SEARCH

SEARCH BY CITATION

Keywords:

  • anti-IL-33;
  • cigarette smoke;
  • lung inflammation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

The mechanism by which cigarette smoke (CS) causes chronic obstructive pulmonary disease (COPD) is poorly understood. Interleukin-33 (IL-33) is a pleiotropic cytokine predominantly expressed in lung tissue and can elicit airway inflammation in naive mice. We tested the hypothesis that IL-33 is induced by CS and contributes to CS-mediated airway inflammation in a mouse model of CS-induced COPD. Groups of mice were exposed to CS three times per day for 4 consecutive days. The expression levels of IL-33 and ST2 were markedly enhanced in the lung tissue of mice inhaling CS. Exposure to CS also induced neutrophil and macrophage infiltration and expression of inflammatory cytokines (IL-1β, tumour necrosis factor-α, IL-17), chemokines (monocyte chemoattractant protein-1) and mucin 5, subtypes A and C in the airways. More importantly, all of these CS-induced pathogenic changes were significantly inhibited by treatment with neutralizing anti-IL-33 antibody delivered intranasally. Hence, our results suggest that IL-33 plays a critical role in CS-mediated airway inflammation and may be a therapeutic target in CS-related diseases, including COPD.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Cigarette smoke (CS) is a principal environmental risk factor closely associated with the development and exacerbation of a wide-range of inflammatory diseases, in particular chronic obstructive pulmonary disease (COPD),[1, 2] which is characterized by progressive and irreversible airflow limitation.[1-4] Pathological features of COPD include inflammation, goblet cell metaplasia, remodelling of the airways and alveolar destruction.[2-5] There is currently no cure for COPD and it is predicted to become the third leading cause of global death by 2020.[4] More effective therapeutic strategies are critically needed to control this treatment-refractory disease. The mechanism by which CS contributes to the pathogenesis of COPD remains to be elucidated. Current evidence suggests that CS has a wide range of biological and toxic effects on structural and immune cells in the airways.[1, 2, 5-7] As such, inhalation of CS can drive the recruitment and activation of inflammatory immune cells, in particular neutrophils and macrophages, which induce the production of an array of inflammatory mediators including cytokines and chemokines in the airways that trigger mucus production, apoptosis of alveolar epithelial cells, matrix degradation, leading to chronic bronchitis and emphysema; all aspects of COPD pathology.[1-5, 8]

Interleukin-33 (IL-33) is a new member of the IL-1 family and signals via ST2.[9] Interleukin-33 is expressed by innate cells in humans and mice, primarily epithelium and endothelium, and can be released when cells sense inflammatory signals or undergo necrosis.[9-11] Current evidence suggests that IL-33 is a pleiotropic cytokine that can orchestrate complex immune responses in immunity and in diseases.[12] ST2 is expressed on most innate cells and IL-33 may therefore play a critical role in innate immunity by directly activating a wide range of innate cells including macrophages, neutrophils and natural killer cells via ST2.[9, 12-17] However, ST2 is also selectively expressed on T helper type 2 (Th2), IL-5+ Th and CD8+ T cells.[9, 18-20] As such it can also induce antigen-specific Th2 responses and CD8 T-cell activation, respectively, in a different context.[18-20] Intriguingly, IL-33 can also promote Th1 and Th17 responses in pro-inflammatory and autoimmune diseases, perhaps indirectly via mast cells.[21, 22]

Interleukin-33 is highly expressed in lung tissue and plays a critical role in respiratory diseases.[9, 18, 23] It is sufficient to elicit airway inflammation, airway hyper-responsiveness and goblet cell metaplasia in allergen-naive mice, and exacerbates asthma-like responses in allergen-exposed mice.[9, 18, 23] However, whether CS can induce IL-33/ST2 expression in the airway and whether the IL-33 system contributes to the pathogenesis of CS-mediated COPD is unknown.

We therefore investigated the role of IL-33 in CS-induced acute airway inflammation in naive mice, a model for smoking COPD.[24, 25] We report that IL-33 and ST2 can be induced in CS-exposed mice, that the IL-33 is able to trigger airway inflammation and mucin expression in the airway, and that these changes can be inhibited by the administration of neutralizing anti-IL-33 antibody. Hence, IL-33 may be a new therapeutic target for CS-mediated respiratory disease including COPD.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Mice

Male C57BL/6 mice were obtained from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China). Mice were housed in sterilized cages with filter tops in specific pathogen-free conditions at Shantou University, China in accordance with animal experimentation guidelines.

