Elsevier

Immunobiology

Volume 223, Issues 6–7, June–July 2018, Pages 486-492
Immunobiology

Elimination of eosinophils using anti-IL-5 receptor alpha antibodies effectively suppresses IL-33-mediated pulmonary arterial hypertrophy

https://doi.org/10.1016/j.imbio.2017.12.002Get rights and content

Abstract

Interleukin (IL)-5 is a critical regulator of eosinophils and a therapeutic target for asthma. The administration of anti-IL-5 or anti-IL-5 receptor (IL-5R) antibodies has been shown to reduce eosinophil counts and ameliorate asthmatic symptoms in studies on animal models of allergy as well as in human clinical trials. In order to explore other potential clinical uses of IL-5R antibodies, we used an animal model of IL-33-mediated pulmonary arterial hypertrophy. We first generated chimeric monoclonal antibodies against the mouse IL-5 receptor α chain (IL-5Rα), which comprised an Fc region from human IgG1 and a Fab region from a previously established anti-mouse IL-5Rα monoclonal antibody. To investigate the role of antibody-dependent cell-mediated cytotoxicity (ADCC), chimeric antibodies that lacked ADCC were prepared. These antibodies recognized IL-5Rα to the same extent as the ADCC-sufficient antibodies. Administration of chimeric antibodies with ADCC resulted in the elimination of eosinophils from the lung and thus suppressed the development of arterial hypertrophy. This effect was attenuated in mice treated with antibodies lacking ADCC. Taken together, the results of this study provided a potential use for anti-IL-5Rα antibodies in the treatment of arterial hypertrophy, which leads to pulmonary hypertension.

Introduction

Interleukin (IL)-5 is a critical cytokine that regulates eosinophil biology (Takatsu, 2011). IL-5 is produced primarily by group 2 innate lymphoid cells (ILC2s) and CD4+ T helper type 2 (Th2) cells (Licona-Limon et al., 2013). In animal models of allergic diseases or helminth infection, IL-5 plays critical roles in proliferating eosinophil progenitors in the bone marrow (Iwasaki et al., 2005; Yamaguchi et al., 1988), promoting eosinophil recruitment (Mattes and Foster, 2003; Pope et al., 2001; Yang et al., 2003), and enhancing eosinophil survival in local inflammatory sites (Simon et al., 1997; Simon, 2006). IL-5 signaling is transmitted through the IL-5 receptor (IL-5R), which comprises IL-5Rα that determines the specificity to IL-5 and the common beta chains (Kusano et al., 2012; Patino et al., 2011; Tavernier et al., 1991). IL-5 and IL-5R have been shown to be therapeutic targets for the treatment of asthma, and clinical trials with anti-IL-5 antibodies and anti-IL-5Rα antibodies have shown promising results (Cabon et al., 2016; FitzGerald et al., 2016; Koike et al., 2009). Because anti-IL-5Rα antibodies deplete eosinophils through antibody-dependent cell-mediated cytotoxicity (ADCC) (Kolbeck et al., 2010), they have been clinically used for treating severe asthma in humans (FitzGerald et al., 2016).

One of the pivotal mediators of allergic reactions including eosinophilia is IL-33. It belongs to the IL-1 cytokine family and initiates Th2 response by enhancing the production of Th2 cytokines, including IL-5 and IL-13, from ILC2 (Cayrol and Girard, 2014; Ohno et al., 2012). At the mucosal surface, IL-33 is maintained in a steady state in the nuclei of epithelial cells and released when pathogens or foreign substances cause tissue damages. This alerts the host to danger, and IL-33 is therefore termed alarmin. Recently, IL-33 was reported to be involved in wide range of immune responses besides allergies, including chronic inflammation and connective tissue diseases (Liew et al., 2010; Oboki et al., 2010; Pei et al., 2014).

Pulmonary arterial hypertension (PAH) is caused by severe occlusive arterial hypertrophy of small arteries. This causes high pulmonary arterial pressure, ultimately resulting in progressive right ventricular failure. Recent studies have revealed that chronic inflammation is one of the key factors in the development of PAH (Price et al., 2012). Interestingly, IL-33 signaling was also suggested to be involved in the pathogenesis of PAH (Carlomagno et al., 2013). We attempted administering IL-33 chronically, and established an animal model with occlusive arterial hypertrophy resembling that observed in patients with PAH (Ikutani et al., 2017). In this model, IL-5 from ILC2 as well as eosinophils played crucial roles in the development of pulmonary arterial hypertrophy.

In the present study, we generated anti-mouse IL-5Rα monoclonal antibodies through genetic engineering and investigated their therapeutic effects using an animal model of IL-33-induced arterial hypertrophy. The anti-IL-5Rα antibody effectively prevented the development of arterial hypertrophy. This effect was largely dependent on ADCC and the anti-IL-5Rα antibody was more effective than an anti-IL-5 antibody. This study reported a potential therapy for arterial hypertrophy using anti-IL-5Rα antibodies.

Section snippets

Cloning of sequences in the antibody variable region of hybridoma H7

Total RNA was extracted from hybridoma cells that produced H7 using RNeasy Mini Kit (QIAGEN, Hilden, Germany). The first strand cDNA was then synthesized using SMARTer RACE cDNA Amplification Kit (Takara Bio, Tokyo, Japan). The nucleotide sequences of antibody VL and VH were amplified using PrimeSTAR Max DNA polymerase (Takara Bio, Tokyo, Japan) using the 5′-universal primer (attached to the kit) and rat immunoglobulin light and heavy chain gene-specific primers. The nucleotide sequences of the

Generation of H7 chimeric antibodies with two types of fc regions

A rat antibody against mouse IL-5Rα (H7) (Hitoshi et al., 1990; Yamaguchi et al., 1990) was used to develop chimeric antibodies. The obtained sequences of H7 VL and VH are shown in Fig. 1. The original VL had one free Cys (Cys87 on framework L3), which may have caused the poor physical stability of the recombinant antibodies. An additional VL was therefore prepared with the Cys87 substituted with Ser (denoted as H7(CS) hereafter) to avoid disulfide mispairing and deterioration of biological

Discussion

In this study, we evaluated a series of novel antibodies, which recognize IL-5Rα, as potential candidates for therapy against pulmonary arterial hypertrophy. The results revealed that anti-IL-5Rα antibodies were effective in suppressing lung eosinophilia and concomitant pulmonary arterial hypertrophy. Because both ADCC-sufficient and -lacking antibodies recognized IL-5Rα and neutralized IL-5 signaling to a similar extent in vitro, the effect to eliminate eosinophils in vivo was dependent on

Disclosures

This study was supported by Kyowa Hakko Kirin.

Funding

This work was supported by JSPS KAKENHI (Grant Number 24390119 to K. T and Grant Number 26860319 to M.I.) and Kyowa Hakko Kirin.

Acknowledgments

We are grateful to Toyama Prefecture (Japan) for supporting our laboratory.

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