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Published ahead of print on April 21, 2005, doi:10.1165/rcmb.2005-0022OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 97-104, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0022OC

Interleukin-17 Induces Hyperresponsive Interleukin-8 and Interleukin-6 Production to Tumor Necrosis Factor-{alpha} in Structural Lung Cells

Arjen van den Berg, Mathys Kuiper, Mieke Snoek, Wim Timens, Dirkje S. Postma, Henk M. Jansen and René Lutter

Department of Pulmonology and Laboratory of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam; and Departments of Pathology and Pulmonology, University of Groningen, Groningen, The Netherlands

Correspondence and requests for reprints should be addressed to Arjen van den Berg, AMC, Lab. Exp. Immunology, room G1-140, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail: a.vandenberg{at}amc.uva.nl


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Lung epithelial cells contribute to local inflammation by the production of pro-inflammatory mediators like interleukin (IL)-8 and IL-6. Although their production depends on gene transcription, previous studies showed that post-transcriptional mechanisms modulate IL-8 and IL-6 production. Human lung epithelial cells turn from normoresponsive into hyperresponsive IL-8– and IL-6–producing cells when their IL-8 and IL-6 mRNA degradation is reduced. We hypothesized that IL-17, a mediator predominantly released by memory T cells and present in airways of individuals with asthma, would modulate rather than induce IL-8 and IL-6 production by both human lung epithelial cells and fibroblasts. We show here for both cell types that IL-17 was a weak stimulus of IL-8 and IL-6 production, but markedly enhanced IL-8 and IL-6 responses to another stimulus, such as tumor necrosis factor-{alpha}. This modulatory effect of IL-17 was paralleled by a reduced IL-8 and IL-6 mRNA degradation, with no effect on IL-8 and IL-6 gene transcription. In conclusion, IL-17 particularly affects post-transcriptional regulation of IL-8 and IL-6 expression leading to enhanced IL-8 and IL-6 responses to secondary stimuli, and is only a weak proinflammatory stimulus by itself. This poses the interesting concept that by releasing IL-17 from memory T cells, the adaptive immune system instructs lung structural cells as part of the innate immune system to respond more vigorously.

Key Words: chemokines • cytokines • inflammation • lung


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Structural cells in the lung, like epithelial cells and fibroblasts, can direct and perpetuate local inflammation by the production of inflammatory mediators such as interleukin (IL)-8 and IL-6. Previous studies into the production of IL-8 and IL-6 by lung epithelial cells have shown that both gene transcription and mRNA degradation are important regulatory mechanisms (1, 2). Strikingly, the dose–response curves for IL-8 and IL-6 production as a function of the concentration of tumor necrosis factor (TNF)-{alpha} or other stimuli are significantly steeper for cells with a reduced IL-8 and IL-6 mRNA degradation (3, 4). Thus reducing mRNA degradation turns these cells from normoresponsive into hyperresponsive cells, which may lead to a more pronounced inflammation.

Interleukin-17A (IL-17), the prototype of six related mediators comprising IL-25 (i.e., IL-17E) and IL-17B to IL-17F, is predominantly secreted by memory CD4+ and CD8+ T cells (5). IL-17 acts as a proinflammatory mediator as it promotes the release of other proinflammatory mediators like the neutrophil chemoattractant IL-8 by epithelial cells and fibroblasts (6). There is some clinical and experimental evidence now that IL-17 may be implicated in the pathophysiology of inflammatory diseases (7). Increased local levels of released IL-17 have been reported for a number of chronic inflammatory diseases such as allergic asthma (8, 9), rheumatoid arthritis (10, 11) and inflammatory bowel disease (12). In asthma, these findings pointed to a role for IL-17 in neutrophil influx, which is a prominent feature of asthma exacerbations and of severe asthma. In support of this, recent studies with a mouse model of allergic asthma showed that blocking-antibodies to IL-17 prevented the ovalbumin-induced neutrophil influx in sensitized mice (13).

