Evaluation of the anti-inflammatory effect of ... - Wiley Online Library

Respirology (2008) 13, 488–497

doi: 10.1111/j.1440-1843.2008.01278.x

ORIGINAL ARTICLE

Evaluation of the anti-inflammatory effect of infliximab in a mouse model of acute asthma Figen DEVECI,1 M. Hamdi MUZ,1 Nevin ILHAN,2 Gamze KI˙RKI˙L,1 Teyfik TURGUT1 AND Nusret AKPOLAT3 Departments of 1Chest Diseases, 2Biochemistry and Clinical Biochemistry, and 3Pathology, Faculty of Medicine, Firat University, Elazig, Turkey

Evaluation of the anti-inflammatory effect of infliximab in a mouse model of acute asthma DEVECI F, MUZ MH, ILHAN N, KI˙RKI˙L G, TURGUT T, AKPOLAT N. Respirology 2008 13: 488–497 Background and objective: To evaluate the potential role of anti-tumour necrosis factor (TNF)-a mAb (infliximab) on the inflammatory response in a mouse model of acute asthma. Methods: BALB/c mice received intraperitoneal (i.p.) ovalbumin (OVA) on days 0 and 14, 100 mg of OVA intranasally on day 14 and 50 mg of OVA intranasally on days 25, 26 and 27. The low-dose (2.5 mg/kg) and high-dose (6.25 mg/kg) infliximab groups received i.p. infliximab before each i.p. sensitization and on challenge days 1, 6, 13, 20 and 27. The control group received i.p. injections of normal saline with alum on days 0 and 14 and normal saline without alum on days 14, 25, 26 and 27. Results: There were statistically significant decreases in the numbers of BAL fluid (BALF) neutrophils, eosinophils, as well as lung eosinophils in both the low- and high-dose infliximab groups when compared with the control OVA sensitized/challenged group. The lower dose of infliximab did not alter lung neutrophil counts, but a marked decrease was seen with the high dose of infliximab. After treatment with low and high doses of infliximab, BALF levels of regulated on activation normal T cell expressed and secreted (RANTES), granulocyte macrophage-colony stimulating factor (GM-CSF), TNF-a, IL-6, macrophage inflammatory protein (MIP)-2, and levels of RANTES, IL-4, GM-CSF, TNF-a, IL-6 and MIP-2 in lung tissue were significantly decreased when compared with the control OVA sensitized/challenged group. There was a significant decrease in BALF IL-4 only in the high-dose infliximab group. Conclusions: These results show that an anti-TNF-a mAb has a considerable anti-inflammatory effect on allergen-induced lung inflammation in an animal model of acute asthma. Key words: anti-tumour necrosis factor alpha, asthma, chemokine, cytokine.

INTRODUCTION Asthma is one of the most common disorders encountered in clinical medicine. Current therapies for asthma focus on optimal control of symptoms but there is a significant population of severe asthmatics that does not respond well to standard therapies.1–3 Therefore, there is a need for innovative therapies Correspondence: Figen Deveci, Department of Chest Diseases, Faculty of Medicine, Firat University, 23119, Elazig, Turkey. E-mail: [email protected] Received: 24 July 2007; invited to revise: 21 August 2007; 5 October 2007; 10 October 2007; revised: 12 September 2007; 10 October 2007; 16 October 2007; accepted: 16 October 2007 (Associate Editor: Takahide Nagase).

aimed at prevention of structural changes in the airways. Based on the current understanding of the pathophysiology of asthma, the development of mAb therapeutics could be useful and anti-tumour necrosis factor (TNF)-a antibodies have previously been tested in the treatment of inflammatory lung diseases.4 Systemic and lung levels of pro-inflammatory cytokines such as TNF-a are reported to be increased in patients with asthma.5,6 It is well established that TNF-a enhances the inflammatory response through induction of other pro-inflammatory cytokines (IL-6, IL-1) and recruitment of effector cells.5 Novel approaches for the treatment COPD and asthma that involve inhibition of TNF-a and other inflammatory mediators are currently under investigation. Recently, Berry et al.7 study showed that etanercept may have beneficial effects in patients with refractory

