Eosinophil Function Research Articles

Distinct functions of eosinophils in severe asthma with type 2 phenotype: clinical implications.

Asthma is commonly recognized as a heterogeneous condition with a complex pathophysiology. With advances in the development of multiple medications for patients with asthma, most asthma symptoms are well managed. Nevertheless, 5% to 10% of adult asthmatic patients (called severe asthma) are in uncontrolled or partially controlled status despite intensive treatment. Especially, severe eosinophilic asthma is one of the severe asthma phenotypes characterized by eosinophilia in sputum/blood driven by type 2 immune responses. Eosinophils have been widely accepted as a central effector cell in the lungs. Some evidence has demonstrated that persistent eosinophilia in upper and lower airway mucosa contributes to asthma severity by producing various mediators including cytokines, chemokines and granule proteins. Moreover, extracellular traps released from eosinophils have been revealed to enhance type 2 inflammation in patients with severe asthma. These novel molecules have the ability to induce airway inf lammation and hyperresponsiveness through enhancing innate and type 2 immune responses. In this review, we highlight recent insight into the function of eosinophil extracellular traps in patients with severe asthma. In addition, the role of eosinophil extracellular vesicles in severe asthma is also proposed. Finally, current biologics are suggested as a potential strategy for effective management of severe eosinophilic asthma.

Epithelial cytokine IL-33 and sub-epithelial matrix protein tenascin C compartmentalize eosinophil phenotype and function

Abstract In allergic inflammation, eosinophils interacting with epithelial barriers experience exposure to epithelial cytokines and the extracellular milieu of airway remodeling. To better understand eosinophil activation biology in allergic tissue, we modeled the synergistic and opposing regulation of naïve eosinophils by both sub-epithelial extracellular matrix protein tenascin C (TNC), a key player in epithelial regeneration, and IL-33, an epithelial damage-induced cytokine. IL-5-differentiated bone marrow-derived murine eosinophils were cultured with recombinant TNC (25 ug/ml), IL-33 (100 ng/ml), or both for 24 hours and profiled by flow cytometry, qPCR, and DiffQuick staining. IL-33 alone significantly induced IL-4 and integrin CD11b expression, which was antagonized by co-culture with TNC. Conversely, TNC alone significantly promoted CD34 and intra-epithelial docking integrin CD103 expression, which was suppressed by IL-33 addition. Both treatments synergized in promoting expression of trans-epithelial migration marker CD11c. TNC promoted resident morphology while IL-33 induced an activated phenotype. This illustrates how microenvironmental cues may progressively shape eosinophils trafficking from sub-epithelial space across epithelial barriers.

Eosinophil functions in allergic diseases: an evolving paradigm

Eosinophil functions in allergic diseases: an evolving paradigm

Unraveling the connection between eosinophils and obesity

Obesity affects more than 650 million adults worldwide and is a major risk factor for a variety of serious comorbidities. The prevalence of obesity has tripled in the past forty years and continues to rise. Eosinophils have recently been implicated in providing a protective role against obesity. Decreasing eosinophils exacerbates weight gain and contributes to glucose intolerance in high fat diet-induced obese animals, while increasing eosinophils prevents high-fat diet-induced adipose tissue and body weight gain. Human studies, however, do not support a protective role for eosinophils in obesity. More recent animal studies have also reported conflicting results. Considering these contradictory findings, the relationship between eosinophils and obesity may not be unidirectional. In this mini-review, we summarize a recent debate regarding the role of adipose tissue eosinophils in metabolic disorders, and discuss local and systemic effects of eosinophils in obesity. Given that adipose eosinophils play a role in tissue homeostasis, more research is needed to understand the primary function of adipose tissue eosinophils in their microenvironment. Therapeutic interventions that target eosinophils in adipose tissue may have the potential to reduce inflammation and body fat, while improving metabolic dysfunction in obese patients.

The emerging roles of eosinophils in mucosal homeostasis.

