Definition of immunoglobulin A receptors on eosinophils and their enhanced expression in allergic individuals. R C Monteiro, … , G L Gartland, H Kubagawa J Clin Invest. 1993;92(4):1681-1685. https://doi.org/10.1172/JCI116754. Research Article Fc alpha receptors (Fc alpha R), detected by the binding of IgA and by anti-Fc alpha R antibodies, were found to be differentially expressed on eosinophils and neutrophils. Neutrophils were the major granulocyte population expressing Fc alpha R, and they expressed much higher levels of Fc alpha R than eosinophils. The expression of Fc alpha R by eosinophils could be upregulated approximately threefold by Ca2+ ionophore treatment in a dose- and time-dependent manner. This effect, which was blocked by a chelating agent, was not duplicated by other cellular stimuli. Eosinophils in allergic individuals displayed enhanced Fc alpha R expression, whereas neutrophils did not. The Fc alpha R on eosinophils had a higher molecular mass (70-100 kD) than those identified on neutrophils (55-75 kD). However, removal of N-linked carbohydrates from Fc alpha R of eosinophils and neutrophils revealed a major protein core of 32 kD for both cell types. The data indicate that expression of Fc alpha R molecules with a characteristic glycosylation pattern is upregulated on eosinophils in allergic individuals. Find the latest version: https://jci.me/116754/pdf Definition of Immunoglobulin A Receptors on Eosinophils and Their Enhanced Expression in Allergic Individuals Renato C. Monteiro, * Robert W. Hostoffer, Max D. Cooper,* James R. Bonner, G. Larry Gartland, and Hiromi Kubagawa Division of Developmental and Clinical Immunology, Departments of Pediatrics, Medicine, Pathology, and Microbiology, and the Comprehensive Cancer Center, University ofAlabama at Birmingham; and the *Howard Hughes Medical Institute, Birmingham, Alabama 35294 Abstract binding to specific Fc7yR and FcER present on the cell surface (3, 4). However, IgA is the most abundant Ig isotype in the Fca receptors (FcaR), detected by the binding of IgA and by secretions (5) where eosinophils carry out many of their effec- anti-FcaR antibodies, were found to be differentially expressed tor functions. It has been shown that IgA can bind to eosino- on eosinophils and neutrophils. Neutrophils were the major phils (6, 7), which suggests they may possess IgA receptor(s), granulocyte population expressing FcaR, and they expressed but FcaR on eosinophils has not been identified and character- much higher levels of FcaR than eosinophils. The expression of ized. FcaR by eosinophils could be upregulated approximately An FcaR on monocyte/macrophages and granulocytes has threefold by Ca2" ionophore treatment in a dose- and time-de- been defined as a variably glycosylated protein of 55-75 kD (8, pendent manner. This effect, which was blocked by a chelating 9) that can bind IgA I and IgA2 antibodies via their Fc regions agent, was not duplicated by other cellular stimuli. Eosinophils (9, 10). The open reading frame of the recently identified in allergic individuals displayed enhanced FcaR expression, FcaR gene encodes a transmembrane protein of approxi- whereas neutrophils did not. The FcaR on eosinophils had a mately 30 kD, which has six potential sites for N-linked glyco- higher molecular mass (70-100 kD) than those identified on sylation in its extracellular region ( 11). The selective expres- neutrophils (55-75 kD). However, removal of N-linked carbo- sion of FcaR by myeloid lineage cells has been confirmed by hydrates from FcaR of eosinophils and neutrophils revealed a analysis of FcaR mRNA expression ( 11 ) and FcaR molecules major protein core of 32 kD for both cell types. The data indi- identified by monoclonal antibodies specific for native and re- cate that expression of FcaR molecules with a characteristic combinant FcaR protein (12-14). glycosylation pattern is upregulated on eosinophils in allergic In this report, we have used the natural IgA ligand and individuals. (J. Clin. Invest. 1993. 92:1681-1685.) Key words: anti-FcaR mAbs to analyze the expression, regulation, and bio- Fca receptor * IgA receptor * Fc receptor - eosinophil - allergy chemical nature of FcaR on eosinophils. FcaR molecules were detected on eosinophils from all normal individuals following in vitro activation, whereas fresh eosinophils from allergic indi- Introduction viduals frequently expressed FcaR. The FcaR molecules ex- pressed by eosinophils differ from those on neutrophils and Eosinophils may play important roles in protection against par- macrophages in that the former have a higher content of N- asitic diseases and in mediating inflammation in allergic indi- linked carbohydrate moieties. viduals ( 1 ). These functions are linked to the eosinophil's abil- ity to release biologically active factors following antigen acti- Methods vation. Several granule-derived proteins are released in the ensuing degranulation process. These include highly cytotoxic Subjects. Heparinized blood samples were obtained from 45 adult indi- factors, such as eosinophil cationic protein, major basic pro- viduals (27 male and 18 female). 