© 2008 The Authors Journal compilation © 2008 Asian Pacific Society of Respirology

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asthma. Infliximab is another monoclonal antiTNF-a antibody with demonstrated efficacy in autoimmune diseases, including Crohn’s disease and rheumatoid arthritis.8–10 Therefore, there is a strong rationale for the potential use of the anti-TNF-a mAb, Remicade, in asthma therapy. Mouse models are commonly used for studying the pathophysiology and clinical manifestations of disease, as well as for evaluating new agents for the treatment of asthma.11,12 In this study, we used a mouse model of acute asthma to examine the effects of the anti-TNF-a mAb, Remicade, on the asthmatic inflammatory response.

METHODS Experimental design

from the heart, and BAL was performed. All blood samples were centrifuged (1500 g, 4°C, 5 min) and serum was stored at -80°C until analysed.

BAL BAL was performed using normal saline instilled through the tracheostomy tube. A total volume of 4.5 mL was instilled in nine successive aliquots of 0.5 mL. Cytospin slides of BAL cells were prepared using a cytocentrifuge (Cytospin model II; Shandon, Pittsburgh, PA, USA) and stained with MayGrünwald-Giemsa stain. A differential count of at least 200 cells was performed. The remaining BAL fluid (BALF) was centrifuged (300 g, 4°C, 10 min) and the supernatants were collected and stored at -70°C until measurement of TNF-a, IL-4, IL-6, macrophage inflammatory protein (MIP)-2, regulated on activation normal T cell expressed and secreted (RANTES) and granulocyte macrophage-colony stimulating factor (GM-CSF) by ELISA.

BALB/c mice (6–8 weeks old, male) were obtained from the Experimental Research Unit of Firat (Euphrates) University, Faculty of Medicine. The protocols used for sensitization and challenge of mice to mimic acute allergic asthma, have been described previously.12 The experimental protocol was approved by the institutional ethics committee of Firat (Euphrates) University. BALB/c mice were seperated into four groups as follows: the control group (n = 10) received intraperitoneal (i.p.) injections of normal saline with alum on days 0 and 14 and intranasal (i.n.) normal saline without alum on days 14, 25, 26 and 27; the ovalbumin (OVA) group (n = 10) received i.p. injections of 0.2 mL (100 mg) OVA complexed with alum (0.4 mL of a 4 mg/mL suspension of Al(OH)3) on days 0 and 14. On days 14, 25, 26 and 27, mice were anaesthetized with an i.p. injection of 0.2 mL ketamine (0.44 mg/mL)/xylazine (6.3 mg/mL) in normal saline before receiving a dose of 100 mg OVA in 0.05 mL normal saline on day 14 and doses of 50 mg OVA in 0.05 mL normal saline on days 25, 26 and 27.

Mouse TNF-a, IL-4, IL-6, GM-CSF, RANTES and rat MIP-2 were measured in BAL supernatants using commercially available ELISA kits (Biosource International, Camarillo, CA, USA) according to the manufacturer’s protocols. Antibodies specific for mouse TNF-a, IL-4, IL-6, GM-CSF, RANTES and rat MIP-2 were coated onto microtitre strips wells and the samples, including standards of mouse TNF-a, IL-4, IL-6, GM-CSF, RANTES and rat MIP-2, were pipetted into the wells and incubated. After washing and development, the intensity of colour was measured at 450 nm.

Remicade treatment

Lung histology

The low-dose treatment group (n = 10) received a once weekly 0.1 mL i.p. injection of 1 mg/mL (2.5 mg/ kg) Remicade (infliximab, Centocor BV, Leiden, the Netherlands) before each i.p. sensitization and challenge on days 1, 6, 13, 20 and 27. The high-dose treatment group (n = 10) received a once weekly 0.25 mL i.p. injection of 1 mg/mL (6.25 mg/kg) Remicade before each i.p. sensitization and challenge on days 1, 6, 13, 20 and 27. The same volume of vehicle was administered to control mice. Two different doses of infliximab were used in order to determine the dose response. The doses were chosen on the basis of previous trials of infliximab in rheumatoid arthritis and Crohn’s disease,13,14 and the protocol used in those studies was adopted. Infliximab has a serum half-life ranging between 8 and 10 days.15 Twenty-four hours after the last OVA challenge, the mice were killed by i.p. injection of 0.2 mL sodium pentobarbital (60 mg/kg), blood samples were drawn

After BAL, lungs were removed from the mice, fixed with neutralized buffered formalin and embedded in paraffin. Lung tissue was cut in 5-mm sections and stained with HE and Giemsa. The sections were examined on an Olympus BX-50 microscope, eosinophils and neutrophils from three different peribronchial areas were counted and mean cell counts were calculated.

