Original article Alteration and acquisition of Siglecs during in vitro maturation of CD34+ progenitors into human mast cells Mast cells (MC) are pro-inflammatory, multifunctional immune cells that contribute to a variety of allergic and innate immune responses (1). For example, in allergy, apart from their classical role in eliciting acute responses including anaphylaxis, MC also participate in chronic forms of allergic inflammation by releasing mediators that alter vascular permeability and inflammatory cell recruitment, activation and survival. Studies on human MC have been hampered in part because of the difficulty in obtaining large numbers in high purity. Recently, however, new methodologies for generating human MC have facilitated advancements in our understanding of their biology. Among these is a greater understanding of the MC transcriptome (2, 3) and surface phenotype (4, 5). It is now well-accepted that besides FceRI, these cells also express a broad array of molecules involved in cell adhesion, migration, and signal transduction (6). Sialic acid-binding immunoglobulin-like lectins (Sig- lecs) are a family of transmembrane I-type lectins characterized by the presence of an N-terminal V-set immunoglobulin domain that binds sialic acid (7). A unique characteristic of many Siglecs is the presence of conserved cytoplasmic sequences containing tyrosine motifs [e.g. immunoreceptor tyrosine-based inhibitory motifs (ITIMs)], suggesting that these molecules possess inhibitory functions (7). Therefore, it is possible that depending on which Siglecs MC express, there may be a number of potential opportunities for inhibiting MC function based on activation or engagement of these receptors. We have reported that Siglec-8 is expressed on human MC, as well as eosinophils and basophils (8). There are other previous reports of mature tissue-derived MC expressing CD33 (Siglec-3) (9) and the HMC-1 MC line expressing Siglec-5 (CD170) (10), but there has never been a systematic study of all Siglecs expressed by human MC. This manuscript reports the results of a comprehensive analysis of expression of all known Siglecs using culture- derived human MC, initially at the mRNA level (using gene arrays) and then validating positive results by Using human mast cells (MC) derived by culture of CD34+ peripheral blood precursors, a comprehensive study was performed of expression of 11 known Siglecs. Analysis was initially performed at the mRNA level using gene arrays. Positive results were then validated at the protein level using indirect immuno- fluorescence and flow cytometry, and for some Siglecs, Western blot analysis was also used. Culture-derived MC expressed mRNA for CD22 (Siglec-2), CD33 (Siglec-3), Siglec-5, Siglec-6, Siglec-8 and Siglec-10. Flow cytometry confirmed surface expression of all these molecules except for CD22 and Siglec-10, where levels were low or undetectable. However, Western blotting was able to detect MC expression of CD22 and Siglec-10, suggesting that these proteins were mostly cytoplasmic. CD34+ precursor cells from peripheral blood constitutively expressed surface CD33, Siglec-5 and Siglec-10. As they matured into MC, their constitutive levels of CD33 changed little, Siglec-5 and Siglec-10 declined, and Siglec-6 and Siglec-8 appeared de novo, all in parallel with accumulation of histamine and other MC markers, such as surface expression of FceRIa, and CD51. Phenotypic analysis of LAD-2 MC yielded a similar pattern of Siglec expression except that CD22 expression was particularly prominent. Finally, immunohistochemistry confirmed expression of these same Siglecs by mature tryptase-positive MC in human lung tissues. These data demonstrate an exten- sive and previously unappreciated pattern of Siglec expression on human MC. Whether engagement and signaling through these inhibitory Siglecs can impact MC biology will require further investigation. H. Yokoi 1 , A. Myers 1 , K. Matsumoto 2 , P. R. Crocker 3 , H. Saito 2 , B. S. Bochner 1 1 Department of Medicine, Division of Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2 Department of Allergy and Immunology, National Research Institute for Child Health and Development, Tokyo, Japan; 3 Division of Cell Biology and Immunology, The Wellcome Trust Biocentre, University of Dundee, Dundee, UK Key words: facs analysis; innate immunity; mast cells. Dr Bruce Bochner Johns Hopkins Asthma and Center 5501 Hopkins Bayview Circle Baltimore MD 21224 USA Accepted for publication 12 March 2006 Abbreviations: MC, mast cells; Siglec, sialic acid-binding immuno- globulin-like lectin; G-CSF, granulocyte-colony stimulating factor; IL, interleukin; SCF, stem cell factor; PBS, phosphate-buffered saline; ITIM, immunoreceptor tyrosine-based inhibitory motif. Allergy 2006: 61: 769–776 Copyright Ó Blackwell Munksgaard 2006 ALLERGY DOI: 10.1111/j.1398-9995.2006.01133.x 769