American Journal of Respiratory Cell and Molecular Biology

Interleukin (IL)-8 is a C-X-C chemokine that potently chemoattracts and activates neutrophils. We determined whether IL-8 could be produced by human airway smooth muscle cells in culture and examined its regulation. TNF-α stimulated IL-8 mRNA expression and protein release in a time- and dose-dependent manner, whereas IFN-γ alone had no effect. Both cytokines together did not induce greater IL-8 release compared to TNF-α alone. IL-1β was more potent in inducing IL-8 release and, together with TNF-α, there was a synergistic augmentation of IL-8 release. IL-8 release induced by TNF-α and IFN-γ was partly inhibited by the Th-2-derived cytokines IL-4, IL-10, and IL-13, as well as by dexamethasone. In addition to its contractile responses, airway smooth muscle cells have synthetic and secretory potential with the release of IL-8 and subsequent recruitment and activation of neutrophils in the airways. Release of IL-8 can be modulated by Th-2-derived cytokines and corticosteroids.

Airway smooth muscle has been regarded as having mainly contractile properties because of its ability to shorten in response to many contractile inflammatory mediators, leading to a reduction in airway caliber. Airway smooth muscle can also respond to cytokines and growth factors released from resident and/or infiltrating proinflammatory cells by undergoing mitogenesis (1). However, it is not known whether airway smooth muscle cells can act as effector cells in initiating or perpetuating airway inflammation by expressing and releasing inflammatory products such as chemotactic cytokines or chemokines.

Interleukin (IL)-8 is a member of the C-X-C chemokine subfamily of cytokines. It was identified following the original description of a neutrophil chemotactic agent in the supernatants of lipopolysaccharide (LPS)- or phytohemagglutinin-stimulated human monocytes (2, 3). IL-8 induces the full pattern of responses of activated neutrophils. It induces shape change and migration, exocytosis of stored proteins, and respiratory burst resulting in the release of superoxide anions or hydrogen peroxide of neutrophils (4). IL-8 is produced by inflammatory cells such as monocytes/macrophages (3), eosinophils (5), and by a variety of resident cells such as airway epithelial cells (6) and endothelial cells (7).

We have shown that human airway smooth muscle cells (HASMCs) in culture can express and release the C-C chemokine RANTES (8). This indicates that airway smooth muscle not only has contractile function but can also secrete inflammatory products that participate in the airway inflammatory response. We postulated that HASMCs could also express and release C-X-C chemokines such as IL-8. In the present study, we demonstrate that HASMCs in culture can be stimulated by tumor necrosis factor α (TNF-α) and IL-1β to express and release IL-8, and that this effect can be inhibited by cytokines such as these derived from Type 2 helper T cell (Th-2 cells): IL-4, IL-10, and IL-13. Dexamethasone had a similar inhibitory effect. Our results reinforce the view that HASMCs can participate in the chemoattraction and activation of neutrophils and of other inflammatory cells.

HASMC Culture

Human bronchial smooth muscle was obtained from the lobar or main bronchus at lung resection from patients of either sex, undergoing surgery for carcinoma of the bronchus, as described previously (9, 10). Once in culture, human airway smooth muscle cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing fetal calf serum (FCS) (10%, vol/vol) supplemented with sodium pyruvate (1 mM), l-glutamine (2 mM), nonessential amino acid mixture (1:100), gentamicin (50 μg/ml), and amphotericin B (1.5 μg/ml). All cultures were maintained in a humidified atmosphere at 37°C in air/CO2 (95:5%, vol/vol). Fresh medium was replaced every 72 h. As revealed by immunofluorescence techniques for both smooth muscle actin and myosin, more than 95% of the cells displayed the characteristics of smooth muscle cells in culture (10).

Cell Stimulation

Confluent human airway smooth muscle cells (passage 3– 8) were growth arrested by FCS deprivation for 72 h in DMEM supplemented with sodium pyruvate (1 mM), nonessential amino acids (1:100), l-glutamine (2 mM), gentamicin (50 μg/ml), amphotericin B (1.5 μg/ml) (GIBCO, Paisley, UK), insulin (1 μM), transferrin (5 μg/ml), ascorbic acid (100 μM), and bovine serum albumin (BSA, 0.1%) (Sigma, Poole, UK). After 72 h, cells were stimulated in fresh FCS-free medium containing cytokines IL-1β, TNF-α, interferon γ (IFN-γ) alone or in combination in a concentration- and time-dependent manner, and in the presence of different concentrations of IL-4, IL-10, IL-13 (R&D Laboratories, Oxford, UK), and dexamethasone (Sigma). In these experiments, dexamethasone and IL-4, IL-10, and IL-13 were preincubated for 2 h prior to addition of TNF-α, IFN-γ, and the mixture of three cytokines (cytomix).

