FIGURE 1.

IL-33 simultaneously occupies multiple sub-cellular domains. A, IL-33 displays nuclear and cytoplasmic localization in wild type primary cells. The subcellular localization of IL-33 was interrogated by immunofluorescence epifluorescent microscopy utilizing two different anti-IL-33 antibodies. Rat neonatal cardiac fibroblasts (RNCF), human skin fibroblasts (HSF), and human coronary endothelial cells (HCEC) in culture were probed with either a commercially available anti-IL-33 antibody (upper panels) or our previously characterized anti-IL-33 antibody (lower panels). IL-33 signal is pseudocolored green, with anti-tubulin staining (denoting the cytoplasmic space) pseudocolored red. Note our anti-IL-33 antibody does not react with rat IL-33 under immunofluorescence protocols. Scale bars represent 10 μm. B, small epitope tags allow proper modeling of wild type IL-33 localization. In contrast to fluoroprotein-tagged IL-33, an HA tag at either the amino (HA-IL-33) or C (IL-33-HA) terminus permitted nuclear (arrowheads) and cytoplasmic (arrows) localization of stably expressed IL-33 in fibroblasts as seen by confocal microscopy in a pattern mimicking the wild type condition. Scale bars represent 10 μm. C, IL-33 occupies nuclear and cytoplasmic positions in vivo. Lentivirus harboring HA-IL-33 as well as a non-fusion GFP reporter was injected into the left ventricle (upper panels) or instilled into the trachea (lower panels) of anesthetized mice. Upon genomic integration and protein expression, cardiac myocytes revealed strong cytoplasmic HA-IL-33 staining (probed via an anti-HA antibody) in infected cells demarked by GFP expression. Respiratory epithelial cells of terminal bronchioles displayed nuclear and cytoplasmic immunohistochemical staining of HA-IL-33 in cells demarked by GFP expression (arrowheads). Images are representative of 2–3 animals per group and 6–10 microscopic fields per animal. Scale bars represent 10 μm.