Introduction

Statins are 3-hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors and are frequently used to treat hyperlipidemia and cardiovascular diseases. Statins have also been proposed as novel drugs for treating inflammatory diseases, including asthma,1, 2, 3, 4, 5, 6, 7 because of their anti-inflammatory and immunomodulatory properties in cardiovascular diseases.8 Although these functions of statins have already been investigated in some animal and human studies,2, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 the anti-inflammatory mechanisms of statins and the interrelationships of these mechanisms are far from being completely understood, and some studies have obtained contradictory results.

Asthma is a chronic airway inflammatory disease that is associated with a large influx of inflammatory cells from the systemic circulation into airway tissues. Lymphocytes have a central role in the development of allergic inflammation.19 CD4+ T cells, especially Th2 cells, are effector cells that have been identified in both bronchoalveolar lavage (BAL) fluid and airway tissues in asthma patients, and the transfer of Th2 cells followed by airway allergen challenge in mice induces airway hyper-responsiveness (AHR) and airway eosinophilia.20, 21 CD8+ T cells also act as effector cells in the airway and have an important role in the development of AHR and airway inflammation following allergen challenge.22

The inflammatory cell influx in asthma is triggered by endothelial cell adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1).23, 24 The infiltration of inflammatory cells into airways is associated with elevated VCAM-1 expression in endothelial cells, which is enhanced by Th2-mediated cytokines.25

A variety of mechanisms have been proposed to explain the potent anti-inflammatory and immunomodulatory effects of statins. The administration of statins has been shown to suppress inflammation via a reduction in secretory cytokines and chemokines, such as IL-1, IL-6, IL-8, IL-12 and tumor necrosis factor-α, in asthma patients,8 and the inhibitory effects of simvastatin on mitogen-activated protein kinases, small G proteins and the pro-inflammatory transcription factor nuclear factor κB have also been observed in a mouse asthma model.2 However, the underlying interactions of the different immune cells, including the role of cytokines, remain unclear and there are conflicting reports of these interactions. Therefore, this study sought to thoroughly investigate previous reports, in the light of our findings, to gain insight into the inhibitory mechanisms of statins. We focused on cellular influx in an OVA-specific mouse model of allergic asthma and systemic simvastatin administration.

Materials and methods

Animals

Six-week-old, female BALB/c mice weighing 20±2 g were purchased from Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed under specific pathogen- and ovalbumin (OVA)-free conditions and maintained on a 12-h light–dark cycle with food and water ad libitum. All experimental animals used in this study were kept under a protocol approved by the Institutional Animal Care and Use Committee of Ajou University (AMC-152 (IACUC-152)).

Sensitization, challenge and simvastatin treatment

The experimental protocol for allergen sensitization and challenge was modified from previously described methods.26 Briefly, BALB/c mice were sensitized intraperitoneally with 10 μg OVA (Fisher Scientific, Pittsburgh, PA, USA) emulsified in 1 mg alum (Imject Alum; Pierce, Rockford, IL, USA) on days 0 and 14. The mice were then subjected to airway allergen challenges by exposure to OVA aerosols (1% in saline) for 20 min on days 28, 29 and 30 with an ultrasonic nebulizer (NE-U22, Omron, Kyoto, Japan). Simvastatin (40 mg kg−1; Sigma-Aldrich, St Louis, MO, USA) was then prepared by dissolving 4 mg simvastatin in 100 ml ethanol and 150 ml of 0.1 N NaOH and then incubating this solution at 50 °C for 2 h. The pH was adjusted to 7 with HCl, and the total volume was 1 ml. The 4 mg ml−1 stock solution was diluted in sterile phosphate-buffered saline. Then, 100 μl simvastatin solution was injected intraperitoneally 30 min before the 1% OVA challenge on days 28–30. Control mice were given phosphate-buffered saline as sham sensitization. All experiments in our study were repeated three times, and each group of mice consisted of eight or nine animals.

Airway resistance measurements

The airway resistance to inhaled methacholine (MCh; Sigma-Aldrich) was measured using the flexiVent System (SCIREQ, Montreal, Quebec, Canada) 48 h after the last OVA challenge, as previously described.27 Briefly, the mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (100 mg kg−1), and then a cannula was inserted via tracheotomy. The mice were connected to a computer-controlled small-animal ventilator and ventilated with a tidal volume of 10 ml kg−1 at a frequency of 150 breaths per min and a positive end-expiratory pressure of 2 cm H2O to achieve a mean lung volume close to that during spontaneous breathing. After the baseline measurement, each mouse was challenged with increasing concentrations (0, 1.56, 3.12, 6.25 and 12.50 mg ml−1) of MCh aerosol, and the peak airway responses to the inhaled MCh were recorded.