The mechanisms by which IL-17 induces the expression of proinflammatory mediators may be cell type–dependent, and appear to involve gene transcription (14, 15), and possibly modulation of mRNA processing (16, 17). We hypothesized that IL-17, by reducing mRNA degradation, would modulate rather than induce IL-8 and IL-6 production by human lung epithelial cells and possibly also that by fibroblasts. Relevant human lung epithelial and fibroblast cell lines and primary cells were exposed to IL-17 alone or in combination with other stimuli, addressing IL-8 and IL-6 production and the underlying mechanisms. Our findings indicate that IL-17 in particular enhances IL-8 and IL-6 responses by these structural cells to other proinflammatory stimuli.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Culture
The human lung mucoepidermoid carcinoma derived cell line NCI-H292 (CRL 1848; American Type Culture Collection [ATCC], Manassas, VA) and the human lung adenocarcinoma–derived Calu-3 cells (HTB 55; ATCC), were cultured and propagated as described before (2, 4). For IL-6 and IL-8 production, 3 x 105 cells were plated and grown overnight in 500 µl in 24-wells plates. For isolation of mRNA and nuclear extracts, 15 x 105 cells were plated and grown overnight in 2.5 ml in 6-well plates. Primary bronchial epithelial cells (NHBE; Cambrex Bio Science Verviers, Belgium) were cultured and propagated as recommended by the supplier. Of these cells, 0.2 x 105 and 1 x 105 were plated in 48- and 12-well plates for measurement of cytokine production and mRNA analyses, respectively. Cells were used between passages 3 and 5.

The human lung tissue derived fibroblast lines MRC-5 (ATCC CCL 171) and WI-38 (ATCC CCL 75) were cultured and propagated as recommended by the supplier. Primary lung fibroblast were obtained from explants of noninvolved peripheral parenchymal lung tissue from three patients undergoing resective surgery for pulmonary carcinoma (described in Ref. 18). The isolated cells were characterized as fibroblasts by morphologic appearance and expression patterns of specific proteins investigated with immunocytochemistry. Fibroblasts were spindle-shaped and elongated, with characteristic staining patterns for vimentin (Dakopatts, Glostrup, Denmark). The cultures showed no immunoreactivity for keratin (Euro-Diagnostica, Apeldoorn, The Netherlands). Cells were cultured in Ham's F12 medium (BioWhittaker Europe BV, Verviers, Belgium) with 10% fetal calf serum, supplemented with L-glutamine (2 mM; Gibco BRL), streptomycin (100 µg/ml; Bio Whittaker), and penicillin (100 U/ml; BioWhittaker) and were used between passages 3 and 5.

For cytokine release and isolation of RNA, 2.5 x 105 cells/ml were plated and grown overnight in 250 µl in 48-well plates or in 1 ml in 12-well plates, respectively.

Determination of IL-6 and IL-8 Protein
Cells were exposed for 18 h to doses of TNF-{alpha} (rhTNF-{alpha}; R&D Systems, Minneapolis, MN), lipopolysaccharide (LPS; Sigma, St. Louis, MO), IL-17 (rhIL-17; R&D Systems), and IL-1ß (rhIL-1ß; Genzyme Diagnostics, Cambridge, MA) up to 5 ng/ml, 1 µg/ml, 100 ng/ml, and 100 U/ml, respectively.

The amount of IL-6 and IL-8 in culture supernatants was measured by sandwich ELISA, as described previously (3).

mRNA Analysis
Cells were stimulated with IL-17 (10 ng/ml), TNF-{alpha} (5 ng/ml), LPS (1 µg/ml), and IL-1ß (1 U/ml). Total RNA was extracted with TriZol (Invitrogen, Paisley, UK) and the amount of IL-6, IL-8, and GAPDH mRNA was determined by dotblotting and hybridization with specific 32P-labeled probes for IL-6, IL-8, and GAPDH, which have been extensively validated for specificity in our samples by Northern blot as described (1, 2). Blots were quantified using a phosphorimager, and variable loading was corrected for by expressing mRNA levels relative to that of the housekeeping gene GAPDH. mRNA decay experiments were performed 1, 3, and 5 h after stimulation by blocking gene transcription with 5 µg/ml actinomycin-D (Boehringer Mannheim, Mannheim, Germany). After various periods of time, remaining mRNA was determined as described above.

