Intracellular Staining by Flow Cytometry
|Detection of IL‑33 in HUVEC Human Cells by Flow Cytometry. HUVEC human umbilical vein endothelial cells were stained with Rat Anti-Human IL‑33 APC‑conjugated Monoclonal Antibody (Catalog # IC3625A, filled histogram) or isotype control antibody (Catalog # IC013A, open histogram). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005). View our protocol for Staining Intracellular Molecules.|
IL-33, also known as NF-HEV and DVS 27, is a proinflammatory protein that may also regulate gene transcription (1-3). DVS 27 was identified as a gene that is upregulated in vasospastic cerebral arteries (1). NF-HEV was described as a nuclear factor that is preferentially expressed in the endothelial cells of high endothelial venules relative to endothelial cells from other tissues (2). IL-33 was identified based on sequence and structural homology with IL-1 family cytokines (3). IL-33 is constitutively expressed in smooth muscle and airway epithelia. It is upregulated in arterial smooth muscle, dermal fibroblasts, and keratinocytes following IL-1 alpha or IL‑1 beta stimulation (1, 3). Similar to IL-1, IL-33 can be cleaved in vitro by Caspase-1, generating an N-terminal fragment that is slightly shorter than the C-terminal fragment (3, 4). The N-terminal portion of full length 32 kDa IL-33 contains a predicted bipartite nuclear localization sequence and a homeodomain-like helix-turn-helix DNA binding domain. By immunofluorescence, full length IL-33 localizes to the nucleus in HUVECs and transfectants (2). The C-terminal 18 kDa fragment, corresponding to mature IL-33, binds and triggers signaling through mast cell IL-1 R4/ST2, a receptor involved in the augmentation of Th2 cell responses (3, 5-7). A ternary signaling complex is formed by the subsequent association of IL-33 and ST2 with IL-1R AcP (8). Stimulation of Th2 polarized lymphocytes with mature IL-33 in vitro induces IL-5 and IL-13 secretion (3). In vivo administration of mature IL-33 promotes increased production of IL-5, IL-13, IgE, and IgA, as well as splenomegaly and inflammatory infiltration of mucosal tissues (3). Full length and mature human IL-33 share 52‑58% aa sequence identity with mouse and rat IL-33. Human IL-33 shares less than 20% aa sequence identity with other IL-1 family proteins.