BAL and lung histology

BAL with 1 ml Hank’s balanced salt solution via the tracheal cannula was performed immediately after assessing the AHR. The number of leukocytes was counted with a hemocytometer, and cell differentiation was performed on CytoSpin slides prepared with Wright-Giemsa stain. After BAL, the lungs were fixed in 10% formalin, embedded in paraffin, and cut into 5-μm sections. The numbers of inflammatory and mucus-containing cells were quantified as described previously.28 Tissue sections were evaluated using ImageJ (National Institutes of Health, Bethesda, MD, USA). To detect inflammatory cells, sections were stained with hematoxylin and eosin, and the number of inflammatory cells per μm2 of perivascular and peribronchial area was determined. In addition, mucus-containing cells were stained with periodic acid–Schiff (PAS) and counterstained with hematoxylin. The number of mucus-containing cells per μm2 of basement membrane was determined.

ELISA

The BAL fluid was centrifuged, and the supernatant was frozen until further analysis. The levels of IL-4, IL-5, IL-13 and interferon-γ in the BAL fluid were measured using a sandwich ELISA according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA). The lower limits of detection were 3 pg ml−1 for IL-4 and IL-13, 7 pg ml−1 for IL-5 and 13 pg ml−1 for interferon-γ.

Isolation of lung mononuclear cells and flow cytometry

Lung mononuclear cells were isolated and purified after collagenase digestion as described previously.29 After purification, the lung mononuclear cells were incubated with antibodies and then analyzed by flow cytometry following the manufacturer’s instructions. The CD3-FITC, CD8-APC and CD4-PE antibodies were purchased from eBioscience.

In vitro cell culture

The human bronchoepithelial cell line BEAS-2B (American Type Culture Collection, Manassas, VA, USA) and human umbilical vein endothelial cells (HUVECs; American Type Culture Collection) were placed in six-well plates (2 × 105 cells per well). The BEAS-2B cells were cultured in RPMI-1640, and the HUVECs were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum under a 5% CO2 humidified atmosphere at 37 °C. After reaching confluence, the culture medium was replaced by serum-free medium with different doses of simvastatin (0.1, 1 and 10 μM) for 24 h. Then, the cells were stimulated with 5 μg ml−1 lipopolysaccharide for 3 h with or without pre-treatment with different concentrations of simvastatin. Then, the adhesion molecule expression was determined by western blotting.

Western blot analysis

Lung tissues were homogenized, and lung lysates were prepared in lysis buffer. Cell extracts were prepared from BEAS-2B cells and HUVECs. Then, the protein samples (25 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked by 5% fat-free milk and then incubated with antibodies targeting the mouse proteins VCAM-1 (sc8304, 1:1000), ICAM-1 (sc1511, 1:1000) or β-actin (sc1616, 1:1000; Santa Cruz Biotechnology, CA, USA).

Data analysis

The results are presented as the means±s.e.m. Data were compared using an unpaired Student’s t-test or one-way analysis of variance using SPSS 18.0 (SPSS, Chicago, IL, USA). Differences were considered statistically significant if the P-value was lower than 0.05.

Results

Simvastatin treatment prevents the development of AHR and airway inflammation

To determine the effects of simvastatin treatment on allergen-induced AHR and airway inflammation, mice were treated with simvastatin during the OVA challenge phase. As shown in Figures 1a and b, vehicle-treated mice showed greater airway responses to MCh, as well as eosinophilia, in BAL fluid following sensitization and challenge with OVA compared with sham-sensitized, OVA-challenged mice. Mice treated with simvastatin developed significantly less airway responsiveness to inhaled MCh and less eosinophilia in BAL fluid than did the vehicle-treated mice (P<0.01).

Figure 1
figure 1

Effect of simvastatin administration on airway responses in an ovalbumin (OVA)-specific asthma model. (a) Changes in lung resistance in response to increasing doses of methacholine (0, 1.56, 3.12, 6.25 and 12.5 mg kg−1) were assessed 48 h after the final challenge and are expressed as a per cent of the baseline (saline) value. (b) Cell composition in bronchoalveolar lavage fluid. Macro, macrophages; Lympho, lymphocytes; Eos, eosinophils; Neu, neutrophils. Control mice received saline as the vehicle. The data are expressed as the mean±s.e.m.; n=9–10 per group. ##P<0.01 vs the phosphate-buffered saline (PBS)/OVA vehicle group; #P<0.05 vs the PBS/OVA group; **P<0.01 vs the OVA/OVA vehicle group; *P<0.05 vs the OVA/OVA group.