Eosinophils are granulocytes, typically implicated as end-stage effector cells in type-II immune responses. They are capable of producing a wide array of pre-formed molecules which render them with vast potential to influence a wide variety of processes. Nonetheless, eosinophil research has traditionally focused on their role in anti-helminthic responses and pathophysiological processes in type-II immune disorders, such as allergy and asthma, where eosinophilia is a hallmark phenotype. However, a number of key studies over the past decade have placed this restricted view of eosinophil function into question, presenting additional evidence for eosinophils as critical regulators of various homeostatic processes including immune maintenance, organ development, and tissue regeneration.

Frontline Science: Superior mouse eosinophil depletion in vivo targeting transgenic Siglec-8 instead of endogenous Siglec-F: Mechanisms and pitfalls.

Eosinophils are important multifunctional granulocytes. When studying eosinophil function and its contribution to diseases, mouse models are often used. Mouse eosinophils selectively express sialic acid-binding immunoglobulin-like lectin (Siglec)-F. Its closest functional paralog on human eosinophils is Siglec-8. These Siglecs are being used to target eosinophils when exploring their mechanistic roles in disease and for potential therapeutic benefit. In order to facilitate preclinical studies of human Siglec-8, we developed transgenic mouse strains expressing human Siglec-8 only on the surface of eosinophils with or without endogenous Siglec-F and have begun characterizing various cellular functions in vitro and in vivo. Eosinophils from Siglec-8+ mice, with or without Siglec-F, responded to Siglec-8 antibody engagement in vitro by up-regulating surface CD11b, whereas Siglec-F antibody had no such effect. Engagement of Siglec-F or Siglec-8 with respective antibodies in vitro resulted in only modest increases in cell death. Administration of rat Siglec-F antibodies to mice led to a significant decrease in Siglec-F surface expression on eosinophils due to internalization, and thus appeared to decrease eosinophil numbers based on Siglec-F+ cells, but with proper gaiting strategies did not in fact result in significant eosinophil depletion. In marked contrast, administration of mouse Siglec-8 antibodies rapidly and effectively depleted eosinophils from blood and spleens of mice, but an F(ab')2 version did not, indicating an Fc-mediated mechanism for eosinophil depletion in vivo. Siglec-8 expressing mice with or without endogenous Siglec-F will be useful to study Siglec-8-based therapeutics, and may be a preferred approach when acute or chronic eosinophil depletion is needed.

Microbial Regulation of Enteric Eosinophils and Its Impact on Tissue Remodeling and Th2 Immunity.

Eosinophils have emerged as multifaceted cells that contribute to tissue homeostasis. However, the impact of the microbiota on their frequency and function at mucosal sites remains unclear. Here, we investigated the role of the microbiota in the regulation of enteric eosinophils. We found that small intestinal (SI) eosinophilia was significantly greater in germ-free (GF) mice compared to specific pathogen free (SPF) controls. This was associated with changes in the production of enteric signals that regulate eosinophil attraction and survival, and was fully reversed by complex colonization. Additionally, SI eosinophils of GF mice exhibited more cytoplasmic protrusions and less granule content than SPF controls. Lastly, we generated a novel strain of eosinophil-deficient GF mice. These mice displayed intestinal fibrosis and were less prone to allergic sensitization as compared to GF controls. Overall, our study demonstrates that commensal microbes regulate intestinal eosinophil frequency and function, which impacts tissue repair and allergic sensitization to food antigens. These data support a critical interplay between the commensal microbiota and intestinal eosinophils in shaping homeostatic, innate, and adaptive immune processes in health and disease.

