ORIGINAL ARTICLE. Tomoe Nishimura 1, Mayumi Saeki 1, Yuji Motoi 1, Noriko Kitamura 1,AkioMori 2, Osamu Kaminuma 1 and Takachika Hiroi 1 INTRODUCTION

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1 Allergology International. 214;63(Suppl 1):29-35 DOI: allergolint.13-oa-635 ORIGINAL ARTICLE Selective Suppression of Th2 Cell-Mediated Lung Eosinophilic Inflammation by Anti-Major Facilitator Super Family Domain Containing 1 Monoclonal Antibody Tomoe Nishimura 1, Mayumi Saeki 1, Yuji Motoi 1, Noriko Kitamura 1,AkioMori 2, Osamu Kaminuma 1 and Takachika Hiroi 1 ABSTRACT Background: The eosinophil is deeply associated with the pathogenesis of bronchial asthma and other allergic diseases. We recently identified a novel eosinophil-specific cell surface molecule, major facilitator super family domain containing 1 (Mfsd1). A monoclonal antibody (mab) against Mfsd1 (M2) showed selective binding and neutralizing activities for eosinophils. However, the relative potency of the blockage of Mfsd1 and other eosinophil-specific molecules for the treatment of allergic diseases has not been evaluated. Therefore, in this study, the effects of M2 and an anti-siglec-f mab on antigen-immunized and antigen-specific Th2 celltransferred murine eosinophilic inflammation models were comparatively investigated. Methods: Ovalbumin (OVA)-specific Th2 cells were differentiated from naïve CD4 + T cells of DO11.1 RAG mice in vitro and cytokine producing activity of the Th2 cells was examined. OVA-immunized and Th2 celltransferred BALB c mice were treated with M2 or anti-siglec-f and challenged with OVA. Then the number of inflammatory cells and the concentration of IL-5 in the bronchoalveolar lavage fluid (BALF) were determined. Results: Antigen-specific Th2 cells produced large amounts of IL-4, IL-5 and IL-13 but not IL-17A or IFN-γ. Administration of M2 significantly suppressed antigen-induced lung eosinophil infiltration both in OVA-immunized and Th2 cell-transferred mice. The potency as well as selectivity of M2 for down-regulating eosinophils was quite similar to that of anti-siglec-f. Both mabs did not affect antigen-induced IL-5 production in the lungs. Conclusions: Mfsd1 as well as Siglec-F could be an effective target to treat eosinophil-related disorders including bronchial asthma. KEY WORDS asthma, eosinophil, Mfsd1, monoclonal antibody, T cell INTRODUCTION Eosinophilic inflammation is a prominent feature of allergic diseases including bronchial asthma. 1-4 The growth, maturation and survival of eosinophils are mediated by Th2 cytokines, such as IL-3, IL-4, IL-5 and GM-CSF. 4 Mature eosinophils secret several cytokines, lipid mediators and toxic granule proteins that induce local inflammation, epithelial fibrosis and airway remodeling. 4 Compared to healthy control, asthmatic patients showed an increase in the number of eosinophils in blood, bronchial mucosa and bronchoalveolar lavage fluid (BALF). 1-3 About 1 years ago, the role of eosinophils in the pathogenesis of bronchial asthma was controversial, since anti-il-5 therapy did not effectively improve the symptoms of 1Allergy and Immunology Project, Tokyo Metropolitan Institute of Medical Science, Tokyo and 2 Clinical Research Center for Allergy and Rheumatology, National Hospital Organization, Sagamihara National Hospital, Kanagawa, Japan. Conflict of interest: No potential conflict of interest was disclosed. Correspondence: Dr. Osamu Kaminuma, Allergy and Immunology Project, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku, Tokyo , Japan. kaminuma os@igakuken.or.jp Received 3 September 213. Accepted for publication 16 February Japanese Society of Allergology Allergology International Vol 63, Suppl 1,