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As shown in Figure 2, simvastatin treatment of sensitized and challenged mice also reduced the levels of IL-4, IL-5 and IL-13 in BAL fluid, but we did not find a significant difference in the interferon-γ level compared with that in the vehicle-treated mice.

Figure 2
figure 2

Effect of simvastatin administration on cytokine levels. Interleukin (IL)-4, IL-5, IL-13 and interferon (IFN)-γ levels in bronchoalveolar lavage fluid were determined by ELISA. The data are expressed as the mean±s.e.m.; n=9–10. ##P<0.01 vs the phosphate-buffered saline (PBS)/ovalbumin (OVA) vehicle group; **P<0.01 vs the OVA/OVA vehicle group; *P<0.05 vs the OVA/OVA vehicle group.

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Histopathological analysis of lung tissue sections revealed that the numbers of inflammatory cells, including eosinophils, in the peribronchial and perivascular areas increased in mice after OVA sensitization and challenge compared with sham-sensitized, OVA-challenged mice (Figure 3A). Similarly, the numbers of PAS+ mucus-containing goblet cells increased in the sensitized and challenged mice (Figure 3B). The administration of simvastatin significantly decreased the numbers of inflammatory cells and PAS+ mucus-containing goblet cells in lung tissue (Figures 3A and B).

Figure 3
figure 3

Effect of simvastatin administration on histology. (A) Lung tissue histology with hematoxylin and eosin and (B) periodic acid–Schiff (PAS) staining. Inflammatory and PAS+ cells in lung tissue were quantified as described in the Materials and methods. (a) Mice sensitized and given nebulized phosphate-buffered saline (PBS; negative control); (b) mice sensitized and given nebulized ovalbumin (OVA; positive control); (c) OVA-challenged mice treated with simvastatin. ##P<0.01 vs the PBS/OVA vehicle group; **P<0.01 vs the OVA/OVA vehicle group; (d) Numbers of perivascular and peribronchial inflammatory cells/1000 μm2 (Figure 3Ad); mucus-containing cells stained with PAS were quantitated and expressed as PAS-positive areas/μm of basement membrane (Figure 3Bd).

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Decrease in CD4+ T-cell number and CD4+/CD8+ T-cell ratio in the lungs of sensitized and challenged mice following simvastatin treatment

As CD4+ and CD8+ T cells are potent effector cells in the development of allergic inflammation, we examined their numbers after simvastatin treatment in sensitized and challenged mice. Lungs from OVA-sensitized and OVA-challenged mice receiving either simvastatin or vehicle were excised, and the lung mononuclear cells were purified. The numbers of CD4+ and CD8+ T cells were determined by flow cytometry. Simvastatin treatment significantly reduced the mean absolute percentage of CD4+ cells (46.44±8.19 vs 40.19±7.11, P<0.05) and the CD4+/CD8+ T-cell ratio (5.48±0.87 vs 4.43±1.31, P<0.05), whereas CD8+ T-cell percentage (8.51±1.28 vs 9.49±1.97, P>0.05) did not significantly differ between simvastatin- and vehicle-treated mice (Table 1).

Table 1 Percentages of CD4+ and CD8+ T cells in lung homogenates after simvastatin administration
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Decreased adhesion molecule levels following simvastatin treatment in vivo and in vitro

Next, we quantified the levels of adhesion molecules in the lungs of sensitized and challenged mice following simvastatin treatment. The expression of the VCAM-1 and ICAM-1 proteins increased in the lungs of sensitized and challenged mice, whereas the levels were reduced significantly in simvastatin-treated mice (Figures 4a and b).

Figure 4
figure 4

Effects of simvastatin administration on the expression of adhesion molecules in mouse lung homogenates. Vascular cell adhesion molecule 1 (VCAM-1; a) and intercellular adhesion molecule 1 (ICAM-1; b). The data are expressed as the means±s.e.m. ##P<0.01 vs the phosphate-buffered saline (PBS)/ovalbumin (OVA) vehicle group; **P<0.01 vs the OVA/OVA vehicle group; *P<0.05 vs the OVA/OVA vehicle group.

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Experiments with human BEAS-2B cells and HUVECs revealed similar decreases in the expression of VCAM-1 and ICAM-1 after simvastatin treatment (Figures 5A and B).

