 0.05 was considered significant. 

Results

S. aureus Induction of IL-8 Production

To determine whether S. aureus induces the synthesis of IL-8 protein in conjunctival cells, primary and Chang cultures were treated with different concentrations of either heat-killed or live bacteria, and IL-8 levels were measured in the supernatants by ELISA. Untreated cells produced small amounts of IL-8, whereas killed S. aureus, at 50 μg/mL, significantly increased IL-8 in both primary and Chang cells (Fig. 1A) . Similar data were obtained with live bacteria (Fig. 1B) . Therefore, in further experiments, killed bacteria were mostly used. These results suggest that S. aureus leads to a release of IL-8 protein in conjunctival cells in a dose-dependent manner. 

S. aureus Induction of IL-8 mRNA

In further experiments, we determined the effects of S. aureus (50 μg/mL) on IL-8 mRNA expression by RT-PCR. There was a low constitutive level of IL-8 transcript in unstimulated cells (Fig. 2 , time 0). In either Chang or primary cells IL-8 mRNA levels significantly increased after the bacterial stimulation, starting at 2 hours and peaking at 3 hours. No increase in mRNA transcript over baseline levels was observed at later times. These data indicate that S. aureus increases IL-8 production at the transcriptional level in a time-dependent manner. 

S. aureus–Induced Mediation of IL-8 Synthesis

Because the increase of IL-8 protein and IL-8 mRNA may be posttranscriptionally regulated, ^{19 20} it was determined whether S. aureus–induced increases in IL-8 mRNA were secondary to an enhanced activity of the IL-8 promoter. Figure 3Ashows that this was the case, since either killed or live S. aureus significantly activated the IL-8 promoter-driven reporter gene expression, peaking at 6 hours. Because IL-8 promoter activity is regulated by p38MAPK, NF-κB, and JNK, specific inhibitors of these pathways were used. Figure 3Bshows that IL-8 promoter activation induced by S. aureus was reduced by SP600125, ²¹ an inhibitor of JNK, but not SB203580 or CAPE, inhibitors of p38MAPK and NF-κB, respectively. ²² SP600125 was effective also in reducing the basal levels of IL-8 promoter activity. 

S. aureus–Induced Activation of c-Jun

To evaluate whether S. aureus induces IL-8 gene transcription through CHOP, a transcription factor involved in the regulation of the IL-8 promoter, ¹⁸ Chang cells were cotransfected with the IL-8 promoter and a plasmid expressing CHOP. Figure 4Ashows that, under these circumstances, CHOP did not increase the basal level of luciferase or modify the activation of IL-8 promoter by S. aureus.  

Because p38MAPK ^{23 24} and NF-κB ²⁵ have been shown to play central roles in IL-8 production, we investigated whether S. aureus was able to activate these signaling pathways. To this end, after microbial challenge, total cell proteins were extracted and subjected to Western blot analysis with antibodies specific for the phosphorylated forms of IκB-α and p38MAPK. As shown in Figure 4B , no phosphorylation of IκB-α or p38MAPK and its substrate ATF-2 was observed at any of the time points examined. The transcription of the IL-8 gene requires the activation of several other transcription factors, including activator protein (AP)-1. The phosphorylation state of c-Jun is a primary determinant of the activity of AP-1. To determine whether S. aureus induces phosphorylation of c-Jun, an antibody specific to the phosphorylated form of c-Jun was used. As shown in Figure 4C , phosphorylation of c-Jun occurred beginning 30 minutes after treatment of the cells with S. aureus, and continuing up to 120 minutes. The specific inhibitor of JNK SP600125 reduced the S. aureus–induced phosphorylation of c-Jun (Fig. 4D) . A similar kinetics of c-Jun phosphorylation was observed in S. aureus–stimulated primary cells (Fig. 4E) . 

S. aureus Stimulation of the Transactivation Potential of c-Jun

To assess whether the IL-8 transcription induced by S. aureus is mediated through c-Jun transactivation, Chang cells were transiently transfected with the JNK-responsive GAL-c-Jun chimeric transcription factor, consisting of the DNA binding domain of yeast GAL4 and the transactivation domain of c-Jun, together with the luciferase reporter plasmid (pFR-Luc) and a bacterial β-galactosidase expression vector (pSV-nlsLACZ). Cells were then treated with S. aureus for different time intervals. As shown in Figure 5 , the luciferase activity of GAL-c-Jun exhibited a 2.5-fold increase over baseline levels at 2 hours after challenge. No activation was observed at 4 hours. These effects required the c-Jun transactivation domain, because treatment with S. aureus failed to induce the reporter expression in cells transfected with a plasmid encoding the GAL4 DNA–binding domain (dbd) only (residues 1-147) and lacking the transactivation domain of c-Jun. 