Measurement of TNF-a, IL-4, IL-6, MIP-2, RANTES and GM-CSF in BAL fluid

Quantitation of TNF-a, IL-4, IL-6, MIP-2, RANTES and GM-CSF in lung tissues The lungs were removed, washed with isotonic saline and stored at -80°C until homogenization. Lung tissue samples were homogenized with 1/10 cold TrisHCl buffer (0.1 mM, pH 7.5) and homogenates were centrifuged at 3000 g, 4°C for 5 min. Aliquots of the homogenates were assayed for TNF-a, IL-4, IL-6,


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Figure 1 The number of (a) eosinophils and (b) neutrophils in BALF. aP < 0.001 versus control group, bP < 0.001 versus ovalbumin (OVA) sensitized/challenged group, cP < 0.01 versus low-dose Remicade treatment group, dP < 0.001 versus lowdose Remicade treatment group. Group 1, control group; group 2, OVA sensitized/challenged group; group 3, low-dose Remicade treatment group; group 4, high-dose Remicade treatment group.

GM-CSF, RANTES and MIP-2 using commercially available ELISA kits (Biosource International).

Quantitation of TNF-a, IL-4, IL-6, MIP-2, RANTES and GM-CSF in serum Serum cytokine levels were measured as described above, according to the manufacturer’s recommendations.

Statistical analyses Results were expressed as mean ⫾ SD. The Kruskall Wallis and Mann–Whitney U-tests were used to compare the variables. All statistical analyses were performed using SPSS version 11.0 software. P-values ⱕ0.05 were considered statistically significant.

RESULTS Lung tissue and BALF were evaluated histologically on day 28 by light microscopy to assess allergeninduced airway inflammation. There was prominent eosinophil infiltration of the bronchial interstitium and the number of BALF eosinophils was markedly increased. These inflammatory changes were absent in the control group.

Effects of infliximab on inflammatory cell counts and inflammatory cell infiltration induced by OVA challenge The numbers of BALF eosinophils and lung eosinophils and neutrophils were increased in the OVA

group compared with the control group. In the lowdose Remicade treatment group there was a statistically significant decrease in the numbers of BALF neutrophils, eosinophils and lung eosinophils when compared with the OVA group. Likewise in the highdose Remicade treatment group, there was a statistically significant decrease in the numbers of BALF neutrophils, eosinophils and lung neutrophils and eosinophils, when compared with the low-dose Remicade treatment group. It is noteworthy that the low dose of infliximab did not alter lung neutrophil counts, but a marked decrease was seen with the high dose. In the high-dose Remicade treatment group, there were marked decreases in the BALF neutrophil and eosinophil counts and lung eosinophil and neutrophil counts compared with the low-dose Remicade treatment group (Figs 1–3).

Effects of infliximab on cytokine and chemokine levels in BALF, lung tissue and serum BALF, serum and lung tissue were obtained from all groups 24 h after OVA challenge. OVA challenge resulted in significantly increased BALF and lung tissue levels of RANTES, IL-4, GM-CSF, TNF-a, IL-6 and MIP-2. The serum RANTES, IL-4, GM-CSF and MIP-2 levels were also significantly increased in the OVA group compared with the control group. After treatment with the low dose of Remicade, BALF RANTES, GM-CSF, TNF-a, IL-6, MIP-2, lung tissue RANTES, IL-4, GM-CSF, TNF-a, IL-6, MIP-2 and serum IL-4, GM-CSF, TNF-a and MIP-2 were significantly decreased when compared with the OVA group. There was no significant difference in the levels of BALF IL-4