2 Nishimura T et al. asthmatic patients. 5,6 However, recent clinical studies for targeting eosinophils, including the newly designed anti-il-5 therapy, have shown that this cell type is exactly involved in an exacerbation of asthmatic symptom. 7-9 To identify and or neutralize eosinophils, antibodies (Abs) against cell surface molecules selectively expressed on eosinophils is useful. In addition to the receptor for IL-5, several eosinophil-specific molecules such as Siglec-F and CCR3 have been reported Administration of monoclonal Abs (mabs) against Siglec-F, CCR3 and IL-5 receptor α induced a significant reduction of eosinophils recruited in the allergic tissues Major facilitator super family domain containing 1 (Mfsd1) was originally identified as a yeast TPO1 gene, which belongs to the major facilitator super family. 18 It was suggested that Mfsd1 is a transporter which uptakes organic anions such as indomethacin and fluorescein. 19 By means of microarray analysis over the mrna library established from a series of murine white blood cells, we previously identified that Mfsd1 was selectively expressed on eosinophils. 2 Flow cytometric analysis revealed that a mab against Mfsd1, M2, was reactive with eosinophils in the BALF of antigen-immunized and -challenged mice. 2 However, the ability of anti- Mfsd1 therapy to treat local eosinophilic inflammation in allergic diseases has not been elucidated. We have recently established a new eosinophilic inflammation model completely dependent on Th2 cells. Upon antigen challenge, massive eosinophil accumulation in the lungs accompanied by significant bronchial hyperresponsiveness was observed in mice transferred with in vitro-differentiated antigenspecific Th2 cells In this model, we have already confirmed that the blockage of IL-5 as well as CCR3 effectively suppressed the infiltration of eosinophils. 21,22 Therefore, by employing the Th2 cell transfer model as well as commonly used antigen immunization model, we here comparatively investigated the effects of M2 and anti-siglec-f mab on antigeninduced eosinophilic inflammation in the lungs. The mechanisms of these effects were further explored by analyzing cytokine levels in the BALF. METHODS ANIMALS Six-week-old female BALB c mice were purchased from Japan SLC (Shizuoka, Japan). DO11.1 RAG-2 - mice were generated and maintained for antigenspecific Th2 cell preparation as described previously. 23 The experimental protocols were approved by the Animal Use and Care Committee of Tokyo Metropolitan Institute of Medical Science. ANTIBODIES M2 was established as described previously. 2 Rat IgG Ab and anti-siglec-f mab were purchased from Inter-Cell Technologies (FL, USA) and R & D Systems (MN, USA), respectively. IN VITRO POLARIZATION OF TH2 CELLS Ovalbumin (OVA)-specific naïve CD4 + T cells were isolated from splenocytes of DO11.1 RAG mice by positive selection using CD4 microbeads and magnetic cell sorting system (Miltenyi, Bergisch Gladbach, Germany). Cells were cultured with X-rayirradiated splenocytes in DMEM-F12 HAM medium (Sigma-Aldrich, MO, USA) supplemented with 1% fetal bovine serum. At the start of culture,.3 μm synthetic OVA peptide, 1 U ml recombinant IL- 2 (Shionogi, Osaka, Japan), 1 U ml recombinant IL- 4 (PeproTech, NJ, USA) and 1 μg ml anti-ifn-γ mab (R4-6A2, ebioscience, CA, USA) were added as described previously. 23 Seven days after the stimulation, cells were harvested and used for the adoptive transfer. To determine the integrity of polarization, the resulting Th2 cells (1 1 5 ) were incubated with X-ray-irradiated splenocytes (2 1 5 )withorwithout.3 μm OVA peptide for 24 h. The concentrations of cytokines in the culture supernatants were determined by a bead-based multiplex immunoassay system using Bio-plex Pro Mouse Cytokine Assay (Bio- Rad Laboratories, CA. USA), according to the manufacturer s instructions. Detection limits of IL-1β, IL-4, IL-5, IL-13, IL-17A and IFN-γ were 375, 25, 2, 55, 15 and 3 pg ml, respectively. AIRWAY EOSINOPHILIC INFLAMMATION MOD- ELS In the antigen immunization model, mice were sensitized by intraperitoneal injection of 2 μg OVA (Sigma-Aldrich) emulsified with 2.25 mg alum (Thermo Fisher Scientific, MA, USA) on days, 7, 14 and 21 of the experiment. On days 35-37, mice were challenged 3 times by daily intratracheal administration of 375 μg OVA dissolved in 25 μl saline or saline alone. Twenty-four hours before the first challenge and 5 h after the last challenge, 2 mg kg M2, 4 mg kg anti-siglec-f mab or control rat IgG was administrated intravenously (Fig. 1A). In Th2 cell transfer model, polarized Th2 cells (2 1 7 ) were intravenously injected in each BALB c mouse on day, and then mice were challenged by intratracheal administration of OVA or saline on day 1. Two hours before and 24 h and 48 h after the challenge, mice were intravenously injected three times with M2, anti-siglec- F or control rat IgG (Fig. 1B). In both models, the most effective dose and timing of Ab administration were determined by serial preliminary experiments. BAL was performed 72 h after the last challenge and inflammatory cells in the BALF were classified by means of morphological criteria as described previously. 2,23 The concentrations of IL-4, IL-5, IL-13 and eotaxin in the BALF were determined by ELISA with 3 Allergology International Vol 63, Suppl 1, 214