Airway Smooth Muscle Proliferation

To determine any potential effect of IL-1β on airway smooth muscle (ASM) proliferation, ASM at confluence and growth arrested by FCS deprivation was incubated with [3H]thymidine at a final concentration of 1 μCi ml−1 to allow this incorporation into DNA. Cultures were washed twice in phosphate-buffered saline to rinse loosely associated radioactive tracer from the wells. Acid-soluble radioactivity was removed by a 20-min treatment with 5% trichloroacetic acid at 4°C, followed by washing the cultures twice in 95% ethanol. The remaining material, which represented the acid-insoluble pools, was solubilized by a 30-min incubation with 2% Na2CO3 in 0.1 M NaOH. The radioactivity was determined by liquid scintillation counting.

Northern Blot Analysis

Total cellular RNA was extracted from adherent cells using a modification of the method of Chomczynski and Sacchi (11). Following two phenol–chloroform extractions and an isopropanol precipitation, RNA samples were stored overnight and washed twice with ethanol (75%, vol/ vol; BDH, Poole, UK) and dissolved in RNase-free water. A total of 20 μg of cytoplasmic RNA was separated by electrophoresis on a 1% agarose gel containing 7.5% formaldehyde and transferred to a nylon Hybond-N membrane (Amersham, Bucks, UK) and fixed by ultraviolet irradiation. Filters were then hybridized with a 32P-labeled human IL-8 cDNA probe using a multiprime DNA labeling kit (Amersham). The IL-8 probe was a 750-bp PstI–BamHI cDNA fragment. Filters were then washed at a final stringency of 0.1× SSC and 0.1% sodium dodecyl sulfate (SDS) at 55°C and exposed at −70°C on Kodak (Rochester, NY) XS 1 100 film for 3–5 d. Probes were stripped by incubating the blot in a 50% formamide solution at 70°C for 2 h before hybridization with a 32P-labeled

1272-bp rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe. Densitometric quantification of the Northern blots was performed by laser densitometry (Protein & DNA Imageware System, Discovery Series, New York, NY). Specific RNA levels are expressed as the ratio of IL-8 to GAPDH mRNA.

Reverse Transcriptase-Polymerase Chain Reaction

Total cellular RNA was prepared as for Northern analysis. Reverse transcription of 1 μg of total RNA was performed using avian myeloblastosis virus (AMV) reverse transcriptase (15 U), a 1 mM of dATP, dCTP, dGTP, and dTTP, 0.4 μg oligo(dT)15 primer, 30 U RNase inhibitor, 5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), and 0.1% Triton X-100 in a total volume of 40 μl (all from Promega, Southampton, UK). Oligo(dT) and dissolved RNA were incubated at 65°C for 10 min and placed on ice for 5 min. The remaining ingredients were then added and samples were incubated at 42°C for 60 min followed by 10 min at 85°C. The cDNA was subsequently diluted to a final volume of 400 μl in nuclease-free water.

Ten microliters of the cDNA solutions were used. The polymerase chain reaction (PCR) was performed using a 7.5 pM concentration of forward and reverse primers; dATP, dGTP, dTTP, and dCTP at a final concentration of 0.2 mM each; Taq polymerase, 1.5 U; MgCl2, 1.5 mM; KCl, 50 mM; Tris-HCl (pH 9.0), 10 mM; and 0.1% Triton X-100 in a final volume of 30 μl. Primers for IL-8 were GTGCCGGTCGAACCTTCAGTA and CTCTTCAAAAACTTCTCCCGACTCTTAAGTATT, giving a product of 298 bp. Primers for GAPDH were TCTAGACGGCAGGTCAGGTCCACC and CCACCCATGGCAAATTCCATGGCA, giving a product of 598 bp. The PCR was carried out in a Techne multiwell thermocycler (Techne, Cambridge, UK) at 95°C for an initial 5 min followed by 24 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 60 s. Final extension was for 10 min at 72°C. The number of cycles was chosen after determination of the linear phase of the product amplification curve from serial sampling with increasing cycles of amplification. Products were distinguished by electrophoresis on a 2% agarose ethidium bromide-stained gel and then visualized and photographed using ultraviolet luminescence. The relative abundance of the product was assessed using laser densitometry measured from the photographic negative and expressed as a ratio of the IL-8 band to the GAPDH band.

IL-8 Radioimmunoassay

IL-8 was measured as described previously (12). All samples were assayed in duplicate, and nonspecific binding was determined by incubation of the radiolabeled ligand under identical conditions but in the absence of the anti-human IL-8 antiserum.

