Alteration and acquisition of Siglecs during in vitro maturation of CD34+ progenitors into human mast cells

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 immunofluorescence 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 FcepsilonRIalpha, 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 extensive 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.