3 Anti-Mfsd1 Suppresses Lung Eosinophilia A B Day OVA + alum immunization Ab administration OVA challenge BAL Day Th2 cell transfer OVA challenge Ab administration BAL Fig. 1 Timeline of the experimental protocol. Mice were sensitized 4 times with OVA plus alum once a week. On days 35-37, mice were challenged by intratracheal injection with OVA. Ab administration was performed on days 34 and 37. BAL was performed 72 h after the last challenge (A). After the transfer of polarized Th2 cells on day, mice were challenged with OVA on day 1. Ab was administrated three times on days 1, 2 and 3. BAL was performed 72 h after the last challenge (B). mouse ELISA kits (ebioscience) according to the manufacture s protocol. Detection limits of these assay systems were 16, 8, 16, 31 pg ml, respectively. STATISTICAL ANALYSIS The results were presented as arithmetic mean ± SEM. Statistical analysis was performed by one-way ANOVA and Dunnett s multiple comparison test. p <.5 was considered to indicate statistical significance. RESULTS Concentration (ng/ml) Stimulation - + N.D. N.D. N.D. N.D. N.D. N.D. IL-1β IL-4 IL-5 IL-13 IL-17A IFN-γ Fig. 2 Cytokine production by Th2 cells in vitro. Polarized Th2 cells were stimulated with OVA peptide or were left unstimulated. Twenty-four hours later, the concentrations of cytokines in the culture supernatants were measured by the bead-based multiplex immunoassay system. Data are expressed as mean ± SEM of triplicate cultures. The results shown are representative of two separate experiments. N.D., not detectable. CHARACTERIZATION OF POLARIZED TH2 CELLS IN VITRO To evaluate the polarization state of in vitro-cultured antigen-specific CD4 + T cells under Th2 differentiation conditions, cells were stimulated with OVA peptide, and then concentrations of various cytokines in the culture supernatants were determined. As shown in Figure 2, antigen-stimulated Th2 cells secreted large amounts of IL-4, IL-5 and IL-13, however these cytokines were not detectable without stimulation. IL-17A was produced very little only by stimulated cells. Weak but constitutive IL-1β production was observed, whereas IFN-γ was not detectable with or without antigen stimulation. These findings suggest that antigen-specific T cells used for adoptive transfer in this study were appropriately differentiated into Th2 cells. EFFECTS OF M2 AND ANTI-SIGLEC-F mab ON LUNG EOSINOPHIL INFILTRATION The effects of M2 and an anti-siglec-f mab on antigen-induced lung eosinophil accumulation were comparatively investigated in antigen-immunized mice. The number of eosinophils in the BALF was significantly increased upon OVA challenge (Fig. 3). Administration of M2 as well as anti-siglec-f significantly suppressed the infiltration of eosinophils. The accumulation of other inflammatory cells, such as lymphocytes, neutrophils and monocytes macrophages in the lungs was also observed following antigen challenge, though it was not statistically significant. Moreover, these inflammatory cells other than eosinophils were not affected by administration of M2 or anti-siglec-f (Fig. 3). Next, we examined the ability of M2 to downregulate Th2 cell-mediated lung eosinophilia. Th2 cell-transferred mice were administrated with each mab and were challenged with OVA. Similar to the case of the antigen immunization model, massive eosinophil infiltration into the lungs was occurred, and the eosinophilia was significantly suppressed by M2 and anti-siglec-f (Fig. 4). The numbers of lymphocytes, neutrophils, monocytes macrophages and total cells in the BALF of M2- and anti-siglec-fadministrated mice were not significantly different from those of control rat IgG-administrated mice. Both in antigen immunization and Th2 cell transfer models, the potency as well as selectivity of M2 and anti-siglec-f for down-regulating antigen-induced lung eosinophilia was equivalent. Allergology International Vol 63, Suppl 1,