22 had severe symptoms of allergic tein, and eosinophil-derived neurotoxin ( 1). rhinitis and/or asthma. These allergic individuals were further selected One degranulation mode is initiated via Fc receptors on the basis of acute wheal and flare reactions in response to two or more allergens when tested by the prick method with extracts of house (FcR)' in an antibody-dependent cell cytotoxic manner (2). dust mite, grass pollen, ragweed pollen, tree pollen, and animal danders Both IgG and IgE antibodies promote this reaction cascade by ( 15). The other 23 individuals had no history of allergy and were skin- test negative. None of the allergic subjects had received systemic corti- costeroids or other medication at the time of blood collection. Address reprint requests to Dr. Hiromi Kubagawa, 378 Tumor Insti- Reagents. The following mouse antibodies were used: A3 (-YIK), tute, University of Alabama at Birmingham, Birmingham, AL 35294. A59 (' Il K), A62 (l1K), and A77 (y IK) mAbs specific for the FcaR Dr. Monteiro's current address is INSERM U25, H6pital Necker, 161, ( 13), the 32.2 (-Y I K) mAb specific for the FcyR I (CD64) (Medarex, rue des Sevres, 75743 Paris Cedex 15, France. Dr. Hostoffer's current Inc., W. Lebanon, NH), the IV.3 ('y2b) mAb specific for the FcyRII address is Rainbow Babies & Children's Hospital, 2074 Abington (CD32w) (ATCC), the 3G8 ('YIK) mAb specific for the Fc-yR III Road, Cleveland, OH 44106. (CD16) (16), the W8E7 (-y2aK) anti-CD1O mAb, the LeuM1 (,UKK) Received /br publication 25 February 1993 and in revised fiorm II anti-CD 15 mAb, and the My4 (y 1K) anti-CD 14 mAb (Coulter Corp., May 1993. Hialeah, FL). Control antibodies included an irrelevant mouse IgG2a (Becton Dickinson & Co., Mountain View, CA), JH3 (-YlK) anti-Id 1. Abbreviation used in this paper: FcR, Fc receptors. ( 17), the Cla(MK) anti-chicken Ia mAb ( 18), and the MOPC 141 ( y2b) myeloma. FITC-labeled goat anti-mouse Ig antibodies lacking cross- J. Clin. Invest. reactivity with human Ig were from Southern Biotechnology Asso- © The American Society for Clinical Investigation, Inc. ciates (Birmingham, AL). The human IgA myeloma proteins and 0021-9738/93/10/1681/05 $2.00 F( ab')2 fragments of the corresponding goat anti-Id antibodies are de- Volume 92, October 1993, 168 1-1685 scribed elsewhere ( 19). Eosinophil Fc Receptor 1681 Isolation ofeosinophils and neutrophils. Red cells and granulocytes scatter and side light scatter characteristics determined by flow were first separated from mononuclear cells by centrifugation over Fi- cytometric analysis (21, 22). A forward-side scatter population coll/Hypaque (Pharmacia Fine Chemicals, Piscataway, NJ) density distinct from neutrophils was first observed when eosinophils gradients. Granulocytes were isolated from the red cell pellet by differ- were partially purified on metrizamide gradients. Sorting for ential sedimentation in 1.5% dextran in PBS (9). All reagents used for this population resulted in an enrichment in eosinophils to granulocyte isolation were prepared in pyrogen-free distilled water. > 98% purity as determined by microscopic evaluation of were using Neutrophils and eosinophils of normal density separated by stained cells. These forward-side scatter characteristics were a discontinuous metrizamide gradient (20). Purity of eosinophils var- ied from 60-90%, whereas neutrophils were > 99% pure as determined then used to establish analytical gates for immunofluorescence by morphological characteristics following Giemsa staining. In some analysis of eosinophils and neutrophils. The eosinophils were experiments, the eosinophils were further enriched (> 99.5%) by fluo- also distinguishable from neutrophils by their lack of or rela- rescence-activated cell sorting, where eosinophils were selected on the tively weak expression of cell surface CD10, CD 14, CD 15, basis of their light scatter characteristics and nonreactivity with anti- CD 16, and CD64 antigens, whereas both cell types expressed CD16 mAb (21, 22). Eosinophils were cultured for 1-18 h in RPMI CDw32 and CD1 lb antigens. 1640 supplemented with 25% of autologous human serum (and peni- To evaluate FcaR expression on blood granulocyte subpo- cillin and streptomycin) in the presence or absence of various concen- pulations, purified eosinophils and the purified neutrophils trations of Ca2" ionophore (Ionomycin; Calbiochem, San Diego, CA). were examined for binding