Isolation of Nuclear Extracts and Electrophoretic Mobility Shift Assay
Nuclear extracts were obtained after 1 h stimulation with 5 ng/ml TNF-{alpha}, 1 µg/ml LPS, and 10 ng/ml IL-17, as described (1, 2). Protein contents was measured using a protein assay kit (Biorad, Hercules, CA). Two micrograms of protein of the nuclear extracts were incubated with 32P-labeled oligonucleotides at 4°C for 1 h and separated on a 4% nonreducing polyacrylamide gel at slowly increasing voltages (60–220 V). Bands were identified by cold oligo competition and by supershift using 1 µg of antibodies against p65 for nuclear factor (NF)-{kappa}B, c-fos and c-jun for activator protein (AP)-1, and C/EBP-ß (Santa Cruz Biotechnology Inc., Santa Cruz, CA). The intensity of the bands was quantified using a phosphorimager. The following oligonucleotides were used in the electrophoretic mobility shift assay (EMSA): nuclear factor (NF)-{kappa}B, 5'-TTGCAAATCGTGGAATTTCCTCTGACATAA-3'; AP-1, 5'-TTAAGTGTGATGACTCAGGTTTAA-3'; C/EBP, 5'-TTAAAGGACGTCACATTGCACAATCTTAATAA-3'.

Transfection of IL-6 and IL-8 Promoter Chloramphenicol Acetyltransferase Constructs
NCI-H292 cells were grown to 70% confluence in 6-well plates and transfected with 5 µg of chloramphenicol acetyltransferase (CAT) reporter vectors driven by the wild-type IL-8 (19) or IL-6 promoter (20), as described (1, 2). Cells were stimulated with 5 ng/ml TNF-{alpha}, 10 ng/ml IL-17, or a combination of both, 24 h after transfection and 18 h before cell lysis. CAT production was measured by CAT ELISA (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions, and data were normalized for total protein content.

Statistical Analysis
IL-8 and IL-6 protein production and EMSA data were analyzed by independent samples t test or ANOVA combined with post hoc Bonferroni test. mRNA half-life was estimated by linear regression. Differences were considered significant at P <= 0.05 (in the figures, if error bars are not visible they are contained within the symbol).


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
IL-17 Amplifies IL-8 and IL-6 Responses to TNF-{alpha} and LPS in Lung Epithelial Cells and Lung Fibroblasts
IL-17 by itself (up to 100 ng/ml) induced low amounts of IL-6 and IL-8 in NCI-H292 and Calu-3 (data not shown) lung epithelial tumor cell lines as well as in normal human bronchial epithelial cells (NHBE). However, IL-17 markedly enhanced IL-6 and IL-8 responses by lung epithelial cells to a concentration range of TNF-{alpha} (Figure 1 and Table 1). Interestingly, the effect of IL-17 was most pronounced with primary cells. NCI-H292 cells, which were studied most extensively, showed a dose-dependent effect of IL-17 for the enhanced IL-8 response (log-transformed data with 5 ng/ml of TNF-{alpha}: r = 0.998), with a maximal effect at 100 ng/ml. No dose-dependent effect of IL-17 on IL-6 responses was observed.



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  		Figure 1. IL-17 augments IL-8 and IL-6 production to TNF-{alpha} in lung epithelial cells. NCI-H292 (A and B) and NHBE (C and D) cells were exposed for 18 h to 0, 0.05, 0.5, and 5 ng/ml TNF-{alpha} without IL-17 (shaded square), or with 1 ng/ml IL-17 (inverted open triangle), 10 ng/ml IL-17 (shaded triangle), or 100 ng/ml IL-17 (open triangle). Please note that curves for IL-17 in B overlap. IL-8 and IL-6 were measured by ELISA and expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by ANOVA and Bonferroni post test. (A and B: triplicate samples, n = 3; C and D: triplicate samples, a representative experiment of n = 2 is shown). If no error bars are visible they are contained within the symbol.

 

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  		TABLE 1. Effect of IL-17 on IL-6 and IL-8 production by lung epithelial cells relative to that without IL-17*

 
We also determined whether IL-17 affected IL-6 and IL-8 responses by lung fibroblasts (Figure 2 and Table 2). Again, IL-17 alone induced small amounts of IL-6 and IL-8 in the human lung fibroblast cell lines MRC-5 (Figures 2A and 2B) and WI-38 (not shown), as well as in primary human lung fibroblasts (Figures 2C and 2D). Similar to that for lung epithelial cells, IL-17 markedly enhanced the IL-6 and IL-8 responses by these lung fibroblasts to a concentration range of TNF-{alpha}. The effect of IL-17 appeared concentration-dependent in the range from 0–10 ng/ml of IL-17 (Figures 2A and 2B; at 5 ng/ml of TNF-{alpha}, r = 0.97 and 0.95 for IL-6 and IL-8, respectively).