4 Nishimura T et al. Cell number ( 1 5 /BALF) ** Challenge/Ab Saline/None OVA/Rat IgG OVA/M2 OVA/Anti-Siglec-F Total cells Lymphocytes Neutrophils Eosinophils *** Monocytes/ Macrophages * Fig. 3 Effects of M2 and anti-siglec-f mab on antigen-induced eosinophil infi ltration in antigen-immunized mice. Antigen-immunized mice were challenged with OVA or saline. Twenty-four hours before the first challenge and 5 h after the last challenge, each Ab was administrated intravenously. The number of cells in the BALF was determined 72 h after the last challenge. Data are expressed as mean ± SEM of 4-6 mice. *p <.5, **p <.1 and ***p <.1, compared with OVA/Rat IgG group. 6 Challenge/Ab Saline/None Cell number ( 1 5 /BALF) 4 2 OVA/Rat IgG OVA/M2 OVA/Anti-Siglec-F Total cells Lymphocytes Neutrophils Eosinophils Monocytes/ Macrophages *** ** ** Fig. 4 Effects of M2 and anti-siglec-f mab on antigen-induced eosinophil infi ltration in Th2 cell-transferred mice. Th2 cell-transferred mice were challenged with OVA or saline. Each Ab was administrated intravenously three times 2 h before, and 24 h and 48 h after the antigen challenge. The number of cells in the BALF was determined 72 h after the last challenge. Data are expressed as mean ± SEM of 4-6 mice. **p <.1 and ***p <.1, compared with OVA/Rat IgG group. 32 Allergology International Vol 63, Suppl 1, 214

5 Anti-Mfsd1 Suppresses Lung Eosinophilia IL-5 concentration (pg/ml) Ab Challenge N.D. - Rat IgG M2 Siglec-F Saline OVA Fig. 5 IL-5 production in lungs of Th2 cell-transferred mice. Th2 cell transfer, antigen challenge and BAL were performed as described in Figure 1. The concentration of IL-5 in the BALF was measured by ELISA. Data are expressed as mean ± SEM of 4-6 animals. N.D., not detectable. EFFECTS OF M2 AND ANTI-SIGLEC-F mab ON IL-5 PRODUCTION To investigate whether the suppression of the eosinophil accumulation by M2 or anti-siglec-f was caused by the down-regulation of eosinophil active cytokines, the effects of these mabs on the production of IL-5 in the BALF were investigated. An apparent increase in the level of IL-5 was observed in the BALF of Th2 cell-transferred and OVA-challenged mice (Fig. 5). However, administration of M2 or anti-siglec-f did not affect the production of IL-5. Not only eosinophilactive chemokines such as eotaxin but also other Th2 cytokines such as IL-4 and IL-13 were not detectable in these experimental conditions. Therefore, it is most likely that the inhibition of eosinophil infiltration by M2 and anti-siglec-f was due to their direct effects on eosinophils, rather than to the down-regulation of eosinophil-active cytokines chemokines. DISCUSSION The recruitment of eosinophils into the target organs is one of the critical pathogenetic mechanisms of allergic diseases. In this study, significant suppression of antigen-induced lung eosinophil infiltration was achieved by administration of the mab, M2, against a novel eosinophil-specific cell surface molecule, Mfsd1, in two different allergic inflammation models. The efficacy of M2 was comparable to that of anti- Siglec-F mab and was not due to the inhibition of IL-5 production. The effect of M2 was highly selective on eosinophils. Thus, both in antigen immunization and Th2 transfer models, accumulation of eosinophils but not lymphocytes, neutrophils or monocytes macrophages in the BALF was significantly affected by M2. These findings are consistent with our previous observations that mrna expression level of Mfsd1 was much higher in eosinophils than in B cells, T cells, monocytes macrophages, neutrophils, basophils and dendritic cells. 2 In addition, we also demonstrated that more than 97% of M2-positive cells in the BALF of antigen-immunized and -challenged mice were CCR3 + Siglec-F + eosinophils. 