Charcot-Leyden crystal protein/galectin-10 interacts with cationic ribonucleases and is required for eosinophil granulogenesis

The human eosinophil Charcot-Leyden crystal (CLC) protein is a member of the Galectin superfamily and is also known as galectin-10 (Gal-10). CLC/Gal-10 forms the distinctive hexagonal bipyramidal crystals that are considered hallmarks of eosinophil participation in allergic responses and related inflammatory reactions; however, the glycan-containing ligands of CLC/Gal-10, its cellular function(s), and its role(s) in allergic diseases are unknown. We sought to determine the binding partners of CLC/Gal-10 and elucidate its role in eosinophil biology. Intracellular binding partners were determined by ligand blotting with CLC/Gal-10, followed by coimmunoprecipitation and coaffinity purifications. The role of CLC/Gal-10 in eosinophil function was determined by using enzyme activity assays, confocal microscopy, and short hairpin RNA knockout of CLC/Gal-10 expression in human CD34+ cord blood hematopoietic progenitors differentiated to eosinophils. CLC/Gal-10 interacts with both human eosinophil granule cationic ribonucleases (RNases), namely, eosinophil-derived neurotoxin (RNS2) and eosinophil cationic protein (RNS3), and with murine eosinophil-associated RNases. The interaction is independent of glycosylation and is not inhibitory toward endoRNase activity. Activation of eosinophils with INF-γ induces the rapid colocalization of CLC/Gal-10 with eosinophil-derived neurotoxin/RNS2 and CD63. Short hairpin RNA knockdown of CLC/Gal-10 in human cord blood-derived CD34+ progenitor cells impairs eosinophil granulogenesis. CLC/Gal-10 functions as a carrier for the sequestration and vesicular transport of the potent eosinophil granule cationic RNases during both differentiation and degranulation, enabling their intracellular packaging and extracellular functions in allergic inflammation.

Effects of β2-adrenergic agonists on house dust mite-induced adhesion, superoxide anion generation, and degranulation of human eosinophils.

BackgroundEven in subjects who are not sensitized to house dust mite (HDM), allergic symptoms can be clinically aggravated by exposure to dust. We previously reported that Dermatophagoides farinae (Df), an important HDM, or Der f 1, a major allergen of Df, induced the effector functions of eosinophils, which may be an important mechanism for HDM-induced symptoms in nonsensitized patients. In a clinical setting, β2-adrenergic agonists, such as salbutamol and formoterol, are used for the treatment of asthma attacks or exacerbation to release the airway obstruction. Several reports have suggested that some β2-adrenergic agonists have an anti-inflammatory capacity.ObjectiveIn this study, we investigated whether β2-adrenergic agonist could modify the Df- or Der f 1-induced activation of eosinophils.MethodsBlood eosinophils obtained from healthy donors were preincubated with either formoterol (1 μM), salbutamol (1 μM), or buffer control and then stimulated with Df extract (1 μg/mL) or Der f 1 (100 pg/mL). Eosinophil adhesion to intercellular adhesion molecule (ICAM)-1 was measured using eosinophil peroxidase assays. Generation of superoxide anion (O2−) was examined based on the superoxide dismutase-inhibitable reduction of cytochrome C. Eosinophil-derived neurotoxin (EDN) concentrations in cell media were measured by enzyme-linked immunosorbent assay.ResultsFormoterol, but not salbutamol, suppressed the Df- or Der f 1-induced eosinophil adhesion to ICAM-1. Furthermore, formoterol, but not salbutamol, suppressed Df-induced O2− generation or EDN release. Neither formoterol nor salbutamol suppressed spontaneous eosinophil adhesion, O2− generation, or EDN release.ConclusionThese findings suggested that formoterol, but not salbutamol, suppressed Df- or Der f 1-induced eosinophil activation when used at the same concentration. Therefore, formoterol could potentially be used for the treatment of bronchial asthma via both bronchodilation and anti-inflammatory effect.

Eosinophils in the Tumor Microenvironment.