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  		Figure 2. IL-17 enhances TNF-{alpha}–induced IL-8 and IL-6 production by lung fibroblasts. MRC-5 (A and B) and primary (C and D) lung fibroblasts were exposed for 18 h to 0, 0.05, 0.5, and 5 ng/ml TNF-{alpha} without IL-17 (shaded squares) or with 1 ng/ml IL-17 (inverted open triangles) or 10 ng/ml IL-17 (shaded triangles). IL-8 and IL-6 were measured by ELISA. Data are presented as mean ± SEM, n = 3 (triplicate samples) for A and B. C and D are representative of three experiments with cells from three different donors (duplicate samples). Statistics are as described in legends to Figure 1. **P < 0.01, ***P < 0.001 versus TNF-{alpha} alone.

 

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  		TABLE 2. Effect of IL-17 (10 ng/ml) on IL-6 and IL-8 production by lung fibroblasts relative to that without IL-17*

 
To better view the effect of IL-17 on the dose–response curves for the primary cells, we expressed the effect of IL-17 relative to that without IL-17 for each dose of TNF-{alpha} (Figure 3). Whereas IL-17 showed the largest effect on TNF-{alpha}–induced IL-8 response by NHBE, in TNF-{alpha}–stimulated lung fibroblasts IL-17 more profoundly enhanced the IL-6 response (see also Tables 1 and 2). In addition, it is also clear that the extent of the effect of IL-17 varies with the dose of TNF-{alpha}.



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  		Figure 3. Effect of IL-on IL-8 and IL-6 production by primary cells. Data from Figures 1 (A) and 2 (B) were used and expressed relative (± SEM) to that of responses by TNF-{alpha} alone. IL-8 induction is shown in white bars, IL-6 induction in black bars.

 
We also tested whether IL-17 amplified IL-6 and IL-8 responses to proinflammatory stimuli other than TNF-{alpha}. IL-17 (10 ng/ml) increased the LPS-induced IL-8 and IL-6 production 2.5-fold in NCI-H292 cells (not shown). Calu-3, NHBE cells, and lung fibroblasts did not produce significant amounts of IL-8 and IL-6 in response to LPS, and no enhancement was observed by co-incubation with IL-17 (data not shown). IL-1ß (10 U/ml) potently stimulated NCI-H292 lung epithelial cells and MRC-5 lung fibroblasts to produce IL-8 (30- and 400-fold over basal secretion, respectively) and IL-6 (20- and 130-fold, respectively), but IL-17 did not augment IL-1ß–induced responses (data not shown).

IL-17 Increases TNF-{alpha}–Induced IL-8 and IL-6 mRNA Expression in Time
To provide a clue as to the mechanism of IL-17–enhanced IL-8 and IL-6 responses to TNF-{alpha}, the expression of IL-8 and IL-6 mRNA were determined over time (Figures 4A and 4B). NCI-H292 cells exposed to 5 ng/ml TNF-{alpha} showed a rapid increase of IL-8 and IL-6 mRNA levels, peaking at 1 h followed by a rapid decline to near basal levels, which is known to be facilitated by active degradation of these mRNAs (1, 2). Exposure to TNF-{alpha} plus IL-17 (10 ng/ml) resulted in a rapid increase of IL-8 and IL-6 mRNA expression, with similar kinetics as for TNF-{alpha} alone. However, the decline of IL-8 mRNA levels and to a lesser extent that of IL-6 was slower than with TNF-{alpha} alone, indicative of a reduced IL-8 and IL-6 mRNA degradation. In line with the low levels of IL-6 and IL-8 induced by IL-17 (Figure 1), IL-17 (10 ng/ml) induced a small and relatively slow increase in IL-8 and IL-6 mRNA levels (Figures 4A and 4B), peaking around 2 h and followed by a slow decline. Comparing the dashed curve, i.e., the sum of the curves for TNF-{alpha} and that of IL-17, with the curve for combined exposure shows a statistically significant increase at the mRNA level. The effect of IL-17 on the IL-8 mRNA expression was more pronounced than that for IL-6, in line with the more pronounced effect of IL-17 on the IL-8 response by lung epithelial cells (Figure 1).



