Intracellular Staining by Flow Cytometry
|Detection of IL‑27 in Human PBMCs by Flow Cytometry. Human peripheral blood mononuclear cells (PBMCs) were stained with Mouse Anti-Human IL‑27 APC‑conjugated Monoclonal Antibody (Catalog # IC25261A, filled histogram) or isotype control antibody (Catalog # IC003A, open histogram). View our protocol for Staining Intracellular Molecules.|
IL-27 is a non-covalent heterodimeric ligand molecule that belongs to the IL-6/IL-12 family of long type I cytokines. It is composed of EBI3 (EBV-induced gene 3), a 34 kDa glycoprotein that is related to the p40 subunit of IL-12 and IL-23, and p28, the 28 kDa glycoprotein that is related to the p35 chain of IL-12. The human EBI3 gene encodes a 229 amino acid (aa) precursor that contains a 20 aa signal peptide and 209 aa mature protein. The mature region contains two potential N-linked glycosylation sites, two fibronectin type III domains, and two pairs of conserved cysteine residues with a WSXWS-like motif that places the molecule in the hematopoietin receptor family. Although p40, the EBI3 counterpart in IL-12, is known to form homodimers, there is no evidence to date that EBI3 also homodimerizes. Human EBI3 is 61% aa identical to mouse EBI3. The human p28 gene encodes a 243 aa precursor that contains a 28 aa signal sequence and 215 aa mature region. The mature region is characterized by the presence of four alpha -helices, placing it in the IL-6 family of helical cytokines. Human p28 is 74% aa identical to mouse p28. IL-27 is expressed by monocytes, endothelial cells and dendritic cells. IL-27 binds to and signals through a heterodimeric receptor complex composed of WSX-1 (TCCR) and gp130. Evidence suggests IL-27 interacts only with WSX-1. IL-27 has both anti- and proinflammatory properties. As an anti‑inflammatory, IL-27 seems to induce a general negative feedback program that limits T and NK-T cell activity. At the onset of infection, IL-27 induces an IL‑12 receptor on naïve CD4+ T cells, making them susceptible to subsequent IL-12 activity that generates Th1 cells at the expense of Th2 and Th17 cells. Finally, IL-27 upregulates both MHC-II and chemokine (CXCL9; CXCL10) expression on vascular endothelium, suggesting a role for IL-27 in vascular inflammation.