Cigarette smoke exposure

Male mice (= 12 per group) were exposed passively to CS in the atmosphere of a Perspex chamber using a modified method.[24, 25] Briefly, groups of mice were exposed to the smoke of nine cigarettes (Reference Cigarette 1R5F; University of Kentucky, Lexington, KY) for three separate 1-hr periods per day for four consecutive days. Control groups of mice were exposed to ambient room air for the same time period. The mice were killed on day 5.

Intranasal administration of anti-IL-33

One hour before the first smoke exposure on days 2 and 4, mice were anaesthetized lightly by intraperitoneal injection of ~ 50 μl 2% sodium pentobarbitone, then were administrated intratracheally purified anti-IL-33 polyclonal antibody 100 μg/mouse or PBS, both diluted in PBS.[26]

Bronchoalveolar lavage (BAL) was performed in some mice using a tracheal cannula.[19] Briefly, the trachea was exposed and the cannula was inserted via a small transverse cut, and lungs were lavaged in situ with three aliquots (500 μl) of ice-cold PBS. The total number of viable cells and the cell differential counts in the BAL fluid were determined by cell counting and by microscopy of cyto-centrifuge slide preparations stained with haematoxylin & eosin.

Histological analysis and immunohistochemistry

Lung tissues (which had not been lavaged) were fixed in 10% buffered formalin solution and embedded in paraffin using standard methods. Sections (5-μm) were stained with haematoxylin & eosin to detect cellular infiltration. For each animal, 10 fields at a magnification of × 200 were captured in a blinded fashion using an image analyser platform (Olympus Corporation, Tokyo, Japan). Peribronchial and perivascular inflammation was scored using a semiquantitative scoring system as described.[15]

Immunohistochemistry of lung tissue was performed with a rabbit polyclonal anti-mouse IL-33 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rat anti-mouse Mac-3 and Ly-6G antibodies (Biolegend, San Diego, CA), respectively. All of these proteins were detected in the lung sections with an IgG Streptavidin Biotin Complex kit (Boster, Wuhan, China) and developed with DAB substrate according to the manufacturer's protocol (Dako, Glostrup, Denmark). The immunohistochemical staining was scored on a four-point scale as follows: 0 = none, 1 = weak, 2 = moderate and 3 = intense.[24]

Reverse transcription-PCR

Total RNA in mouse lung tissue was isolated using TRIzol (Invitrogen, Carlsbad, CA) and reverse transcription was performed using a PrimeScript II 1st strand cDNA Synthesis Kit (TakaRa, Dalian, China) according to the manufacturer's protocol. The cDNA was amplified with paired primers using Premix Ex Taq™ Hot Start Version (TakaRa) according to the manufacturer's protocol. The endogenous β-actin gene was used as an reverse transcription (RT-) PCR control. The PCR products underwent electrophoresis in 1·2% agarose gels and were stained with SYBR safe dye, then visualized digitally with a UV illuminator. The band intensities were semi-quantified using gel-pro analyzer 3·2 software (Media Cybernetics, Rockville, MD).

Statistical analysis

Data are presented as mean ± SEM. Differences between groups were determined by one-way analysis of variance followed by the Bonferroni post hoc test for multiple comparisons or the two-tailed Student's t-test as appropriate.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Cigarette smoke induces IL-33 and ST2 expression in lung tissue

We initially determined whether CS can induce IL-33 and ST2 in naive mice. Groups of mice were exposed or not to CS three times a day for 4 days. Interleukin-33 protein levels in the lung tissue were determined by immunohistochemistry. We found that the IL-33 detected by immunohistochemistry was increased (< 0·01) in the lung tissue of mice exposed to CS compared with control mice exposed to the room air (Fig. 1a). The morphological appearance of IL-33-positive cells suggested that these might be alveolar macrophages and epithelial cells (Fig. 1b). The CS also significantly enhanced IL-33 and ST2 mRNA expression (< 0·05 for each) in the lung tissues when compared with that in the control mice (Fig. 1c). Hence, CS is able to induce lung IL-33 and ST2 expression in naive mice.

image

Figure 1. Cigarette smoke (CS) induces interleukin-33 (IL-33) and ST2 expression in the lung tissue. Groups of mice (= 5) were exposed to CS or room air three times per day for 4 days and lung tissues were harvested on day 5. (a) IL-33 protein expression on lung tissue was determined by immunohistochemical staining (original magnification, 100×) and scored as described in Materials and methods. (b) The morphology of IL-33-expressing cells in the lung sections was further determined (400×). (c) The expression of IL-33 and ST2 in lung tissues detected by PCR and semiquantified. Values represent means ± SEM. #< 0·05 versus the respective control, *< 0·01 versus the respective control.