Data Analysis

Data are reported as a mean ± SEM. Comparison between groups was performed using the paired t test. A P value of < 0.05 was considered to be significant.

Induction of IL-8 Protein and mRNA Expression by TNF- α and IFN- γ

HASMCs were stimulated for 24 h with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) or with both TNF-α and IFN-γ. TNF-α alone induced IL-8 protein and mRNA, as measured on Northern analysis, whereas IFN-γ had no significant effect. There was no further induction of IL-8 protein and mRNA with the combination of both cytokines (Figure 1).

Dose effect.

HASMCs were plated on 24-well plates and stimulated either with TNF-α or IFN-γ alone at concentrations of 0, 1, 3, 10, 30, and 100 ng/ml for 24 h. In addition, TNF-α (10 ng/ml) or IFN-γ (10 ng/ml) was added to each concentration of the alternate cytokine in order to test any potential dose-dependent synergistic effect of one cytokine on the other. IFN-γ alone had no significant effect up to a concentration of 100 ng/ml, whereas TNF-α induced the release of IL-8, with the highest effect being seen at 100 ng/ml. In the presence of TNF-α (10 ng/ml), IFN-γ did not induce a dose-dependent increase in IL-8 production. Similarly, there was no potentiating effect of IFN-γ (10 ng/ml) on the response to increasing concentrations of TNF-α (Figure 2).

Time course.

HASMCs were cultured in the presence of TNF-α or IFN-γ or TNF-α plus IFN-γ at 10 ng/ml each. There was a time-dependent increase in IL-8 release after stimulation with TNF-α and with the combination of TNF-α and IFN-γ (Figure 3). There was no synergistic effect of TNF-α and IFN-γ on IL-8 release. The time course of IL-8 release after stimulation with TNF-α plus IFN-γ was similar to that of TNF-α alone. IFN-γ alone had no effect on IL-8 release. There was a persistent increase in IL-8 mRNA as measured by reverse transcriptase (RT)-PCR between 12 and 96 h, with a peak expression at 24 h (Figure 3).

Effect of Different Combinations of IL-1 β , TNF- α , and IFN- γ on IL-8 Release

HASMCs were stimulated for 24 h with TNF-α (10 ng/ml) or IFN-γ (10 ng/ml) in the presence of IL-1β (10 ng/ml) or with a combination of the three cytokines (cytomix). The stimulatory effect of IL-1β and IFN-γ was similar to that of IL-1β alone. Cytomix was significantly less effective than IL-1β and TNF-α, indicating that IFN-γ had inhibitory effects when combined with IL-1β and TNF-α on IL-8 release (Figure 4).

HASMCs were stimulated with IL-1β (0, 4, 20, and 100 ng/ml) for 24 h. All concentrations of IL-1β stimulated IL-8 release, with the 4-ng/ml concentration already having a submaximal effect (Figure 5). Cytomix induced IL-8 release in a time-dependent fashion up to 48 h (Figure 5).

Effect of IL-1 β on ASM Proliferation

IL-1β (10 ng/ml) decreased [3H]thymidine incorporation by 36.3 ± 14.8% (n = 3); cytomix (IL-1β, IFN-γ, and TNF-α, at 10 ng/ml each; n = 3) also decreased it by 18.6 ± 9.3%.

Effects of IL-4, IL-10, IL-13, and Dexamethasone on IL-8 Release

HASMCs were stimulated with TNF-α plus IFN-γ (10 ng/ ml each) in the presence of IL-4, IL-10, IL-13, or dexamethasone. IL-4, IL-10, and IL-13 significantly reduced IL-8 production with a maximal effect observed at 10 ng/ ml for IL-4 and IL-10, and 1 ng/ml for IL-13 (Figure 6). Dexamethasone inhibited IL-8 release by 26.5 ± 12.9% at 10−6 M (P < 0.001) together with a reduction in IL-8 mRNA as assessed by Northern analysis (Figure 1). After stimulation with cytomix dexamethasone (10−6 M) inhibited IL-8 release by 41.2 ± 6% (Figure 6).

We have shown that human airway smooth muscle cells in culture are able to express IL-8 mRNA and release IL-8 protein in response to TNF-α and IL-1β but not to IFN-γ. This effect is concentration- and time-dependent. IL-1β was more potent than TNF-α and it synergized with TNF-α in terms of IL-8 release. The Th-2-derived cytokines IL-4, IL-10, and IL-13 inhibited the release of IL-8 protein. Similarly, IL-8 release was inhibited by dexamethasone. Our results suggest that airway smooth muscle may contribute to airway inflammation by releasing the neutrophil chemoattractant and activating cytokine, IL-8.

