Induction of RALDH2 in human basophils by MC-derived and recombinant IL-3. (A,B) IgE-activated MCs induce RALDH2 in basophils. (A) Resting or IgE-activated MCs (± α-IgER; 100 ng/mL anti-FcϵRIα mAb) were cocultured in transwells with basophils for 24 hours. Basophil (Ba) proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie (left panel). The gel was scanned using Luminescent Image Analyzer (LAS3000; Fuji, Valhalla, NY). The integrated intensities (light arbitrary units [LAU]) of the stained protein bands were evaluated by densitometric measurements using AIDA software (Raytest, Straubenhardt, Germany) (right panel). The profile of noninduced proteins is depicted in blue; the peak corresponding to up-regulated RALDH2, in green. MS analysis of the peptides of the 55-kDa region of the gel is shown in Table S1. (B) Resting or activated MCs (± α-IgER) were cocultured in transwells with basophils for 24 hours (Ba from MC cocultures). Ba alone: Basophils were cultured separately without MCs with or without 10 ng/mL IL-3 and/or α-IgER, as indicated. (C) Neutralizing anti–IL-3 antibody abrogates MC-induced RALDH2 expression in basophils. Basophils were stimulated with IL-3 or with MC supernatants for 24 hours in the absence or presence of neutralizing anti–IL-3 antibody (± α-IL-3; 10 μg/mL), as indicated. MC supernatants were derived from resting or IgE receptor–activated (α-IgER) MCs. Untreated basophils (buffer) are shown as control. (B,C) RALDH2 expression in basophil extracts assessed by Western blotting. (D) Immunofluorescence analysis of RALDH2 expression in basophils. Following culture for 20 hours with or without IL-3, basophils were stained with anti-RALDH2 Abs (green, middle panels) and DNA was stained with DAPI (blue, left panels). Images were acquired using constant settings on a Nikon Eclipse E600 fluorescence microscope (Nikon, Tokyo, Japan) equipped with a 60×/1.4 NA oil-immersion objective lens using FITC and DAPI filter sets. A Nikon DXM 1200 digital camera was used to capture images. Merged images (right panels) were created in Adobe Photoshop 8.0.1 (Adobe Systems, San Jose, CA) without any alteration of the original digital images. Priming growth factors (E), chemotactic agonists (F), and immunoregulatory and proinflammatory cytokines (G,H) do not stimulate or modulate RALDH2 expression. In all experiments, basophils were cultured for 24 hours in the absence (buffer) or presence of the stimuli indicated. Stimuli were used as follows: IL-3 and IL-1β (10 ng/mL); IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, and TSLP (20 ng/mL); IL-5, NGF, and GM-CSF (50 ng/mL); IL-10, IL-18, and TNF-α (100 ng/mL); C5a (10 nM); MCP-1 and eotaxin (100 nM); and IFN-α and IFN-γ (1000 U/mL). Data in the different panels were from separate experiments using basophils isolated from different donors. All experiments were repeated at least 3 times showing identical results. However, the weakly immunoreactive bands shown in panel G were not a consistent finding, and we thus can neither confirm nor exclude a marginal RALDH2 induction by some factors.