Figure 5
figure 5

Effects of simvastatin administration on the expression of adhesion molecules in human cell lines. BEAS-2B cells (A) and HUVECs (B). (a) Expression of vascular cell adhesion molecule 1 (VCAM-1); (b) expression of intercellular adhesion molecule 1 (ICAM-1). Cells were pre-treated with different doses of simvastatin (0.1, 1 and 10 μM) for 24 h and exposed to 5 μg ml−1 lipopolysaccharide (LPS) for 6 h with or without simvastatin pre-treatment. ##P<0.01 vs the phosphate-buffered saline/ovalbumin (OVA) vehicle group; **P<0.01 vs the OVA/OVA vehicle group; *P<0.05 vs the OVA/OVA vehicle group.

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Correlations between CD4+ T-cell numbers and adhesion molecule levels

To investigate the association between CD4+ T-cell numbers and adhesion molecule levels in lung homogenates, a Spearman correlation analysis was conducted. The percentage of CD4+ T cells showed a significant positive correlation with the expression of VCAM-1 (P=0.013, R=0.645) and ICAM-1 (P=0.036, R=0.563) in simvastatin-treated mice (Figures 6a and b). It should be noted that the percentage values used were the percentage of total cells.

Figure 6
figure 6

Correlations between CD4+ T-cell numbers and adhesion molecule levels. Correlation between CD4+ T cells and vascular cell adhesion molecule 1 (VCAM-1; a); correlation between CD4+ T cells and intercellular adhesion molecule 1 (ICAM-1; b).

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Discussion

We set out to accomplish two goals in this paper. The first was based on previously published information and sought to clarify in detail the effects of simvastatin on allergen-induced AHR airway inflammation in asthma. The animal studies and clinical trials of statins are summarized in Tables 2 and 3, respectively. The second goal, which was more important, was to focus on the effect of simvastatin on cellular influx in an OVA-specific mouse model of allergic asthma.

Table 2 Studies about the effects of simvastatin in animal models of asthma and airway inflammation
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Table 3 Clinical trials that investigated the effects of simvastatin in asthma patients
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Over the past few decades, there has been a huge increase in the prevalence of asthma in all age groups, especially in developed countries, despite the successful introduction of various asthma medications.30 Currently, mainstream asthma treatment is focused on anti-inflammatory drugs, as these drugs are the most effective therapeutic agents for many asthma patients. However, the many patients who are still unable to achieve optimal asthma control account for a significant portion of the vast worldwide health-care costs for asthma,31 which suggests that the identification of potential targets for therapeutic intervention is an important goal in asthma research.

Because tremendous costs and huge efforts are required to develop a new drug, many existing drugs that are already being used to target one disease are being studied to explore their potential as therapeutics for other diseases. This concept, known as drug repositioning or drug repurposing, has become an increasingly important part of the drug development process. Statins are one such group of drugs. As competitive inhibitors of 3-hydroxy-3-methylglutaryl co-enzyme A reductase, statins have a lipid-lowering capacity and reduce both cardiovascular-related morbidity and mortality in patients with or without coronary disease.32 In addition, statins regulate inflammatory processes by interrupting the mevalonate pathway.9 For example, statin treatment inhibits the progression of aneurysms associated with degenerative atherosclerosis33 and diminishes inflammation and related anemia in end-stage renal disease.34 McKay et al.18 were among the earliest investigators to suggest the possibility of simvastatin for the treatment of allergic airway disease.

Several studies found that the administration of a statin attenuated AHR, inflammatory cell number and Th2 cytokine levels in mouse models of allergic asthma,2, 3, 9, 10 although other studies showed contradictory results.11, 12 Tschernig et al.12 found that the relative and absolute numbers of neutrophils, eosinophils and lymphocytes were only partially reduced after statin treatment. However, they found significantly reduced CD4+ T-cell numbers in most statin-treated mice, although they did not report significant changes in the asthma-related parameters. These controversial results were repeated in clinical studies.5, 7, 13, 35, 36 Given the complexity of human studies, various factors might contribute to the controversial results, such as the different characteristics and treatments of the patients. Nevertheless, a recent, large population-based study found that statin exposure was associated with decreased oral corticosteroid dispensing and asthma-related emergency department visits or hospitalizations.35 It has been reported by Zeki et al.9 that simvastatin may exert its effects through modulation of the mevalonate pathway, whereas Xu et al.10 reported that simvastatin attenuated airway inflammation, remodeling and AHR by inhibiting the RhoA pathway in a dose-dependent manner. Furthermore, the latter proposed the possible administration of simvastatin by inhalation rather than oral administration. This finding is supported by our findings that simvastatin attenuates airway responsiveness and allergic inflammation by reducing the T-cell influx as a result of decreased expression of cell adhesion molecules.