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in the OVA group compared with the low-dose Remicade treatment group. Likewise, in the high-dose Remicade treatment group, BALF RANTES, IL-4, GM-CSF, TNF-a, IL-6, MIP-2, lung tissue RANTES, IL-4, GM-CSF, TNF-a, IL-6, MIP-2 and serum IL-4, GM-CSF, TNF-a, IL-6 and MIP-2 were significantly decreased when compared with the OVA group. In addition, BALF IL-4 levels also decreased significantly with high-dose Remicade compared with the OVA group (Figs 4–7). In the high-dose Remicade treatment group, the levels of BALF RANTES (P < 0.01), IL-4 (P < 0.05), GM-CSF (P < 0.05) and the levels of lung tissue RANTES (P < 0.05), TNF-a (P < 0.05), as well as serum GM-CSF (P < 0.001) and TNF-a (P < 0.05) were markedly decreased when compared with the lowdose Remicade treatment group.

DISCUSSION This study demonstrated that infliximab reduced inflammatory cell infiltration and the levels of several cytokines and chemokines in BALF and lung tissue in a mouse model of acute asthma.

‘First wave’ cytokines such as TNF-a and IL-6 may act on epithelial cells to release ‘second wave’ cytokines, including GM-CSF, IL-8 and RANTES, which then amplify the inflammatory response and lead to the influx of secondary cells such as eosinophils, which may themselves release multiple cytokines.16,17 Increased levels of GM-CSF in the epithelium of bronchial biopsy specimens and RANTES in plasma and BALF have been reported in asthma.18–22 GM-CSF can induce the synthesis and release of a number of cytokines, including TNF-a from monocytes,16 and RANTES expression and release by airway epithelium in vivo may in turn be upregulated by TNF-a.23 The CXC chemokines such as MIP-2 play important roles in allergy and asthma, both at the level of pulmonary cell recruitment and systemic immune responses.24 In addition, levels of MIP-1a and MIP-2 mRNA were increased in a new mouse model of allergic lung inflammation.25 Chemokines are strongly chemotactic for neutrophils, induce their activation and degranulation, and could be secreted by epithelial cells through their activation by TNF-a, leading to an amplification of the inflammatory response.26 IL-4 promotes the development of Th2-like CD4 T cells

Figure 2 The number of (a) eosinophils and (b) neutrophils in lung tissue. aP < 0.001 versus control group, bP < 0.001 versus ovalbumin (OVA) sensitized/challenged group, cP < 0.01 versus control group, dP < 0.001 versus low-dose Remicade treatment group. Group 1, control group; group 2, OVA sensitized/challenged group; group 3, low-dose Remicade treatment group; group 4, high-dose Remicade treatment group.

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Figure 3 Histological appearance of lung tissue in ovalbumin (OVA) sensitized/challenged mice, Remicade treated and control mice. (a) Normal appearance of bronchioles, arteries and veins in control group (HE, ¥400). (b,c) Prominent eosinophil infiltration (arrows) in the bronchial interstitium in the OVA sensitized/challenged group (b: HE, ¥400; c: HE, ¥1000). (d) Mild infiltration of inflammatory cells around the bronchioles in the low-dose Remicade treatment group (HE, ¥400). (e) Low-dose Remicade treatment decreased eosinophil infiltration (thick arrow) in the lung interstitium but there were small numbers of neutrophils (thin arrow) in the lung interstitium (HE, ¥400). (f,g) High-dose Remicade treatment decreased eosinophil and especially neutrophil infiltration in the lung interstitium around the airways (f: HE, ¥400; g: HE, ¥1000).


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Figure 4 The levels of (a) RANTES, IL-4 and (b) GM-CSF, TNF-a, IL-6 in BALF in all groups. aP < 0.001 versus control group, b P < 0.01 versus OVA sensitized/challenged group, cP < 0.001 versus OVA group, dP < 0.05 versus OVA group. Group 1, control group; group 2, OVA sensitized/challenged group; group 3, low-dose Remicade treatment group; group 4, high-dose Remicade treatment group. GM-CSF, granulocyte macrophage-colony stimulating factor; OVA, ovalbumin; RANTES, regulated on activation normal T cell expressed and secreted; TNF-a, anti-tumour necrosis factor (TNF)-a.

