Cd300f Associates with IL-4 Receptor Α and Amplifies IL-4–Induced Immune Cell Responses
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CD300f associates with IL-4 receptor α and amplifies IL-4–induced immune cell responses Itay Moshkovitsa, Danielle Karo-Atara, Michal Itana, Hadar Reichmana, Perri Rozenberga, Netali Morgenstern-Ben-Barucha, Dana Shika, Aroa Ejarque-Ortizb, Alon Y. Hershkoc, Linjie Tiand, John E. Coligand, Joan Sayósb, and Ariel Munitza,1 aDepartment of Clinical Microbiology and Immunology, The Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel; bImmunobiology Group, Centre d’Investigacions en Bioquímica i BiologiaMolecular en Nanomedicina-Nanomedicine Program, Hospital Universitari Vall d’Hebrón, Institut de Recerca, Universitat Autònoma de Barcelona, Barcelona 08035, Spain; cLaboratory of Allergy and Clinical Immunology, Department of Medicine, The Herbert Center of Mast Cell Disorders, Meir Medical Center, Kfar Saba 44261, Israel; and dReceptor Cell Biology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852 Edited by Warren J. Leonard, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, and approved June 4, 2015 (received for review April 24, 2015) IL-4 receptor (R) α, the common receptor chain for IL-4 and IL-13, is IL-4Rα chain possesses an intrinsic immunoreceptor tyrosine- a critical component in IL-4– and IL-13–mediated signaling and sub- based inhibitory motif (ITIM), which can suppress IL-4 (and sequent effector functions such as those observed in type 2 in- likely IL-13) signaling (8). In addition, stress-induced phospho- flammatory responses. Nonetheless, the existence of intrinsic protein 1 (STIP1) homology and U box-containing protein 1 pathways capable of amplifying IL-4Rα–induced responses remains (STUB1) interacts with IL-4Rα and targets it for degradation, thus unknown. In this study, we identified the myeloid-associated Ig re- terminating IL-4 or IL-13 signaling (9). It is unknown whether an ceptor CD300f as an IL-4–induced molecule in macrophages. Subse- additional receptor system exists that may act to amplify IL-4Rα quent analyses demonstrated that CD300f was colocalized and signaling and subsequent IL-4/IL-13–induced responses. −/− physically associated with IL-4Rα. Using Cd300f cells and recep- CD300 family members consist of nine transmembrane gly- tor cross-linking experiments, we established that CD300f ampli- coprotein receptors, which are expressed by a variety of immune fied IL-4Rα–induced responses by augmenting IL-4/IL-13–induced cells including eosinophils, dendritic cells, macrophages, and B signaling, mediator release, and priming. Consistently, IL-4– and −/− cells (10). The only CD300 family members that possess ITIMs aeroallergen-treated Cd300f mice displayed decreased IgE pro- in their intracellular domains are CD300f and CD300a, and are duction, chemokine expression, and inflammatory cell recruitment. thus potentially capable of suppressing immune cell activation Cd300f−/− Impaired responses in mice were not due to the inability by recruitment of phosphatases (10). Importantly, despite its to generate a proper Th2 response, because IL-4/IL-13 levels were known inhibitory activities (11, 12), CD300f can also exert cel- Cd300f−/− markedly increased in allergen-challenged mice, a finding lular activation and is required for phagocytosis of apoptotic cells that is consistent with decreased cytokine consumption. Finally, via recruitment of p85α of the PI3K signaling pathway (13, 14). CD300f expression was increased in monocytes and eosinophils The finding that the genetic loci (human chromosome 17q22-25) obtained from allergic rhinitis patients. Collectively, our data high- α– of CD300 members are under strong positive evolutionary se- light a previously unidentified role for CD300f in IL-4R induced lection suggests potent immune regulatory roles for these mole- − − immune cell responses. These data provide new insights into the mo- cules (15). Indeed, recent studies using Cd300f / mice revealed lecular mechanisms governing IL-4Rα–induced responses, and may key roles for CD300f in governing the activation of inflam- provide new therapeutic tools to target IL-4 in allergy and asthma. matory myeloid cells, mast cells, and eosinophils (11, 12, 16). However, the overall physiological function of CD300f is still IL-4 receptor | eosinophil | macrophage | CD300f | inflammation largely unknown. nterleukin (IL) 4 and IL-13 play pivotal roles in shaping the Significance Inature of type 2 immune responses. IL-4 is required for in- duction of IgE antibodies by B cells and the subsequent de- + α – – velopment of naïve CD4 T cells into Th2 cells (1). Furthermore, IL-4 receptor (R) is a critical component in IL-4 and IL-13 IL-4 and IL-13 can activate multiple cells of the myeloid lineage, mediated signaling and subsequent effector functions such as those including macrophages, dendritic cells, and