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  		Figure 4. IL-17 enhances TNF-{alpha}–induced IL-8 and IL-6 mRNA expression in time. NCI-H292 (A and B) and MRC-5 (C and D) cells were exposed to 10 ng/ml IL-17 (inverted shaded triangles), 5 ng/ml TNF-{alpha} (shaded squares), or both (shaded triangles). The dashed curve represents the calculated additional effect of IL-17 and TNF-{alpha}. RNA was isolated at indicated time points, isolated, dotblotted, and hybridized. The signal was quantified using a phosphorimager. Data are presented as fold-induction (duplicate samples; mean ± SEM) of the ratio IL-8 or IL-6 mRNA over that of GAPDH. Statistics are as described in legend to Figure 1.

 
Similar experiments with MRC-5 fibroblasts showed that co-exposure to TNF-{alpha} and IL-17 resulted in a significant 1.5 ± 0.05 fold increase of IL-8 mRNA that leveled off at five hours and remained high thereafter (Figure 4C). Virtually similar results were obtained for IL-6 mRNA (Figure 4D), but with a 4.2- ± 0.3-fold increase of IL-6 mRNA upon co-exposure to IL-17, showing a more profound increase than for IL-8 mRNA. This is in line with the larger effect of IL-17 on the TNF-{alpha}–induced IL-6 protein production as compared with that for IL-8 (Figure 2). As for IL-6 and IL-8 mRNA kinetics in airway epithelial cells (Figure 4A and B), IL-17 particularly affects mRNA expression after the initial peak, suggestive of mRNA stabilization in MRC-5 cells.

IL-17 Does Not Augment IL-8 or IL-6 Gene Transcription in Lung Epithelial Cells
IL-17 was reported to induce NF-{kappa}B activation in a number of cell types (14, 15). Previous experiments with NCI-H292 cells showed that NF-{kappa}B and, to a lesser extent, AP-1 are involved in IL-8 gene transcription, whereas C/EBP is involved in IL-6 gene transcription (1, 2). To assess the role of nuclear translocation and promoter binding of the relevant transcription factors by IL-17 in NCI-H292 cells, EMSAs were performed. NCI-H292 cells were stimulated for 1 h with TNF-{alpha} (5 ng/ml) or LPS (1 µg/ml), with or without IL-17 (10 ng/ml). Figure 5A shows that both TNF-{alpha} and LPS induced activation of NF-{kappa}B, and to a higher extent than by stimulation with IL-17. The combination of IL-17 with TNF-{alpha} or LPS did not further increase NF-{kappa}B activation, indicating that the IL-17–enhanced IL-8 response was not due to increased activation of NF-{kappa}B. AP-1 recruitment (Figure 5B) was only slightly elevated and was not significantly augmented by IL-17 with TNF-{alpha} and LPS. C/EBP recruitment (Figure 5C) was stimulated only weakly by either stimulus, and there was no additional effect of IL-17 on C/EBP activation.



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  		Figure 5. IL-17 does not enhance transcriptional activation in lung epithelial cells. EMSA (A–C): NCI-H292 cells were exposed for 1 h to 5 ng/ml TNF-{alpha}, 100 ng/ml LPS, 10 ng/ml IL-17, or a combination as indicated, followed by preparation of nuclear extracts (A–C). Specific bands, as determined by supershift and competition, are shown in rectangles. Bands were quantified with a phosphorimager as represented in the lower graphs. A representative experiment out of three experiments is shown. Reporter assay (D–E): NCI-H292 cells were transfected with CAT expression vectors in which the IL-8 (D) or IL-6 (E) promoter was cloned. Cells were exposed to 5 ng/ml TNF-{alpha}, 10 ng/ml IL-17, or both for 18 h. CAT production was measured by ELISA and correct for total protein. Data are expressed as fold induction over medium and represent the mean ± SEM of two independent experiments.

 
To assess the transcriptional effect of IL-17 by an alternative approach, we transfected NCI-H292 cells with CAT constructs linked to the IL-8 or the IL-6 promoter. Transfected cells were stimulated for 18 h and CAT production was measured by ELISA. The results showed that TNF-{alpha}–induced IL-8 (Figure 5D) and IL-6 (Figure 5E) gene transcription were not augmented by IL-17, whereas IL-17 weakly activated both promoters. The small reduction in CAT production in the combined exposure to IL-17 and TNF-{alpha} was consistent in both experiments, but did not reach statistical significance.