S. aureus Activation of the IL-8 Promoter via c-Jun

To determine whether c-Jun phosphorylation and transactivation are involved in S. aureus–stimulated IL-8 promoter activation, experiments were performed cotransfecting Chang cells with wt IL-8 promoter and plasmids expressing wt or transdominant negative mutant c-Jun TAM-67. Figure 6shows that the IL-8 promoter activity of wt c-Jun cotransfected and S. aureus–treated cells is significantly higher relative to cells stimulated with S. aureus only or to cells cotransfected with wt c-Jun only. Transdominant negative mutant c-Jun TAM-67, on the contrary, inhibited either the basal level or the S. aureus–induced IL-8 promoter activity. These data indicate that S. aureus induces the IL-8 gene transcription by activating the transactivation potential of c-Jun. 

Role of the AP-1 Binding Site in the IL-8 Promoter

To probe the transcription elements involved in IL-8 induction, wt IL-8, IL-8-97, IL-8-97/mC/EBP, IL-8-97/mNF-κB, or IL-8/AP-1 promoters (Fig. 7A)were transfected into Chang cells before treatment with S. aureus. As shown in Figure 7Bluciferase activity was increased more than twofold only when the cells were transfected with wt IL-8 or IL-8/AP-1 promoters. In contrast, luciferase activity was not significantly increased when the cells were transfected with the IL-8-97/mC/EBP, IL-8-97/mNF-κB, or IL-8-97 promoters. A protein-DNA binding study by EMSA showed that only AP-1 bound to the IL-8 promoter after S. aureus stimulation (Fig. 7C) . 

Discussion

Previous studies have established that conjunctival and corneal epithelial cells produce a variety of proinflammatory factors, including IL-8, after exposure to S. aureus. ^{26 27} It is recognized that IL-8 has a central role in ocular defenses and in the pathophysiology of conjunctivitis, mainly through its ability to recruit neutrophils. ¹⁵ It has been reported that antibodies directed against platelet-activating factor receptor (PAFR), but not against TLR-2, inhibit S. aureus–induced IL-8 release from human conjunctival cells. ²⁶ Little is known, however, about the intracellular signal-transduction pathways involved in IL-8 secretion. A variety of transcription factors, such as CHOP, NF-κB, NF-IL-6, AP-1, and octamer (Oct)-1 have been previously shown to regulate IL-8 gene transcription in response to stimuli different from S. aureus. ^{18 28 29 30 31 32 33 34} However, the molecular mechanisms of S. aureus–induced IL-8 transcription have been studied in epithelial systems other than the conjunctival epithelium. ²⁷ We identified here at least some of the activation events involved in S. aureus–induced IL-8 production by human conjunctival cells. Our data were initially obtained using a cell line (Chang), but were also validated in primary cultures of human conjunctival epithelium. In initial experiments we have shown that IL-8 production follows a marked increase in IL-8 gene expression, as demonstrated by rises in IL-8 mRNA synthesis and promoter activation in conjunctival epithelium stimulated with S. aureus. These data are in agreement with those of a recent study using corneal epithelial cells, ²⁷ concerning the induction of IL-8 synthesis by S. aureus. However, the transduction pathways identified in that study appear to differ, in part, from those described here. In fact, whereas IL-8 production by corneal cells was NF-κB-, p38MAPK-, and JNK-dependent, the present study shows that the sequential activation of JNK and c-Jun followed by the AP-1 binding is responsible for increased IL-8 transcription. The involvement of the AP-1 binding site of IL-8 promoter was documented through transfection experiments with mutant forms of the IL-8 promoter in S. aureus challenged cells. Moreover, we could not detect, using Western blots with phosphospecific antibodies or transfection experiments, NF-κB or p38MAPK activation in conjunctival cells. This is in contrast to findings in other cell types in which IL-8 gene transcription is activated through these pathways. ^{18 29 35 36} Results by us showed that the AP-1 binding site alone was required for optimal IL-8 promoter activity. Indeed, the presence of loss-of-function mutations in the NF-κB and C/EBP sites did not affect S. aureus–induced IL-8 promoter activation. Furthermore, EMSA studies showed that only AP-1 transcription factors bound to IL-8 promoter after S. aureus stimulation. Collectively, our results and those obtained in corneal epithelium ²⁷ indicate that the transcription factors required for S. aureus–induced IL-8 responses may differ in different ocular cell types. Specifically, NF-κB and AP-1 may play a predominant role in corneal and conjunctival epithelial cells, respectively. In addition, the present study highlights an important role of the JNK/c-Jun pathway in S. aureus–induced IL-8 release. 

