IL-17R activation of human airway smooth muscle cells induces CXCL-8 production via a transcriptional-dependent mechanism

doi:10.1016/j.clim.2005.01.014

Clinical Immunology

Volume 115, Issue 3, June 2005, Pages 268-276

https://doi.org/10.1016/j.clim.2005.01.014 Get rights and content

Abstract

Airway neutrophilia has been recognized as a predominant feature of acute lung disorders. While it has been shown that IL-17 induces expression of the CXC chemokines in the airways leading to neutrophil recruitment, the IL-17R expression in human ASM cells and the molecular mechanism by which IL-17 mediates neutrophilic chemo-attractant CXCL-8 (IL-8) production have not been determined. Our study showed that ASM cells express steady state IL-17R protein, mRNA and surface-bound receptor. Interestingly, airway sections from COPD patients revealed IL-17R-positive immunostaining within ASM bundles. IL-17 was capable of stimulating CXCL-8 protein release from ASM cells which was significantly decreased by neutralizing anti-IL-17 mAb. Furthermore, IL-17 induction of CXCL-8 mRNA and protein release from ASM cells was abrogated by transcriptional inhibitor actinomycin D. CXCL-8 promoter reporter analysis using wild type and site specific mutant constructs demonstrated a key role for AP1 and NF-κB binding sites in IL-17-induced CXCL-8 expression. These data demonstrate that IL-17 mediates CXCL-8 expression in ASM cells via a transcriptional mechanism depending on NF-κB and AP-1 pathways. Together, our findings suggest that ASM cells play an important role in airway neutrophilia.

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 (IgG_2b), recombinant human IL-17 and IL-1β were purchased from R&D (Minneapolis, MN). Affinity purified mouse IgG1 (Clone MOPC21), IgG_2b 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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IL-17R activation of human airway smooth muscle cells induces CXCL-8 production via a transcriptional-dependent mechanism

Abstract

Introduction

Section snippets

Reagents and antibodies

Human primary ASM cells express constitutive levels of IL-17R

Discussion

Acknowledgments

J. Allergy Clin. Immunol.

J. Invest. Dermatol.

Immunol. Lett.

Trends Pharmacol. Sci.

J. Biol. Chem.

Cytokine Growth Factor Rev.

J. Allergy Clin. Immunol.

J. Heart Lung Transplant.

J. Heart Lung Transplant.

J. Biol. Chem.

Neutrophils in chronic obstructive pulmonary disease

Novartis Found. Symp.

Increased neutrophil numbers and IL-8 levels in airway secretions in acute severe asthma: clinical and biologic significance

Am. J. Respir. Crit. Care Med.

The many faces of airway inflammation. Asthma and chronic obstructive pulmonary disease. Asthma Research Group

Am. J. Respir. Crit. Care Med.

Bronchial neutrophilia in patients with noninfectious status asthmaticus

Am. J. Respir. Crit. Care Med.

Neutrophilic inflammation in severe persistent asthma

Am. J. Respir. Crit. Care Med.

Decreased steroid responsiveness at night in nocturnal asthma. Is the macrophage responsible?

Am. J. Respir. Crit. Care Med.

T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines

J. Exp. Med.

Human IL-17: a novel cytokine derived from T cells

J. Immunol.

CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus saimiri gene

J. Immunol.