Identification of an Interleukin 17F/17A Heterodimer in Activated Human CD4+ T Cells

Abstract

IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfidelinked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins, as well as that of natural IL-17F and IL-17A derived from activated human CD4؉ T cells, by enzyme-linked immunosorbent assay, immunoprecipitation followed by Western blotting, and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system, and that all forms of the recombinant proteins have in vitro functional activity. Furthermore, we find that in addition to the homodimers of IL-17F and IL-17A, activated human CD4؉ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses.Interleukins 17F 3 and 17A (IL-17A) are closely related members of the IL-17 cytokine family, and share 50% amino acid identity. Studies in the mouse have identified Th17 cells as a distinct CD4ϩ T cell lineage that is defined by the production of IL-17F and IL-17A (1-7). IL-6 and transforming growth factor-␤ (TGF-␤) are required for the differentiation of naïve CD4ϩ T cells to Th17 cells (1,8), which are maintained in the presence of IL-23 and IL-1␤. Conversely, IL-4 and interferon-␥ can inhibit the development of Th17 cells (9, 10). Th17 cells have been implicated in the pathology of mouse autoimmune disease models (2).Expression of IL-17F and IL-17A has been detected in activated human peripheral blood lymphocytes. It has been shown by reverse transcriptase-PCR experiments that the cytokines are expressed in activated human CD4ϩ T cells (11,12). Expression of IL-17F and IL-17A has also been observed in tissue samples from various autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, and asthma (2, 3, 13-22).The crystal structure of IL-17F has been solved and shows that the protein forms a disulfide-linked dimeric glycoprotein (23). IL-17A is also a disulfide-linked homodimeric glycoprotein (24), although crystal structure or data defining the precise subunit interactions are lacking. The IL-17F homodimer includes a classical cysteine knot motif, which is found in the TGF-␤, bone morphogenetic protein, and nerve growth factor superfamilies (25, 26). One difference in the cysteine knot motif of IL-17F compared with the other known cysteine knot protein families is that it only utilizes four cysteines instead of the classical six cysteines to form the knot.There have been reports that some of the cysteine knot family members can exist as heterodimers in vivo. TGF-␤1.2 and -␤2.3 were identified in bovine bone extracts, whereas inhibin and activin AB have been found in gonadal fluids (27...

An IL-17F/A Heterodimer Protein Is Produced by Mouse Th17 Cells and Induces Airway Neutrophil Recruitment

“…Although several studies have examined the effects of mouse IL-17A/A, most comparisons of IL-17A/A and IL-17F/F activity have been reported using human cytokines. In these studies, human IL-17A/A was either more potent or equally as potent as IL-17F/F in inducing certain proinflammatory mediators from epithelial cells in vitro (10,11,23). Because IL-17A/A and IL-17F/F are glycosylated proteins, a recombinant protein may have different activity depending on whether a prokaryotic or eukaryotic cell produced it.…”

“…Recently, human and mouse IL-17A and IL-17F have been reported to exist as a heterodimer (23,24). The regulation of IL-17F/A expression by Th17 cells and its expression relative to IL-17A/A and IL-17F/F have not been reported.…”

“…(Hereafter, we use IL-17A/A and IL-17F/F to specifically refer to the homodimeric proteins, IL-17F/A to refer to the heterodimeric protein, and IL-17A or IL-17F to refer to all proteins containing the given molecule.) Mass spectrometry analysis of natural IL-17F/A heterodimer produced by primary human CD4 ϩ T cells has shown the existence of interchain disulfide-linked peptides containing a peptide from IL-17F and one from IL-17A (23). More recently, mouse IL-17F/A has been detected in conditioned medium from Th17 cells (24).…”

“…The coexpression of IL-17A and IL-17F by Th17 cells further supports this possibility. The existence of human IL-17F/A heterodimer has recently been demonstrated using biochemical and physiochemical methods (23). (Hereafter, we use IL-17A/A and IL-17F/F to specifically refer to the homodimeric proteins, IL-17F/A to refer to the heterodimeric protein, and IL-17A or IL-17F to refer to all proteins containing the given molecule.)…”

Inflammatory cytokines in pediatric obstructive sleep apnea

“…[20,21] The interaction is as follows: IL-17A and IL-17F form homodimers or heretodimers that bind to the IL-17 receptor complex on inflammation-related cells such as macrophages, epithelial cells, and endothelial cells. [22,23] The activated inflammatory cells produce various cytokines including IL-1, IL-6, and TNF-a. The stimulation of these cytokines and inflammatory cells leads to inflammatory responses such as neutrophil recruitment, tissue destruction, and neovascularization.…”