Eosinophils are rare blood-circulating and tissue-infiltrating immune cells studied for decades in the context of allergic diseases and parasitic infections. Eosinophils can secrete a wide array of soluble mediators and effector molecules, with potential immunoregulatory activities in the tumor microenvironment (TME). These findings imply that these cells may play a role in cancer immunity. Despite these cells were known to infiltrate tumors since many years ago, their role in TME is gaining attention only recently. In this chapter, we will review the main biological functions of eosinophils that can be relevant within the TME. We will discuss how these cells may undergo phenotypic changes acquiring pro- or antitumoricidal properties according to the surrounding stimuli. Moreover, we will analyze canonical (i.e., degranulation) and unconventional mechanisms (i.e., DNA traps, exosome secretion) employed by eosinophils in inflammatory contexts, which can be relevant for tumor immune responses. Finally, we will review the available preclinical models that could be employed for the study of the role in vivo of eosinophils in cancer.

Mechanisms of eosinophilic inflammation.

Eosinophils play roles in the pathogenesis of various diseases. In order to accumulate within sites of inflammation, eosinophils must adhere to, and migrate across the microvasculature. These processes are largely controlled by type 2-immune responses; interleukin (IL)-4 and IL-13 induce the expression of endothelial adhesion molecule vascular cell adhesion molecule-1 (VCAM-1), a representative adhesive ligand for eosinophils, while also stimulating generations of CC chemokines from structural cells, including epithelial cells. VCAM-1 and CC chemokines synergistically induce transmigration of eosinophils to the tissue inflammation site. Another type 2 cytokine, IL-5, prolongs survival, and enhances the effector functions of eosinophils. Recently, accumulating evidence has established that corticosteroid-resistant group 2 innate lymphoid cells are cellular sources for IL-5. Another immunological mechanism that may be contributing to eosinophilic inflammation involves type 1 immune system-associated molecules such as interferons and IP-10. In addition to these immune pathways, lipid mediators, such as cysteinyl leukotrienes, directly provoke the infiltration and activation of eosinophils. Extracellular matrix proteins including periostin also induce the adhesion and activation of eosinophils. Finally, activated neutrophils can also induce eosinophil transmigration. In summary, various mechanisms are involved within eosinophilic inflammation, and effective therapeutic strategies targeting these pathways should be established.

Bioinformatic analysis of eosinophil activity and its implications for model and target species.

Eosinophils are important immune cells that have been implicated in resistance to gastrointestinal nematode (GIN) infections in both naturally and experimentally infected sheep. Proteins of particular importance appear to be IgA-Fc alpha receptor (FcαRI), C-C chemokine receptor type 3 (CCR3), proteoglycan 3 (PRG3, major basic protein 2) and EPX (eosinophil peroxidase). We used known human nucleotide sequences to search the ruminant genomes, followed by translation to protein and sequence alignments to visualize differences between sequences and species. Where a sequence was retrieved for cow, but not for sheep and goat, this was used additionally as a reference sequence. In this review, we show that eosinophil function varies among host species. Consequently, investigations into the mechanisms of ruminant immune responses to GIN should be conducted using the natural host. Specifically, we address differences in protein sequence and structure for eosinophil proteins.

LCP1 triggers mTORC2/AKT activity and is pharmacologically targeted by enzastaurin in hypereosinophilia.