IL-17 Reduces IL-8 and IL-6 mRNA Degradation
To determine whether IL-17 (10 ng/ml) modulated IL-8 and IL-6 mRNA degradation in lung epithelial cells, gene transcription was blocked using actinomycin D (5 µg/ml) 1 h after stimulation, and the decline of IL-8 and IL-6 mRNA with time was quantified. Figure 6 represents results from mRNA half-life experiments with NCI-H292 and NHBE cells. In TNF-{alpha}–stimulated NCI-H292 cells, the half-life of IL-8 and IL-6 mRNA (linear regression over 80 min) was 50 ± 10 and 60 ± 11 min, respectively (Figures 6A and 6B). Co-incubation with IL-17 (10 ng/ml and [not shown] 50 ng/ml) led to a small but consistent (n = 4, triplicate samples) increase of ~ 20–30% in the half-life of TNF-{alpha}–induced IL-8 and IL-6 mRNA, resulting in half-lives of 61 ± 6 and 88 ± 6 min (P < 0.05) for IL-8 and IL-6 mRNA, respectively. Also for NHBE cells (Figures 6C and 6D), there was a consistent but small effect on the IL-8 and IL-6 mRNA half-lives. IL-17 (10 ng/ml and [not shown] 100 ng/ml) increased the IL-8 mRNA half-life from 181 ± 40 to 227 ± 0.53 (1.3-fold) minutes, and for IL-6 mRNA from 123 ± 24 to 234 ± 15 (P < 0.05: 1.9-fold). Similar results were obtained with Calu-3 cells (data not shown). The enhanced response to LPS by IL-17 in NCI-H292 cells was also paralleled by an increased half-life of the corresponding mRNAs. IL-8 mRNA half-life increased from 80 ± 7 to 96 ± 1 min, and for IL-6 mRNA from 80 ± 7 to 242 ± 104 min. Taken together, these results indicate that IL-17 increased IL-8 and IL-6 mRNA half-life in lung epithelial cells.



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  		Figure 6. Half-life of IL-8 and IL-6 mRNA in lung epithelial cells stimulated with TNF-{alpha} and IL-17. NCI-H292 cells (A and B) and NHBE cells (C and D) were exposed to 5 ng/ml TNF-{alpha} (shaded squares) or to TNF-{alpha} and 10 ng/ml IL-17 (shaded triangles). After 1 h, Actinomycin D (ActD; 5 µg/ml) was added to block transcription. RNA was obtained at 0, 40, and 80 min after the addition of ActD, purified, and dotblotted. Blots were hybridized and IL-8 (A and C) and IL-6 (B and D) mRNA was quantified with a phosphorimager. Variable loading was corrected by expressing IL-6 and IL-8 mRNA over that of GAPDH. Data are expressed as mean ± SEM (A and B, n = 3; C and D, n = 2; all triplicate samples). Statistics are as described in legends to Figure 1.

 
mRNA decay in MRC-5 fibroblasts and primary human lung fibroblasts was analyzed 3 h after exposure to TNF-{alpha} with or without IL-17, the time point at which mRNA curves for cells with and without IL-17 start to deviate (cf. Figures 4C and 4D). IL-8 mRNA in both the cell line and primary cells exposed to TNF-{alpha} was quite stable (Figures 7A and 7C), and thus a stabilizing effect of IL-17 was difficult to assess. In contrast, IL-6 mRNA in MRC-5 and primary lung fibroblasts had a half-life of 2.7 ± 0.2 h (Figure 7B) and 2.5 ± 0.4 h (Figure 7D), respectively, which was increased by IL-17 to 61 ± 0.8 h and 4.8 ± 0.4 h (by extrapolation). IL-8 and IL-6 mRNA decay at 5 h after stimulation yielded similar results (data not shown). These data indicate that also for lung fibroblasts, IL-17 stabilizes IL-6 mRNA.



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  		Figure 7. IL-17 stabilizes IL-6 mRNA in lung fibroblasts. MRC-5 (A and B) and primary (C and D) lung fibroblasts were exposed for 3 h to 5 ng/ml TNF-{alpha} without (shaded square) or with 10 ng/ml IL-17 (shaded triangle). IL-8 (A and C) and IL-6 (B and D) mRNA decay was assessed as described in the legend to Figure 6. Data are presented as mean ± SEM. For A and B, one representative experiment of three (all duplicate samples) is shown. C and D show an experiment (duplicate samples, n = 2) with cells from one donor, representative of that found with cells from three donors. Statistics are as described in legend to Figure 1.