The involvement of this pathway was shown by the following: (1) the specific JNK inhibitor SP600125 blocked S. aureus–induced c-Jun phosphorylation and IL-8 promoter activation; (2) S. aureus increased the transactivation potential of c-Jun; (3) cotransfection with a dominant negative mutant c-Jun blocked S. aureus–induced IL-8 transcription. 

In conclusion, the findings show that S. aureus induces IL-8 mRNA transcription in conjunctival cells by activating the JNK pathway and c-Jun transactivation of the IL-8 gene. Moreover, on the basis of our studies, it may be supposed that the IL-8 gene of conjunctival epithelial cells belongs to the subset of the AP-1 target gene, actively repressed by c-Jun in basal conditions and upregulated on signal-induced c-Jun phosphorylation. ³⁷ In this case, S. aureus may be considered as one of the signals able to activate IL-8 gene transcription fully, through this mechanism. These studies increase our understanding of the signaling pathways whereby S. aureus induces conjunctivitis and may have a practical impact not only in the development of new anti-inflammatory drugs, but also in the correct use of those already available. 

 Submitted for publication January 26, 2006; revised June 14, 2006; accepted November 15, 2006.

 Disclosure: I. Venza, None; M. Cucinotta, None; S. Caristi, None; G. Mancuso None; D. Teti, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

IL-8 production in conjunctival cells stimulated with S. aureus. (A) Primary and Chang conjunctival cells were treated with different concentrations of heat-killed S. aureus for 6 hours. (B) Primary and Chang conjunctival cells were treated with different concentrations of live S. aureus for 6 hours. IL-8 protein was measured in culture supernatants by ELISA. Data are as the mean ± SD of the results of five independent experiments. **P < 0.01 S. aureus–treated versus control, untreated cells, based on Student’s two-tailed t-test.

Figure 2.

 

IL-8 and β-actin mRNA expression in conjunctival cells stimulated with S. aureus. Chang and primary cells were incubated for the indicated times with 50 μg/mL of heat-killed S. aureus. Top: The relative densities IL-8 and β-actin (as an internal control) mRNA were measured by RT-PCR. The expected product sizes of IL-8 and β-actin are 289 and 391 bp, respectively. Bottom: IL-8 and β-actin relative densities were calculated by dividing the density of the IL-8 band by the density of the β-actin band at the same time point. Data are the mean ± SD of the results of five independent experiments. **P < 0.01 S. aureus–treated versus untreated cells, based on Student’s two-tailed t-test.

Figure 3.

 

S. aureus induced IL-8 synthesis through IL-8 promoter activation. (A) Chang cells were transfected with wt IL-8 promoter (IL-8 LUC reporter) and stimulated for different times with 50 μg/mL of heat-killed or 3 × 10⁶ CFU/mL of live S. aureus. (B) After transfection, Chang cells were pretreated for 1 hour with 20 μM SB203580, CAPE or SP600126, p38MAPK, NF-κB, and JNK inhibitors, respectively and subsequently stimulated with 50 μg/mL of heat-killed S. aureus for 6 hours. Data are the mean ± SD of results in five independent experiments and are expressed as x-fold luciferase activation. β-Galactosidase levels were determined for transfection efficiency. **P < 0.01 S. aureus–treated versus control cells, °°P < 0.01 SP-treated versus control cells, ^§§ P < 0.01 SP+S. aureus–treated versus S. aureus–treated cells, based on Student’s two-tailed t-test.

Figure 4.