Hypereosinophilia (HE) is caused by a variety of disorders, ranging from parasite infections to autoimmune diseases and cancer. Only a small proportion of HE cases are clonal malignancies, and one of these, the group of eosinophilia-associated tyrosine kinase fusion-driven neoplasms, is sensitive to tyrosine kinase inhibitors, while most subtypes lack specific treatment. Eosinophil functions are highly dependent on actin polymerization, promoting priming, shape change, and infiltration of inflamed tissues. Therefore, we investigated the role of the actin-binding protein lymphocyte cytosolic protein 1 (LCP1) in malignant and nonmalignant eosinophil differentiation. We use the protein kinase C-β (PKCβ) selective inhibitor enzastaurin (Enza) to dephosphorylate and inactivate LCP1 in FIP1L1-platelet-derived growth factor receptor α (PDGFRA)-positive Eol-1 cells, and this was associated with reduced proliferation, metabolic activity, and colony formation as well as enhanced apoptosis and impaired migration. While Enza did not alter FIP1L1-PDGFRA-induced signal transducer and activator of transcription 3 (STAT3), STAT5, and ERK1/2 phosphorylation, it inhibited STAT1Tyr701 and AKTSer473 (but not AKTThr308 ) phosphorylation, and short hairpin RNA knockdown experiments confirmed that this process was mediated by LCP1 and associated mammalian target of rapamycin complex 2 (mTORC2) activity loss. Homeobox protein HoxB8 immortalized murine bone marrow cells showed impaired eosinophilic differentiation upon Enza treatment or LCP1 knockdown. Furthermore, Enza treatment of primary HE samples reduced eosinophil differentiation and survival. In conclusion, our data show that HE involves active LCP1, which interacts with mTOR and triggers mTORC2 activity, and that the PKCβ inhibitor Enza as well as targeting of LCP1 may provide a novel treatment approach to hypereosinophilic disorders.

Eosinophils, the IL-5/IL-5Rα axis, and the biologic effects of benralizumab in severe asthma.

Bronchial asthma is a chronic inflammatory disease characterized, in a percentage of patients, as an eosinophilic inflammation of the airways. Eosinophils are recognized as a proinflammatory granulocyte playing a major role in the T2-high phenotype, which includes severe eosinophilic asthma. Eosinophilic asthma represents the majority of the phenotypic variants clinically characterized by severity and frequent exacerbations. For patients with severe uncontrolled asthma, monoclonal antibodies are used as add-on treatments. Among them, in addition to anti-immunoglobulin E therapy, biologic agents directed toward the interleukin (IL)-5/IL-5Rα axis and, thus, interfering with the pathologic functions of eosinophils, are now available. Unlike the other anti‒IL-5 monoclonal antibodies which exert an indirect effect on eosinophils, benralizumab, an afucosylated IgG1 kappa antibody directed against the α subunit of IL-5R, directly depletes eosinophils and their associated bone marrow progenitor cells through induction of antibody-dependent cell-mediated cytotoxicity, through recruitment of natural killer cells. This article reviews the role of eosinophils in the pathogenesis of bronchial asthma and discusses the potential advantageous biologic effects of benralizumab in comparison with other monoclonal antibodies targeting the IL-5 ligand.

Atomic Force Microscopy Is a Potent Technique to Study Eosinophil Activation.

Eosinophils are multifunctional cells with several functions both in healthy individuals, and those with several diseases. Increased number and morphological changes in eosinophils have been correlated with the severity of an acute asthma exacerbation. We measured eosinophils obtained from healthy controls and individuals with acute asthma using atomic force microscopy (AFM). In the control samples, cells showed more rounded morphologies with some spreading, while activated cells from symptomatic individuals were spreading, and presenting emission of multiple pseudopods. Eosinophils presenting separate granules close to the cells suggesting some degranulation was also increased in asthma samples. In comparison to histopathological techniques based on brightfield microscopy, AFM showed considerably more details of these morphological changes, making the technique much more sensitive to detect eosinophil morphological changes that indicate functional alteration of this cell. AFM could be an important tool to evaluate diseases with alterations in eosinophil functions.

Comparison of IL-33 and IL-5 family mediated activation of human eosinophils.