Download figure to PowerPoint

Anti-IL-33 treatment impairs CS-induced airway inflammation

We next investigated whether the CS-derived IL-33 contributes to the CS-induced airway inflammation in mice by treating the mice with intranasal delivery of neutralizing anti-IL-33 antibody.[26] Morphometric assessment was performed to explore the effects of anti-IL-33 treatment by histological examination of lung sections. The lungs of mice exposed to room air showed a normal parenchyma with normal airways and anti-IL-33 antibody had no significant effect on the airway histology or cytology (Fig. 2a,b,e,f). However, exposure to CS increased the number of inflammatory cells in the lung parenchyma and interstitial space of the airways as well as foci of inflammation and goblet cell metaplasia and hyperplasia (Fig. 2c,g,i). In contrast, treatment with anti-IL-33 significantly reduced inflammatory cell infiltration in the peribronchiolar and alveolar regions as well as goblet cell metaplasia and hyperplasia (Fig. 2d,h,i).

image

Figure 2. Anti-interleukin-33 (IL-33) treatment impairs cigarette smoke (CS) -induced airway inflammation. Groups of mice were exposed to CS or room air as in Fig. 1 and treated with anti-IL-33 antibody (αIL-33) or PBS as control. Mice in room air received PBS (a and e) or anti-IL-33 antibody (b and f). The CS-exposed mouse received PBS (c and g) developed inflammation in the lung but treated with anti-IL-33 impaired lung inflammation (d and h). (a–d) were at original magnification (100×) and e–h were 400×. (i) Histological score of peribronchial and perivascular inflammation. (j) Total cell number in broncholaveolar lavage fluid in different treated mice. Data are mean ± SEM (n = 5 mice/group). #< 0·05 versus the respective control, *< 0·01 versus the respective control, **< 0·05 versus the CS + PBS group, ***< 0·01 versus the CS + PBS group.

Download figure to PowerPoint

Furthermore, CS exposure also increased the total number of cells in BAL fluid (up to ~ 30%) in the mice. However, this increase was significantly inhibited in the CS-exposed mice when they were treated with anti-IL-33 (Fig. 2j).

These results suggest that IL-33 contributes to the CS-induced airway inflammation, which can be effectively inhibited by neutralizing anti-IL-33 antibody in mice.

Anti-IL-33 treatment decreases CS-elicited neutrophil and macrophage infiltration in the lungs

Neutrophils and macrophages are the key cell components in CS-induced airway inflammation and play a critical role in the pathogenesis of COPD.[1, 2, 5, 8] We therefore further assess the effect of anti-IL-33 on the CS-induced neutrophil and macrophage infiltration in lung tissue by immunohistochemical staining of Ly-6G and Mac-3, surface markers for tissue neutrophils and macrophages, respectively.[27, 28]. Ly-6G staining showed a significant increase in the levels of Ly-6G+ neutrophils in the lung tissue of CS-exposed mice compared with controls (< 0·01). Importantly, the CS-induced neutrophil infiltration was effectively prevented by anti-IL-33 treatment (Fig. 3a,b). Similarly, Mac-3 staining showed that CS exposure induced marked macrophage accumulation in lung tissue, and this was also significantly inhibited by anti-IL-33 treatment (Fig. 3c,d). Therefore, the CS-induced inflammatory cell infiltration observed above was, at least in part, due to inducing IL-33.

image

Figure 3. Anti- interleukin-33 (IL-33) treatment decreases cigarette smoke (CS) -elicited neutrophil and macrophage infiltration into the lung. Immunohistochemical staining of Ly-6G protein (a) and Mac-3 protein (c) on lung tissue of different treated mice. Immunohistochemistry score of Ly-6G protein (b) and Mac-3 protein (d) expression levels on lung sections. Data are representative of two experiments (= 5) at original magnification 400 × , means ± SEM. *< 0·01 versus the respective control, **< 0·05 versus the CS + PBS group, ***< 0·01 versus the CS + PBS group.

Download figure to PowerPoint

Anti-IL-33 reduces CS-induced pro-inflammatory gene expression

Exposure to CS triggers the expression of an array of inflammatory cytokines, chemokines and other mediators in the airways.[1, 2, 5, 8] We found that CS could induce IL-33 and ST2 expression in the lung (Fig. 1a,c). We further assessed the impact of anti-IL-33 treatment on the expression of the key inflammatory genes, including IL-33/ST2 in the lung tissue in response to CS. Exposure to CS enhanced the expression of key pro-inflammatory cytokines [IL-33, ST2, tumour necrosis factor-α (TNF-α), IL-1β and IL-17] and chemokine [monocyte chemoattractant protein (MCP)-1], as well as airway mucin 5, subtypes A and C (MUC5AC) (Fig. 4a, b). Critically, the treatment with neutralizing anti-IL-33 antibody significantly abolished CS-induced expression of these key inflammatory mediators (Fig. 4a and b).