 

S. aureus did not activate p38MAPK and NF-κB but activated c-Jun. (A) Chang cells were transfected with the wt IL-8 promoter (IL-8 LUC reporter) and, where indicated, cotransfected with the CHOP expression plasmid and treated with 50 μg/mL of heat-killed S. aureus for 6 hours. Data are the mean ± SD of results of five independent experiments and are expressed as x-fold luciferase activation. β-Galactosidase levels were determined for transfection efficiency. (B) Total cell lysates of Chang cells stimulated for different times with 50 μg/mL of heat-killed S. aureus were loaded on gels (60 μg of protein per lane), subjected to SDS-PAGE, and treated with phospho-specific IκB-α (P-IκB-α), p38 (P-p38), and ATF-2 (P-ATF-2) antibodies. (C) Chang cells were stimulated with 50 μg/mL of heat-killed S. aureus for the indicated times. Total cell lysates were treated as described in (A) and blotted using polyclonal antibodies that recognize the phosphorylated form of c-Jun. (D) Chang cells were pretreated for 1 hour with the specific inhibitor of JNK SP600125 (20 μM) and subsequently incubated with 50 μg/mL of heat-killed S. aureus for the indicated times. Total cell lysates were treated as described in (A) and blotted with a phosphospecific c-Jun antibody. (E) primary cells were stimulated with 50 μg/mL of heat-killed S. aureus for the indicated times. Total cell lysates were treated as in (A) and blotted using polyclonal antibodies that recognize the phosphorylated form of c-Jun. Results shown are representative of those in five independent experiments. The mean densitometry values are depicted as the mean ± SD of results of five independent experiments. All densitometry values were normalized to the endogenous β-actin protein. **P < 0.01 S. aureus–treated versus control cells, ^§§ P < 0.01 SP+S. aureus–treated versus S. aureus–treated cells, based on Student’s two-tailed t-test.

Figure 5.

 

S. aureus induced the transactivation potential of c-Jun. Chang cells were transfected with a GAL-TATA-luciferase reporter gene and expression vectors encoding GAL-c-Jun or, where indicated (GAL), the GAL4 DNA binding domain (dbd), and a bacterial β-galactosidase expression plasmid. After transfection, the cell cultures were treated with 50 μg/mL of heat-killed S. aureus for the indicated times. Data are the mean ± SD of results in five independent experiments and are expressed as x-fold luciferase activation. β-Galactosidase levels were determined for transfection efficiency. **P < 0.01 S. aureus–treated versus control cells, based on Student’s two-tailed t-test.

Figure 6.

 

S. aureus activated IL-8 promoter via c-Jun. Chang cells were transfected with wt IL-8 promoter (IL-8 LUC reporter) and, where indicated, cotransfected with wt or transdominant negative mutant (TAM-67) c-Jun expression plasmids. After transfection, Chang cells were treated with 50 μg/mL of S. aureus for 6 hours. Data are the mean ± SD of results in five independent experiments and are expressed as x-fold luciferase activation. β-Galactosidase levels were determined for transfection efficiency. **P < 0.01 S. aureus–treated or wt c-Jun cotransfected versus control cells, °°P < 0.01 TAM-67 cotransfected versus control cells, ^§§ P < 0.01 wt c-Jun cotransfected+S. aureus–treated versus S. aureus–treated and wt c-Jun cotransfected cells, ##P < 0.01 TAM-67 cotransfected+S. aureus–treated versus S. aureus–treated and wt c-Jun+S. aureus–treated cells, based on Student’s two-tailed t-test.

Figure 7.

 

S. aureus induced IL-8 promoter activation through AP-1. (A) The IL-8 promoter mutants used are shown: black, gray, and white boxes: wild-type sites; striped boxes: mutated sites. (B) Chang cells were transfected with wt IL-8 promoter (IL-8 LUC reporter), IL-8-97, IL-8-97/mC/EBP, IL-8-97/mNF-κB, IL-8/AP-1 promoters, and subsequently stimulated with 50 μg/mL of S. aureus for 6 hours. The graph shows the increase in luciferase activity (x-fold) in cells treated with S. aureus (black boxes) by comparing with those without S. aureus treatment (white boxes). Data are the mean ± SD of results in five independent experiments and are expressed as x-fold luciferase activation. β-Galactosidase levels were determined for transfection efficiency. **P < 0.01 wt IL-8 and IL-8/AP-1–transfected+S. aureus–treated cells versus the relative controls based on Student’s two-tailed t-test. (C) Chang cells were pretreated with 50 μg/mL of S. aureus for 1 hour. The nuclear extracts were incubated with labeled AP-1, NF-κB, or C/EBP probes, to detect the effect of S. aureus on protein-DNA binding. Data representative of results in three experiments are shown.