Eosinophils are the prominent inflammatory cell involved in allergic asthma, atopic dermatitis, eosinophilic esophagitis, and hypereosinophilic syndrome and are found in high numbers in local tissue and/or circulating blood of affected patients. There is recent interest in a family of alarmins, including TSLP, IL-25 and IL-33, that are epithelial-derived and released upon stimulation of epithelial cells. Several genome wide association studies have found SNPs in genes encoding IL-33 to be risk factors for asthma. In two studies examining the direct role of IL-33 in eosinophils, there were differences in eosinophil responses. We sought to further characterize activation of eosinophils with IL-33 compared to activation by other cytokines and chemokines. We assessed IL-33 stimulated adhesion, degranulation, chemotaxis and cell surface protein expression in comparison to IL-3, IL-5, and eotaxin-1 on human eosinophils. Our results demonstrate that IL-33 can produce as potent eosinophil activation as IL-3, IL-5 and eotaxin-1. Thus, when considering specific cytokine targeting strategies, IL-33 will be important to consider for modulating eosinophil function.

Eosinophils in the gastrointestinal tract and their role in the pathogenesis of major colorectal disorders

Eosinophils are currently regarded as versatile mobile cells controlling and regulating multiple biological pathways and responses in health and disease. These cells store in their specific granules numerous biologically active substances (cytotoxic cationic proteins, cytokines, growth factors, chemokines, enzymes) ready for rapid release. The human gut is the main destination of eosinophils that are produced and matured in the bone marrow and then transferred to target tissues through the circulation. In health the most important functions of gut-residing eosinophils comprise their participation in the maintenance of the protective mucosal barrier and interactions with other immune cells in providing immunity to microbiota of the gut lumen. Eosinophils are closely involved in the development of inflammatory bowel disease (IBD), when their cytotoxic granule proteins cause damage to host tissues. However, their roles in Crohn’s disease and ulcerative colitis appear to follow different immune response patterns. Eosinophils in IBD are especially important in altering the structure and protective functions of the mucosal barrier and modulating massive neutrophil influx to the lamina propria followed by transepithelial migration to colorectal mucus. IBD-associated inflammatory process involving eosinophils then appears to expand to the mucus overlaying the internal gut surface. The author hypothesises that immune responses within colorectal mucus as well as ETosis exerted by both neutrophils and eosinophils on the both sides of the colonic epithelial barrier act as additional pathogenetic factors in IBD. Literature analysis also shows an association between elevated eosinophil levels and better colorectal cancer (CRC) prognosis, but mechanisms behind this effect remain to be elucidated. In conclusion, the author emphasises the importance of investigating colorectal mucus in IBD and CRC patients as a previously unexplored milieu of disease-related inflammatory responses.

Butyrate ameliorates allergic airway inflammation by limiting eosinophil trafficking and survival

Lung eosinophilia is a hallmark of asthma, and eosinophils are believed to play a crucial role in the pathogenesis of allergic inflammatory diseases. Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced in high amounts in the gastrointestinal tract by commensal bacteria and can be absorbed into the bloodstream. Although there is recent evidence that SCFAs are beneficial in allergic asthma models, the effect on eosinophils has remained elusive. The role of SCFAs was investigated in human eosinophil function and a mouse model of allergic asthma. Eosinophils were purified from self-reported allergic or healthy donors. Migration, adhesion to the endothelium, and eosinophil survival were studied invitro. Ca2+ flux, apoptosis, mitochondrial membrane potential, and expression of surface markers were determined by using flow cytometry and in part by using real-time PCR. Allergic airway inflammation was assessed invivo in an ovalbumin-induced asthma model by using invasive spirometry. For the first time, we observed that SCFAs were able to attenuate human eosinophils at several functional levels, including (1) adhesion to the endothelium, (2) migration, and (3)survival. These effects were independent from GPR41 and GPR43 but were accompanied by histone acetylation and mimicked by trichostatin A, a pan-histone deacetylase inhibitor. Invivo butyrate ameliorated allergen-induced airway and lung eosinophilia, reduced type 2 cytokine levels in bronchial fluid, and improved airway hyperresponsiveness in mice. These invitro and invivo findings highlight the importance of SCFAs, especially butyrate as a promising therapeutic agent in allergic inflammatory diseases.

A Concept Suggestion on the Effect of Cigarette Smoking in Inflammatory Destruction of Gingiva.