eosinophils (2, 3). observed in allergy. Thus, it is a primary therapeutic target in For example, IL-4/IL-13–activated myeloid cells display an al- diseases such as atopic dermatitis and asthma. Despite extensive studies, it is unknown whether an additional receptor system ternatively activated phenotype, which is associated with the in- α duction of a distinct genetic signature, including the expression exists that may act to amplify IL-4R signaling and subsequent IL-4/IL-13–induced responses. We now report that CD300f is of specific mediators and enzymes (4). Furthermore, IL-4 in- α α– duces rapid eosinophil mediator release and priming (5). Thus, physically associated with IL-4R and potently amplifies IL-4R induced responses in vitro and in vivo. Our results establish IL-4 and IL-13 are primary therapeutic targets in Th2 diseases α such as allergy and asthma. CD300f as a previously unidentified IL-4R coreceptor. To the The majority of studies concerning IL-4 and/or IL-13 have best of our knowledge, this is the first report of an additional focused either on defining the cellular source for these cytokines receptor that serves to amplify the IL-4 signaling pathway. or on the respective expression and function of their receptor Author contributions: I.M., D.K.-A., A.E.-O., L.T., J.E.C., J.S., and A.M. designed research; chains. These studies revealed that the biological functions of I.M., D.K.-A., M.I., H.R., P.R., N.M.-B.-B., D.S., A.E.-O., A.Y.H., L.T., J.E.C., J.S., and A.M. IL-4 largely overlap with those of IL-13 due to the utilization of performed research; I.M., D.K.-A., M.I., H.R., P.R., N.M.-B.-B., D.S., A.E.-O., L.T., J.E.C., shared signaling components such as IL-4Rα, IL-13Rα1, and J.S., and A.M. analyzed data; and I.M., J.E.C., J.S., and A.M. wrote the paper. STAT-6 (6). Importantly, signaling elicited by these receptor The authors declare no conflict of interest. chains is regulated by various mechanisms. For example, differ- This article is a PNAS Direct Submission. ential expression of the common γ-chain and IL-13Rα1 chains in 1To whom correspondence should be addressed. Email: [email protected]. distinct cells renders them responsive to IL-4, IL-13, or both (7). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Furthermore, biochemical studies have demonstrated that the 1073/pnas.1507625112/-/DCSupplemental. 8708–8713 | PNAS | July 14, 2015 | vol. 112 | no. 28 www.pnas.org/cgi/doi/10.1073/pnas.1507625112 Downloaded by guest on September 23, 2021 (Fig. 1E and SI Appendix, Fig. S1). Thus, CD300f is an IL-4– induced molecule in macrophages. CD300f Is Physically Associated with IL-4Rα. Next, we examined the spatial distribution of CD300f in macrophages using confocal microscopy. CD300f was strongly codistributed, and associated with IL-4Rα both under baseline conditions and following IL-4 stimulation (Fig. 2A). Quantitation of colocalized pixels of CD300f and IL-4Rα revealed that 78.85 ± 13.49% of detected CD300f was colocalized with IL-4Rα and, vice versa, 51.55 ± 15.62% of IL-4Rα colocalized to the same region as CD300f (Fig. 2B). A similar colocalization pattern was demonstrated in BM-derived dendritic cells that highly express CD300f (SI Ap- pendix, Fig. S3). Although IL-4 activation increased the expres- sion of CD300f in macrophages, it did not increase the level of colocalization, which was observed under baseline conditions (Fig. 2 and SI Appendix, Fig. S3). Notably, and despite our efforts to precipitate CD300f or IL-4Rα from BM-derived macro- phages, we were unable to pull down any of these proteins (even Fig. 1. IL-4 up-regulates the expression of CD300f in macrophages. (A and B) WT and Cd300f−/− bone marrow-derived macrophages (BMMΦs) were alone) with the currently available commercial antibodies. As an stained for CD300f expression at baseline (A) and following cytokine ac- alternative approach, to definitely demonstrate the physical as- tivation (10 ng/mL, 24 h; A and B). (C and D) Time kinetics and dose- sociation between CD300f and IL-4Rα, we cotransfected HEK- dependent effects of IL-4 on CD300f expression. NA, nonactivated. 293T cells with IL-4Rα– (Fig. 2C; transfected cells are marked (E) Expression of CD300f by naïve and IL-4–treated peritoneal macro- as “+”; nontransfected cells are marked as “-”) and c-Myc–tagged + + phages (CD11b /F4/80 cells) is shown. Data represent n = 3; *P < 0.05, CD300f constructs or empty vectors. Subsequently, the cells were < < ± **P 0.01, ***P 0.001; error bars are mean SEM. lysed, c-Myc–precipitated (IP: anti–c-Myc), and blotted with anti–IL-4Rα (IB: anti–IL-4Rα) or anti–c-Myc (IB: anti–c-Myc) INFLAMMATION – as a surrogate tag for CD300f expression. Notably, c-Myc IMMUNOLOGY AND In this study, we demonstrate that CD300f is an IL-4 induced α molecule in macrophages that is physically associated with IL- coprecipitated with IL-4R only in cells that were cotransfected α with CD300f and IL-4Rα (Fig.