IL-8 gene induction and protein release have been described in many cell types such as airway epithelial cells (6, 13), mononuclear cells (14), and endothelial cells (7, 15), and our current work demonstrates the expression and production of IL-8 by airway smooth muscle cells. IL-1β was more potent than TNF-α in inducing IL-8 protein release, and synergized with TNF-α in increasing IL-8 release. IL-1β and TNF-α have also been shown to increase the expression of IL-8 in other cell types such as airway epithelial cells and fibroblasts (13, 16-18). We did not observe any effect of IFN-γ. However, a synergistic interaction between TNF-α and IFN-γ has been described in airway smooth muscle cells (8) and also in fibroblasts, endothelial cells, and airway epithelial cells for gene expression and protein release of another chemokine, RANTES (19-21). Thus, the ultimate effect of these proinflammatory cytokines on airway smooth muscle cells depends on the particular chemokine generated.

There was a continuing time-dependent increase in IL-8 protein release on exposure to TNF-α or to cytokine mixture up to 48 to 96 h. The parallel increase in IL-8 mRNA as observed on Northern analysis or reverse transcription polymerase chain reaction indicates that the increase in protein release is secondary to IL-8 gene transcription. This is further supported by the observation that glucocorticosteroids inhibited both IL-8 gene expression together with IL-8 protein release to a similar extent. A negative GRE site has been described on the 5′-flanking region of the IL-8 gene (22) and binding of activated glucocorticoid receptor to that site may lead to inhibition of IL-8 gene expression, as has been reported in a human fibrosarcoma cell line (18).

It is possible that the time-dependent increase in IL-8 mRNA and protein expression indicates that this response could be related to increased mitogenesis and changes in airway smooth muscle cell phenotype. Previous studies using similar concentrations of TNF-α have shown that it induces modest proliferation in cultures of human airway smooth muscle (23) and IL-1β has been reported to increase guinea pig airway smooth muscle cell mitogenesis (24). However, in the present study, there was a reduction in proliferation of human airway smooth muscle cells as measured by thymidine incorporation induced by IL-1β and also by the mixture of cytokines. It is therefore unlikely that the increase in IL-8 expression and release observed following cytokine stimulation is due to any induced increase in smooth muscle cell proliferation.

The cytokines IL-4, IL-10, and IL-13 are derived from Th-2 cells, and IL-10 and IL-13 can also be released from monocytes/macrophages. The degree of inhibition observed by these cytokines did not exceed 60%, with IL-10 being the most effective. We have also observed that these three cytokines can inhibit to a similar extent the release of RANTES from airway smooth muscle cells (8) and of macrophage inflammatory protein 1α (MIP-1α) from alveolar macrophages (14, 25, 26). The inhibition observed by these cytokines on the release of IL-8 from airway smooth muscle cells indicates that Th-2 cells or macrophages may interact with airway smooth muscle. Activated T cells have been shown to adhere to airway smooth muscle cells via specific integrins (27).

Our results indicate that the airway smooth muscle cell should not be regarded solely as a specialized cell capable only of contractile responses. Proinflammatory cytokines and several growth factors can modulate airway smooth muscle phenotype and mitogenesis (1) and the resulting increase in airway smooth muscle mass may contribute to airway obstruction and bronchial hyperresponsiveness in asthma (28). It is not known whether IL-8 could have an autocrine role in the airway smooth muscle in controlling mitogenesis. In preliminary studies of immunostaining with an anti-IL-8 antibody, we have observed positive staining in airway smooth cells of the airways in lungs obtained from patients undergoing lung resection for cancer (T. Gilbey and K. F. Chung, unpublished observations). This indicates that IL-8 may be expressed under basal conditions in vivo, and the role of IL-8 in this situation is not known.

The additional secretory potential of airway smooth muscle, particularly in terms of IL-8 and RANTES release, adds another dimension to the putative role of airway smooth muscle in airway inflammation. Airway smooth muscle could contribute directly to the recruitment of inflammatory cells such as neutrophils to the airways by increased expression of IL-8. This could occur through the release of the proinflammatory cytokines TNF-α and IL-1β from monocytes/alveolar macrophages or T cells within the vicinity of airway smooth muscle cells. On the other hand, Th-2 cells may provide cytokines such as IL-4, IL-10, and IL-13 to inhibit IL-8 expression. Our observations support the notion that airway smooth muscle could be a major contributor to the inflammatory features of the airways in diseases characterized by a neutrophilic airway inflammation such as chronic bronchitis and asthma.

This work was supported by a Program Grant from the British Medical Research Council (K.F.C. and P.J.B.), and a grant from the Overseas German Academic Exchange Service (M.J.).

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Address correspondence to: Dr. K. F. Chung, National Heart and Lung Institute, Dovehouse St., London SW3 6LY, UK. E-mail:

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