 
Given that IL-17 failed to enhance IL-6 and IL-8 responses to IL-1ß, the effect of IL-1ß on IL-6 and IL-8 mRNA half-life in MRC-5 fibroblasts was tested. IL-6 and IL-8 mRNA half-life after stimulation with IL-1ß was prolonged compared with that after stimulation with TNF-{alpha}, which was not further increased by IL-17 (data not shown), indicating that IL-17 and IL-1ß may affect IL-6 and IL-8 mRNA half-life via the same pathway.


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Structural lung cells such as epithelial cells and fibroblasts contribute to airway inflammation by the production of inflammatory mediators. Factors modulating the production of these mediators may have profound effects on inflammation. We studied the contribution of IL-17 to IL-6 and IL-8 production by lung epithelial cells and fibroblasts. IL-17 by itself weakly stimulated epithelial IL-6 and IL-8 production, but was 25-fold less potent then TNF-{alpha} at equimolar amounts. Similar findings were obtained for lung fibroblasts. However, IL-17 potently enhanced TNF-{alpha}–induced IL-6 and IL-8 responses in cell lines of both cell types, and even more so of their primary counterparts. Thus, IL-17 predominantly enhances IL-6 and IL-8 responses to TNF-{alpha} rather than acts as a proinflammatory stimulus by itself.

The lower potency of IL-17 compared with that of TNF-{alpha} to induce IL-6 and IL-8 differs from that reported by Jones and Chan for primary bronchial epithelial cells (21), but is in line with other reports for the bronchial epithelial cell line 16-HBE (22), airway smooth muscle cells (23), and several types of fibroblasts (17, 24). The reason for this discrepancy with the results by Jones and Chan is not clear, but may relate to donor differences. Nevertheless, Jones and Chan show, like in the present study, that IL-17 and TNF-{alpha} synergize in IL-8 production, but without addressing the underlying mechanism. The slight increase in IL-6 and IL-8 production by IL-17 alone is paralleled by a slow and transient increase of the respective mRNAs in lung epithelial cells and fibroblasts. The kinetics of IL-6 and IL-8 mRNA are suggestive of a minor increase in transcription followed by a reduced rate of IL-6 and IL-8 mRNA degradation. In line herewith, we observed a modest increase in IL-8 and IL-6 gene transcription by IL-17 alone by EMSA and with promoter constructs. We were unable to determine the half-life of IL-6 and IL-8 mRNA in epithelial cells and fibroblasts exposed to IL-17 alone, as mRNA levels remained too low for accurate measurements.

The enhanced IL-6 and IL-8 responses to TNF-{alpha} by IL-17, however, are not due to a synergy with TNF-{alpha} at the transcriptional level; if anything, there was a small reduction in transcriptional activity. Instead, IL-17 affected the post-transcriptional regulation as reflected by a reduced IL-6 and IL-8 mRNA degradation. Identical effects of IL-17 on mRNA stability for other cell types and transcripts have been described (16, 17, 23, 25, 26), but this is the first report to show similar effects in lung epithelial cells and fibroblasts. The effect of IL-17 on TNF-{alpha}–induced IL-6 and IL-8 responses was most pronounced for IL-6 in lung fibroblasts, and for IL-8 in epithelial cells. The increased IL-6 response by lung fibroblasts may relate to the more pronounced effect of IL-17 on IL-6 mRNA stabilization in fibroblasts than in lung epithelial cells. The increased IL-8 response by lung epithelial cells, however, cannot solely be explained by mRNA stabilization, as IL-6 mRNA in epithelial cells was stabilized to a higher extent than that of IL-8. Although IL-17 exerts its effect apparently by stabilizing IL-6 and IL-8 mRNA, we cannot rule out that IL-17 also affects IL-6 and IL-8 mRNA translation.