Figure 5 The levels of (a) RANTES, IL-4 and (b) GM-CSF, TNF-a, IL-6 in lung tissue in all groups. aP < 0.01 versus control group, bP < 0.05 versus control group, cP < 0.001 versus control group, dP < 0.01 versus OVA sensitized/challenged group, e P < 0.05 versus OVA sensitized/challenged group, fP < 0.001 versus OVA sensitized/challenged group. Group 1, control group; group 2, OVA sensitized/challenged group; group 3, low-dose Remicade treatment group; group 4, high-dose Remicade treatment group. GM-CSF, granulocyte macrophage-colony stimulating factor; OVA, ovalbumin; RANTES, regulated on activation normal T cell expressed and secreted; TNF-a, anti-tumour necrosis factor (TNF)-a.


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Figure 6 The levels of (a) RANTES, IL-4 and (b) GM-CSF, TNF-a, IL-6 in serum in all groups. aP < 0.01 versus control group, b P < 0.001 versus control group, cP < 0.001 versus OVA sensitized/challenged group, dP < 0.05 versus OVA sensitized/ challenged group. Group 1, control group; group 2, OVA sensitized/challenged group; group 3, low-dose Remicade treatment group; group 4, high-dose Remicade treatment group. GM-CSF, granulocyte macrophage-colony stimulating factor; OVA, ovalbumin; RANTES, regulated on activation normal T cell expressed and secreted; TNF-a, anti-tumour necrosis factor (TNF)-a.

Figure 7 The levels of MIP-2 in BALF, lung tissue and serum in all groups. aP < 0.001 versus control group, b P < 0.001 versus ovalbumin (OVA) sensitized/challenged group, cP < 0.01 versus OVA sensitized/challenged group. Group 1, control group; group 2, OVA sensitized/challenged group; group 3, low-dose Remicade treatment group; group 4, high-dose Remicade treatment group.

and inhibits the release of cytokines, including TNF-a, IL-1, IL-12, interferon-g, IL-8 and MIP-1a.16 In the present study, the levels of TNF-a, RANTES, IL-4, GM-CSF, MIP-2 and IL-6 in BALF and lung tissue were increased in the OVA group. IL-6 is a pleiotrophic cytokine with growth regulatory effects on many cells, as well as effects on T lymphocytes, and it may also have anti-inflammatory effects. Increased release of IL-6 was detected from alveolar macrophages of asthmatic patients but its role in asthma remains unclear.16,27 In response to inflammatory mediators such as TNF-a, airway smooth muscle cells synthesize and express cell adhesion molecules28 and these cells also synthesize and secrete a variety of cytokines, including IL-6 and RANTES.29,30 The role of TNF-a in asthma remains controversial with conflicting results in the literature. However, there is increasing evidence that TNF-a is responsible for inflammatory responses in asthma. Broide et al.31 showed decreased pulmonary inflammation in TNF-a receptor knockout mice using an OVA model. In previous studies using the OVA model, some groups have shown that blocking TNF-a does not alter cellular recruitment,32,33 while others have demonstrated a reduction in pulmonary recruitment of inflammatory cells.5,34–36 A recently published study indicates that anti-TNF-a antibody reduces the inflammation and pathophysiology of asthma in a murine model of asthma induced by house dust extract.37 While the presence of eosinophils is