2 The suppression of eosinophil accumulation was also displayed by anti-siglec-f mab. The potency of this mab against lung eosinophil accumulation both in antigen immunization and Th2 transfer models was similar to that of M2. These finding are consistent with a previous report showing that intact as well as F(ab )2 fragments of anti-siglec-f mab similarly suppressed antigen-induced lung eosinophilia in mice. 13 Zimmermann et al. also demonstrated that administration of anti-siglec-f Ab also decreased the number of peripheral blood eosinophils. 14 However, the down-regulation of eosinophils but not neutrophils by anti-siglec-f in this study was not supported by our previous data that mrna of Siglec-F was equivalently expressed in eosinophils and neutrophils. 2 Zhang et al. also reported that weak expression of Siglec-F was observed on mouse neutrophils. 24 On the other hand, the suppressive effect of anti-siglec-f on neutrophil infiltration was not demonstrated by Song et al.. 13 The reason for the contradiction is unclear, although the allergic inflammation models employed in this study especially featured eosinophils. Therefore, in order to elucidate the exact role of Siglec-F on neutrophils, the effect of anti-siglec-f is required to be investigated in some neutrophil-rich inflammation model. Most importantly, it is clearly suggested that Mfsd1 is a critical target for the selective downregulation of eosinophil accumulation in allergic diseases. The eosinophil is crucially involved in an important pathological feature of bronchial asthma, airway remodeling. It was reported that antigen-induced peribronchial fibrosis was diminished by administration of anti-siglec-f due to reduction of eosinophil-derived TGF-β1. 13 Anti-CCR3 mab was also reported to reduce mucus accumulation or subepithelial basement membrane thickness, according to the suppression of eosinophil infiltration. 15 These previous findings suggest the possible management of airway remodeling by anti-mfsd1 Ab. Further investigations are required to elucidate the effect of M2 on antigeninduced airway remodeling models. IL-5 is mainly produced by activated Th2 as well as mast cells and eosinophils, 25,26 and regulates the differentiation, maturation, migration, development, survival, trafficking and effector function of eosinophils. 4 In this regard, it seems that inhibition of the eosinophil accumulation by M2 and anti-siglec-f was contradictory to their ineffectiveness on IL-5 production. However, in a series of our previous investigation employing Th2 cell-transferred mice, the cellular source of IL-5 was mostly designated as antigen-specific T Allergology International Vol 63, Suppl 1,

6 Nishimura T et al. cells already distributed in the lungs before the challenge. 21,27 Consequently, M2 as well as Siglec-F could down-regulate the eosinophilia without affecting the BALF IL-5 level in this study. Eosinophils may contribute as sources of IL-5 in more chronic inflammatory sites in asthma. It was reported that Abs against Siglec-8, a human paralog of Siglec-F, induced caspase- and or reactive oxygen species-dependent apoptosis of eosinophils Although mechanisms of M2 to suppress infiltration of eosinophils are needed to be elucidated, Ushijima et al. reported that Mfsd1 is a membrane transporter which uptake of organic anions. 19 Therefore, delineating the role of this transporter in eosinophil functions is also required. Conclusively, M2 and anti-siglec-f selectively suppressed antigen-induced lung eosinophilic infiltration. Mfsd1 is a potential target to treat eosinophil-related disorders including bronchial asthma. ACKNOWLEDGEMENTS This work was supported by the research grants Agri-Health Translational Research Project from Ministry of Agriculture, Forestry, and Fisheries of Japan to T. Hiroi, and Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science to O. Kaminuma (Grant Number 24551). REFERENCES 1. Bhakta NR, Woodruff PG. Human asthma phenotypes: from the clinic, to cytokines, and back again. Immunol Rev 211;242: Mukherjee AB, Zhang Z. Allergic asthma: influence of genetic and environmental factors. JBiolChem211;286: de Boer JD, Majoor CJ, van t Veer C, Bel EHD, van der Poll T. Asthma and coagulation. Blood 212;119: Kita H. Eosinophils: multifaceted biological properties and roles in health and disease. Immunol Rev 211;242: Leckie MJ, ten Brinke A, Khan J et al. Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and late asthmatic response. Lancet 2;356: Flood-Page PT, Menzies-Gow AN, Kay AB, Robinson DS. Eosinophil s role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. Am J Respir Crit Care Med 23;167: Haldar P, Brightling CE, Hargadon B et al. Mepolizumab and exacerbations of refractory eosinophilic asthma. N Engl J Med 29;36: Nair P, Pizzichini MM, Kjarsgaard M et al. Mepolizumab for prednisone-dependent asthma with sputum eosinophilia. NEnglJMed29;36: Gauvreau GM, Pageau R, Séguin R et al. Dose-response effects of TPI ASM8 in asthmatics after allergen. Allergy 211;66: Sun Z, Yergeau DA, Tuypens T et al. Identification and characterization of a functional promoter region in the human eosinophil IL-5 receptor alpha subunit gene. JBiol Chem 1995;27: Zhang JQ, Biedermann B, Nitschke L, Crocker PR. The murine inhibitory receptor msiglec-e is expressed broadly on cells of the innate immune system whereas msiglec-f is restricted to eosinophils. Eur J immunol 24;34: Ponath PD, Qin S, Post TW et al. Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils. JExpMed1996;183: Song DJ, Cho JY, Lee SY et al. Anti-Siglec-F antibody reduces allergen-induced eosinophilic inflammation and airway remodeling. J Immunol 29;183: Zimmermann N, McBride ML, Yamada Y et al. Siglec-F antibody administration to mice selectively reduces blood and tissue eosinophils. Allergy 28;63: Justice JP, Borchers MT, Crosby JR et al. Ablation of eosinophils leads to a reduction of allergen-induced pulmonary pathology. Am J Physiol Lung Cell Mol Physiol 23;284: Sénéchal S, Fahy O, Gentina T et al. CCR3-blocking antibody inhibits allergen-induced eosinophil recruitment in human skin xenografts from allergic patients. Lab Invest 22;82: Kolbeck R, Kozhich A, Koike M et al. MEDI-563, a humanized anti-il-5 receptor alpha mab with enhanced antibody-dependent cell-mediated cytotoxicity function. J Allergy Clin Immunol 21;125: Mima S, Ushijima H, Hwang HJ et al. Identification of the TPO1 gene in yeast, and its human orthologue TETRAN, which cause resistance to NSAIDs. FEBS Lett 27;581: Ushijima H, Hiasa M, Namba T et al. Expression and function of TETRAN, a new type of membrane transporter. Biochem Biophys Res Commun 28;374: Motoi Y, Saeki M, Nishimura T et al. Establishment of monoclonal antibodies against a novel eosinophil-specific cell surface molecule, major facilitator super family domain containing 1. Immunol Lett 212;147: Kaminuma O, Mori A, Ogawa K et al. Successful transfer of late phase eosinophil infiltration in the lung by infusion of helper T cell clones. Am J Respir Cell Mol Biol 1997;16: Mori A, Ogawa K, Someya K et al. Selective suppression of Th2-mediated airway eosinophil infiltration by lowmolecular weight CCR3 antagonists. Int Immunol 27; 19: Kaminuma O, Ohtomo T, Mori A et al. Selective downregulation of Th2 cell-mediated airway inflammation in mice by pharmacological intervention of CCR4. Clin Exp Allergy 212;42: Zhang M, Angata T, Cho JY, Miller M, Broide DH, Varki A. Defining the in vivo function of Siglec-F, a CD33- related Siglec expressed on mouse eosinophils. Blood 27;19: Robinson DS, Hamid Q, Ying S et al. Predominant TH2- like bronchoalveolar T-lymphocyte population in atopic asthma. NEnglJMed1992;3: Ying S, Durham SR, Corrigan CJ, Hamid Q, Kay AB. Phenotype of cells expressing mrna for TH2-type (interleukin 4 and interleukin 5) and TH1-type (interleukin 2 and interferon gamma) cytokines in bronchoalveolar lavage and bronchial biopsies from atopic asthmatic and normal control subjects. Am J Respir Cell Mol Biol 1995;12: Kaminuma O, Fujimura H, Fushimi K et al. Dynamicsof antigen-specific helper T cells at the initiation of airway eosinophilic inflammation. Eur J Immunol 21;31: Allergology International Vol 63, Suppl 1, 214

7 Anti-Mfsd1 Suppresses Lung Eosinophilia NutkuE,AizawaH,HudsonSA,BochnerBS.Ligationof Siglec-8: a selective mechanism for induction of human eosinophil apoptosis. Blood 23;11: Nutku E, Hudson SA, Bochner BS. Mechanism of Siglec- 8-induced human eosinophil apoptosis: role of caspases and mitochondrial injury. Biochem Biophys Res Commun 25;336: von Gunten S, Vogel M, Schaub A et al. Intravenous immunoglobulin preparations contain anti-siglec-8 autoantibodies. J Allergy Clin Immunol 27;119: Allergology International Vol 63, Suppl 1,