IL-17 also enhanced LPS-induced IL-6 and IL-8 responses in NCI-H292 cells, but failed to enhance IL-6 and IL-8 responses in airway epithelial cells and fibroblasts to IL-1ß, which also is a potent inducer of IL-6 and IL-8 production in these cell types. The most likely explanation is that IL-1ß affects the same post-transcriptional processes as IL-17, in addition to IL-6 and IL-8 gene transcription. Indeed, we found that IL-1ß stabilized IL-6 mRNA in fibroblasts and thus IL-1ß may induce a similar hyperresponsiveness by structural cells as described here for IL-17. A similar failure of IL-1ß to enhance IL-17 effects were reported by Shimada and coworkers (17) and by Henness and colleagues (23). Both IL-17 and IL-1ß have been shown to activate p38 MAPK, a kinase of which its activation is strongly correlated with IL-17- and IL-1ß-induced mRNA stabilization (26, 27; reviewed in Ref. 28). Given these findings, it is likely that activation of p38 MAPK by IL-17 is not further enhanced by IL-1ß, and vice versa.

Taken together, this indicates that IL-17 is a relative poor inducer of IL-8 and IL-6 gene transcription, and predominantly exerts its effect by modulating post-transcriptional processes. IL-17–reduced IL-6 and IL-8 mRNA degradation is paralleled by a hyperresponsive IL-6 and IL-8 production in lung epithelial cells, as shown previously for other conditions (3, 4, 29), and for the first time also in lung fibroblasts. It is important to further clarify what is meant here by hyperresponsive IL-6 and IL-8 production. IL-17 synergizes with TNF-{alpha}, either independent of the dose of the stimulus or dependent on the dose of TNF-{alpha} (as seen, for example, for the fibroblastic IL-6 production). So, in both cases IL-17 enhances IL-6 and IL-8 responses, i.e., hyperresponsive IL-6 and IL-8 production. However, when the extent of synergy varies with the dose of the stimulus, this will change the shape of the dose–response curve, leading for example to a relative further enhanced IL-6 production for fibroblasts at high doses of TNF-{alpha}.

Like IL-6 and IL-8 mRNA, most if not all mRNAs encoding response genes contain a common motif in the 3'-untranslated region of these mRNAs, referred to as AUUUA-repeats or ARE-rich elements. Transfer of these repeats to stable mRNAs facilitates their rapid degradation (30), and thus these repeats are strongly linked to mRNA degradation (31). As IL-17 modulates mRNA degradation, this may be related to the AUUUA-repeats and thus may apply to many mRNAs encoding response genes. This is in line with the data reported by Jones and Chan (21) looking into the IL-17–induced gene expression in primary human bronchial epithelial cells, where several response genes were upregulated.

IL-17 is found in tens of pg in bronchoalveolar lavage fluid and sputum of patients with asthma (32, 33). Epithelial lining fluid, which is sampled by the lavage technique, is diluted ~ 50-fold, and thus levels of IL-17 in epithelial lining fluid may reach up to low ng levels. In addition, it may be envisaged that T cells release IL-17 in close association with structural cells and thus even higher local levels may be reached. It remains to be determined whether IL-17, or indeed other mediators related to IL-17, contribute to chronic inflammation in asthma or other inflammatory diseases. The present results, however, indicate that IL-6 and IL-8 responses by lung epithelial cells and fibroblasts are highly susceptible to IL-17. Moreover, compared with lung epithelial cells, lung fibroblasts on a cell-to-cell basis produce ~ 10-fold more IL-8 and ~ 100-fold more IL-6, underlining their potential as important players in inflammatory processes in the lung. Besides chronic inflammation, an apparently thickened basement membrane and the appearance of myofibroblasts are characteristic of asthmatic airways, which may also come about by activated fibroblasts and epithelial cells.

In summary, IL-17 potently amplifies IL-8 and IL-6 responses to secondary stimuli in both lung epithelial cells and fibroblasts. IL-17 is released by memory T cells, which are part of the adaptive immune system enabling the organism to respond more rapidly upon re-exposure to antigens. Our results indicate that the adaptive immune system by means of IL-17 release may direct the lung innate immune response to act more vigorously.


   Acknowledgments
 
The authors are grateful to Dr. Jeroen Maertzdorf for scientific input, to Dr. T. A. Out and Prof. R. A. W. van Lier for critical reading of the manuscript, and to Nico Ponne for automating the IL-6 ELISA on the Biomek.


   Footnotes
 
This research was sponsored by the Netherlands Asthma Foundation (grant 99.27).

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 12, 2005

Received in final form March 22, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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