IL-17R activation of human airway smooth muscle cells induces CXCL-8 production via a transcriptional-dependent mechanism
Introduction
Airway neutrophilia has been recognized as a predominant feature of lung inflammatory disorders such as Chronic Obstructive Pulmonary Disease (COPD) and severe asthma [1], [2], [3]. Elevated numbers of neutrophils within the inflammatory infiltrate are often associated with disease severity [4]. These cells are believed to contribute to tissue damage through release of granule proteins, reactive oxygen metabolites, pro-inflammatory, and pro-fibrotic cytokines. In addition to eliciting tissue damage, neutrophil-derived pro-inflammatory mediators can perpetuate the inflammatory reaction and lead to chronic changes in airway function [5], [6], [7].
Human interleukin-17 (IL-17) is a homodimeric protein, with pleiotropic biological activities, that can be released by activated CD4+ T cells [8], [9], [10]. IL-17 is able to stimulate production of IL-6, IL-8, GM-CSF, stem cell factor, and prostaglandin E2 from various cell types, such as fibroblasts, keratinocytes, and renal epithelial cells [11], [12], [13], [14]. A potential role of IL-17 in the initiation or maintenance of inflammatory responses is suggested by elevated IL-17 mRNA expression in mononuclear cells from patients with multiple sclerosis [15], in patients with rheumatoid arthritis [16], and patients with systemic lupus erythematosus [17]. Furthermore, studies using a murine model have clearly demonstrated the role of IL-17 network in inducing neutrophil recruitment to the inflammatory sites [18].
IL-8/CXCL-8 belongs to the CXC chemokines subfamily of cytokines and is an important chemoattractant of neutrophils. It has also chemotactic activity for activated T lymphocytes, eosinophils and basophils. Furthermore, CXCL-8 enhances integrin expression in monocytes and their adherence to endothelial cells leading to inflammatory process.
Recent evidence suggests that airway smooth muscle (ASM) cells are a potential source of proinflammatory cytokines and chemokines [19], [20], thereby acting as effector cells in initiating or perpetuating airway inflammation. While it has been shown that IL-17 induces expression of chemokines and cytokines in the airway [21], potential direct effects of IL-17 on ASM cells have not been determined. We hypothesized that ASM cells play a key role in the pathogenesis and maintenance of chronic airway inflammation via IL-17-dependent mechanisms. To investigate the role of IL-17 in this process, we examined the expression of IL-17R by human airway smooth muscle, and the molecular mechanism by which IL-17 influences smooth muscle neutrophilic mediator release, particularly CXCL-8.
Section snippets
Reagents and antibodies
Mouse mAb anti-human IL-17R directed to N-terminal extracellular domain (IgG1), mouse mAb neutralizing anti-IL-17 (IgG2b), recombinant human IL-17 and IL-1β were purchased from R&D (Minneapolis, MN). Affinity purified mouse IgG1 (Clone MOPC21), IgG2b isotype controls, normal human and goat serum were obtained from Sigma (Oakville, Ontario). Goat anti-mouse IgG F(ab′)2 Alexa Fluor 488, ProLong® antifade were obtained from Molecular Probes (Eugene, Oregon). Normal rabbit serum was from Cedarlane
Human primary ASM cells express constitutive levels of IL-17R
We used reverse transcription PCR to search for expression of IL-17R in human primary ASM cells. As shown in Fig. 1, mRNA of IL-17R was detected in all RNA preparations from four different confluent serum fed human ASM cell cultures using specific primers for the IL-17R extra-cellular domain (upper panel) and intracellular domain (middle panel). To further investigate protein expression of IL-17R by human ASM cells, immunocytochemistry was performed with mouse anti-human-IL-17R mAb followed by
Discussion
Airway inflammation is thought to play a central role in the pathogenesis of many diseases including asthma and COPD [3]. A hallmark of COPD and severe asthma is increased numbers of neutrophils within the airway [29]. Although airway smooth muscle cells contribute to the perpetuation of airway wall inflammation [19], [30], the exact mechanism is still unknown. In this study, we investigated the possibility that ASM cells have the potential to contribute to the accumulation of inflammatory
Acknowledgments
We thank Dr. M. Kracht, Institute of Pharmacology, Medical School Hannover, Hannover, Germany for the gift of CXCL-8 promoter constructs.
Abdelilah Soussi Gounni is supported by a CIHR New Investigator Scholarship. This work was supported by grant from the Manitoba Health Research council (MHRC) to ASG. Rahman MS. is supported by a fellowship from CIHR National Training Program in Allergy and Asthma.
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