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recognized as a hallmark of asthmatic inflammation, there is also evidence for TNF-a mediated neutrophil involvement in the pathogenesis of asthma.38 In addition, neutrophilia was seen in the BALF from rats.39 An altered inflammatory cell profile involving neutrophils was especially evident in severe asthma.40,41 In the present study, there were increased numbers of BALF and lung eosinophils as well as lung neutrophils, and significant reductions in inflammatory cell infiltration with infliximab therapy. BALF neutrophil counts were significantly decreased with both lowand high-dose infliximab, and lung neutrophil counts were significantly decreased with high-dose infliximab. Therefore, TNF-a blockade using anti-TNF-a antibody may decrease neutrophilic inflammation in the airways and BALF in this mouse model of asthma. Consistent with previous findings, this study showed elevation of TNF-a levels in BALF and airways.37,42 TNF-a is produced in response to allergic pulmonary inflammation by various cell types, including mast cells, macrophages, neutrophils, eosinophils and epithelial cells.43,44 Large amounts of TNF-a are generated in response to bacteria or parasitic proteins, but physical, chemical and immunological noxious stimuli can all rapidly induce the production and release of TNF-a. Moreover, TNF-a can also be generated as a consequence of the stimulation of a wide range of pro-inflammatory cytokines, including TNF-a itself. For example, mast cells are known to release and respond to TNF-a, indicating a positive autocrine loop leading to augmentation of mast cell activation.45 The present study showed decreased TNF-a levels in the BALF, lung tissue and serum after infliximab treatment. A possible explanation could be the inhibitory effect of infliximab on inflammatory cell recruitment in this asthma model. Kim et al.37 showed that blocking TNF-a significantly decreased the inflammatory response but there was a paradoxical augmentation of Th2 cytokines such as IL-4. In contrast, in the present study IL-4 levels were increased in BALF, lung tissue and serum but were reduced by infliximab. In addition, the data demonstrated that anti-TNF-a antibody treatment reduced the levels of GM-CSF, RANTES and MIP-2. The inhibition of these cytokines and chemokines by infliximab contributed to decreased inflammatory cell recruitment in this mouse model of acute asthma. The present study showed that TNF-a, GM-CSF, RANTES, MIP-2, IL-4 and IL-6 were decreased in both BALF and lung tissue after infliximab treatment. These inhibitory effects of infliximab may result from the decreased secretion of second wave cytokines owing to TNF-a and inflammatory cell recruitment. Ammit et al.46 examined the effect of increased intracellular cAMP on TNF-a-induced RANTES and IL-6 secretion from airway smooth muscle cells, and demonstrated that pretreatment with cAMP-elevating agents inhibited TNF-a-induced RANTES secretion but increased TNF-a-induced IL-6 secretion. Binding of infliximab to TNF-a has been shown to reduce TNF-a bioactivity based on the induction of IL-6.47 Another study investigating GM-CSF production in intestinal cell cultures from Crohn’s disease patients before and after infliximab treatment, showed that

increases in GM-CSF in Crohn’s disease were reduced after infliximab treatment, probably because intestinal T-cell GM-CSF production was reduced.48 These studies showed that the inhibition of TNF-a with infliximab resulted in decreased levels of RANTES, IL-6 and GM-CSF. To our knowledge, there is no study evaluating the effects of infliximab on inflammation and cytokine levels in a mouse model of acute asthma. Studies in animal models of allergic bronchopulmonary inflammation, including investigations in gene-targeted mice deficient in one or more Th2 cytokines, have suggested that the manifestations of asthma may be ameliorated by targeting these mediators.49–51 However, clinical trials using anti-IL-5 and soluble IL-4 receptor have been disappointing.52–54 In a recent clinical study it was suggested that the beneficial effects of etanercept may be confined to patients with refractory asthma.7 In previous studies, allergen induced pulmonary inflammation and airway hyperreactivity were markedly reduced by blocking TNF-a activity in animal models of asthma.37,55 In the present study, pulmonary inflammation as observed by histological evaluation, as well as BALF inflammatory cell and cytokine levels were reduced with infliximab. Additionally, results from other clinical studies showed that TNF-a inhibitors significantly improved asthma symptoms, lung function and airway hyperresponsiveness in patients with severe asthma.7,56,57 A recently published clinical study demonstrated that infliximab caused a decrease in the number of patients with exacerbations of symptomatic moderate asthma, and sputum TNF-a levels were lower with infliximab treatment but infliximab did not affect eosinophil numbers in blood or sputum. Nevertheless there was a trend toward fewer sputum neutrophils at week 8.58 It was concluded that blocking TNF-a activity represents a new approach in asthma therapy.59 The present findings in an acute mouse model of asthma emphasize the potential for inhibition of TNF-a with infliximab as a therapeutic strategy. Decreased neutrophilic inflammation following infliximab therapy may be the most significant result from this study. Neutralizing TNF-a may be beneficial in refractory asthma, but considering the experimental nature of this study, further studies are required to more conclusively demonstrate a benefit in human disease.

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