Dear Editor, Cigarette smoking has destructive effect on periodontal tissue. The rates of loss of periodontal attachment and recession of gingival are higher in smokers than non-smokers (1-2). Previous studies on the inflammatory immune responses in smokers’ periodontitis have mainly focused on the role of neutrophils. Tumor necrosis factor–α, prostaglandin E2 and matrix metalloproteinase-8 have been shown to rise in smokers with periodontitis (3-4). Different functions of mast cells and eosinophils in inflammatory immune responses make them distinctive cells in disease pathogenesis (5-6). In an investigation, our team examined the effect of smoking on mast cells density in chronic periodontitis. The study showed that the mean number of mast cells in smokers was significantly lower compared to the non-smokers. Based on the literature, no research was found regarding the effect of cigarette smoking on eosinophil cells in human periodontitis. Eosinophils and mast cells regulate the hypersensitivity reactions by affecting each other function (5). Thus, in the next study, we examined this issue on the same samples. The results revealed that the number of eosinophil count in smokers was significantly lower than non-smokers. Considering the findings of both studies on decreased number of mast cells and eosinophils in the same samples, it seems that cigarette smoke had an apoptotic function on extra-vascular immune inflammatory related cells in human periodontitis. According to our opinion, with the death of mast cells and eosinophils, a cascade of different events occurs in the microenvironment of gingiva which causes more tissue damage in the smokers. The apoptotic effect of cigarette smoke on gingival connective tissue must be studied in the enzymatic level.The Heme Oxygenase-1 (HO-1)/Carbon Monoxide (CO) system demand to explain the pathogenesis of diseases by using the basic metabolism and enzymatic activities. HO-1 has a regulatory action on inflammatory signaling programs. CO is an end-product of HO-1. CO affects the apoptosis and cellular inflammation by modulating the inflammatory related cytokines. Modulating the HO-1 and application of CO-releasing molecules are new therapeutic strategies in inflammatory diseases (7). Based on our previous findings, we suggest that further study on HO-1/CO can probably determine the effect of cigarette smoke on inflammatory immune cells in human chronic periodontitis. The system can be potentially considered as a therapeutic target in inflammatory disease of periodontium in cigarette smokers. Conflict of Interest The authors declared no conflict of interest regarding the publication of this article.

Critical role of CCL4 in eosinophil recruitment into the airway.

Excessive eosinophil airway infiltration is a clinically critical condition in some cases. Eosinophilic pneumonia (EP) is a pulmonary condition involving eosinophil infiltration of the lungs. Although several chemokines, including eotaxin-1 (CCL11), RANTES (CCL5) and macrophage inflammatory protein 1β (MIP-1β or CCL4), have been detected in bronchoalveolar lavage fluid (BALF) from patients with EP, the pathophysiological mechanisms underlying EP, including potential relationships between eosinophils and CCL4, have not been fully elucidated. To examine the involvement of CCL4 in eosinophilic airway inflammation. We analysed supernatants of activated eosinophils and BALF from 16 patients with eosinophilic pneumonia (EP). Further, we examined the effects of CCL4 on eosinophil functions in vitro and those of anti-CCL4 neutralizing antibody in an in vivo model. We found that purified human eosinophils stimulated with IL-5 predominantly secreted CCL4 and that patients with EP had elevated CCL11 and CCL4 levels in BALF compared with samples from individuals without EP. Because CCL4 levels were more strongly correlated with eosinophil count and expression of eosinophil granule proteins than CCL11, in vitro experiments using purified eosinophils concentrated on the former chemokine. Interestingly, CCL4 acted as a chemoattractant for eosinophils. In a mouse model, administration of a CCL4-neutralizing antibody attenuated eosinophilic airway infiltration and airway hyperresponsiveness. Overall, these findings highlight an important role of CCL4 in the mechanisms underlying eosinophil recruitment into the airway and may provide a novel insight into this potential therapeutic target.