Activated T cells, especially CD4+ T cells, have a pivotal role in the pathophysiology of allergic asthma via the production of cytokines that cause inflammation and promote IgE production.37, 38 The recruitment of CD4+ T cells has been demonstrated in the lungs and BAL fluid of asthma patients.39, 40, 41 Another study demonstrated that CD4+ T-cell activation increased the mRNA expression of Th2-type cytokines and the recruitment of eosinophils in BAL after an allergen inhalation challenge in patients with allergic asthma.42 In addition, the CD4+/CD8+ ratio increased significantly in symptomatic asthma patients compared with healthy controls, and this ratio decreased significantly after treatment.40

One of the crucial points of our study is that we found a significant reduction in CD4+ T-cell numbers and in the CD4+/CD8+ ratio, with attenuated AHR, airway inflammation and Th2 cytokine levels, in simvastatin-treated mice compared with vehicle-treated mice. The attachment of T lymphocytes to endothelial cells is a critical step in lymphocyte migration into the surrounding tissue, and the expression of adhesion molecules is the key process of cellular migration. The expression of adhesion molecules such as ICAM-1 and VCAM-1 is regulated by inflammatory cytokines. The pro-inflammatory cytokine IL-1 can upregulate ICAM-1 expression, and Th2 cytokines such as IL-4 and IL-13 enhance VCAM-1 expression, whereas regulatory cytokines cannot modulate either ICAM-1 or VCAM-1 expression.43 For example, the expression of ICAM-1 in asthma patients was significantly higher than in healthy controls, and more markedly significant differences were observed in severe asthma attacks.44 Thus, methods to reduce ICAM-1 and VCAM-1 expression would be of interest in the development of novel therapeutic methods for asthma. Weitz-Schmidt et al.45 has provided evidence showing the anti-inflammatory effect of simvastatin by inhibiting the interaction between LFA-1 and ICAM-1 in a T-cell lymphoma cell line.

We also found that the expression of ICAM-1 and VCAM-1 was decreased after simvastatin treatment compared with the vehicle-treated group in both homogenized lung and human cell lines in vitro. Our data clearly show that simvastatin treatment directly reduced the expression of cell adhesion molecules, inhibited the migration of inflammatory cells (especially CD4+ T cells) from the blood into the airways and sites of inflammation and reduced both the augmentation of cytokines and eosinophil recruitment. In addition, our results showed an expected positive correlation between CD4+ T-cell numbers and adhesion molecule levels. Therefore, our results support the crucial finding that the downregulation of T-cell-mediated immune responses by simvastatin in asthma occurs via the modulation of cell adhesion molecules, and this new finding opens new horizons for potential further clinical applications. Previous studies lend support to our findings. Tschernig et al.12 showed that simvastatin treatment significantly reduced CD4+ T-cell numbers in a rat asthma model. In rheumatoid arthritis patients, the addition of simvastatin to conventional immunosuppressive therapies significantly improved the clinical, biological and immunological parameters by reducing the Th1/Th2 and CD4/CD8 ratios.46 Another study suggested that one of the mechanisms by which simvastatin reduces inflammation might be by reducing the expression of adhesion molecules in circulating monocytes, as shown in hypercholesterolemic patients.47

Although many experimental and clinical studies have indicated the effectiveness of statins in the treatment of allergic asthma, there are still many controversies, suggesting that there are inter-individual differences in responses, for which genetic differences may be a contributory factor. Therefore, future studies could be designed to identify the genetic changes in specific targets.

To the best of our knowledge, this is the first study to evaluate the effects of simvastatin on CD4+ and CD8+ T cells together with the association of simvastatin with adhesion molecules in asthma. However, one limitation of this study was, as in many other experimental studies, that the dose of simvastatin that was administered (40 mg kg−1) was high compared with the routinely prescribed clinical dose. Further studies are needed to elucidate the detailed interactions between adhesion molecules and T cells and the possible adverse reactions if simvastatin is administered at high doses in humans. In addition, thorough investigations of other statins and their interactions are also necessary.

Overall, our results provide compelling evidence showing that a reduction in T-cell influx as a result of decreased expression of cell adhesion molecules is one of the mechanisms by which simvastatin attenuates airway responsiveness and allergic inflammation; therefore, taking into account the effects on CD4+ and CD8+ T cells, statins should be considered novel therapeutic targets in the future treatment of asthma in humans. Because there is a paucity of understanding concerning the basis for variability in human responses, it is necessary to meticulously determine the effect of statins in clinical trials before they can be adopted clinically.