image

Figure 4. Neutralization of interleukin-33 (IL-33) inhibits cigarette smoke (CS) -induced inflammatory gene expression. The expression levels of IL-33, ST2, IL-17, IL-1β, tumour necrosis factor-α (TNF-α), monocyte chemoattractant protein 1 (MCP-1) and mucin 5, subtypes A and C (MUC5AC) in the lungs of CS-exposed or control mice were analysed by reverse transcription (RT) -PCR. (a) Gel images of PCR bands separated by electrophoresis and (b) result of RT-PCR score. Data are representative of three experiments, means ± SEM. *< 0·05 versus the respective control, **< 0·05 versus the CS + PBS group, ***< 0·01 versus the CS + PBS group.

Download figure to PowerPoint

Our results suggest that these key inflammatory genes may be predominantly induced by IL-33 and that IL-33 neutralization is effective in the control of key pro-inflammatory gene expression in CS-induced airway inflammation.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

How tobacco smoking causes COPD is not fully understood. Data reported in this study reveal a hitherto unrecognized effect and mechanism underlying IL-33 in CS-induced airway inflammation in mice. Our results suggest that CS may primarily trigger IL-33/ST2 production, which then stimulates the synthesis of further key pro-inflammatory cytokines, chemokines and mediators in the airway. Together, IL-33-stimulated cytokines and chemokines subsequently promote the migration and activation of inflammatory cells including neutrophils and macrophages into the lung tissue, leading to airway inflammation and lung damage. Hence, IL-33 may be a key intermediate in CS-mediated airway inflammation in vivo and a new therapeutic target in CS-related diseases.

Our results demonstrated for the first time that CS exposure can trigger the expression of IL-33 and ST2 in the lung tissue in vivo (Fig. 1). Whereas the detailed mechanism is still unresolved, CS may drive IL-33 expression in the lung by at least two possible pathways. (1) It was demonstrated that bacterial lipopolysaccharide is present in high concentrations in tobacco and in CS,[29, 30] which may contribute to the pathogenesis of COPD.[1, 31] Recent findings suggest that lipopolysaccharide can also stimulate the release of IL-33 by airway epithelial cells and macrophages, suggesting that CS may trigger IL-33 production, at least in part, via lipopolysaccharide.[32, 33] (2) It is also known that IL-33 can be induced by inflammatory cytokines, IL-1β and TNF-α.[21] Intriguingly, CS can elicit the production of these cytokines in the lung tissue (Fig. 4). Furthermore, we found that this CS-induced IL-33 expression could be blocked by the neutralization of endogenous IL-33 (Fig. 4), suggesting that the induction of IL-33 expression is an autocrine/paracrine process in the CS context; consistent with previous reports.[21, 34, 35]

Interleukin-33 has been described as an inducer of type II cytokine production and eosinophil-mediated airway inflammation in allergy and asthma.[9, 19] However, using neutralizing antibody, we have demonstrated that IL-33 also contributes to the CS-mediated airway inflammation, which is mainly driven by neutrophils and macrophages and by pro-inflammatory cytokines and chemokines.[1, 2, 5] Indeed, our results showed that IL-33 was able to induce the expression of key pro-inflammatory cytokines (TNF-α, IL-1β and IL-17) and MCP-1 and the recruitment of neutrophils and macrophages in this context (Fig. 3 and 4). This is in agreement with previous reports that IL-33 is a pleiotropic cytokine and can also promote Th1 and Th17 responses in pro-inflammatory diseases[12, 21, 22] and that IL-33 is capable of inducing the migration and activation of neutrophils and macrophage in vivo.[15, 36]

The clinical relevance of IL-33 in COPD is still unclear, although it is known that ST2 expression is elevated in patients with COPD.[37] Given that it is inducible by CS and able to elicit the key pathogenic changes of COPD, the IL-33 system may play a critical role in COPD.

Tobacco smoking affects many respiratory and autoimmune disorders, so IL-33 may therefore have a general effect on all CS-mediated inflammatory conditions. This concept suggests that IL-33 may represent a novel therapeutic target for a range of CS-induced inflammatory diseases.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

This study received financial support from the National Natural Science Foundation of China (81070034) to Mingcai Li, Arthritis Research UK, the Medical Research Council UK and the Wellcome Trust to Damo Xu, and was sponsored by K.C. Wong Magna Fund in Ningbo University. The authors thank Dr Yanchun Zhou, Ms Yan Wu and Ms Liu Liu for their excellent technical support and Ms. Yanfei Xie for assistance in animal work.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References