Conservation of Functional Sites on Interleukin-6 and Implications for Evolution of Signaling Complex Assembly and Therapeutic Intervention

A number of secreted cytokines, such as interleukin-6 (IL-6), are attractive targets for the treatment of inflammatory diseases. We have determined the solution structure of mouse IL-6 to assess the functional significance of apparent differences in the receptor interaction sites (IL-6Rα and gp130) suggested by the fairly low degree of sequence similarity with human IL-6. Structure-based sequence alignment of mouse IL-6 and human IL-6 revealed surprising differences in the conservation of the two distinct gp130 binding sites (IIa and IIIa), which suggests a primacy for site III-mediated interactions in driving initial assembly of the IL-6/IL-6Rα/gp130 ternary complex. This is further supported by a series of direct binding experiments, which clearly demonstrate a high affinity IL-6/IL-6Rα-gp130 interaction via site III but only weak binding via site II. Collectively, our findings suggest a pathway for the evolution of the hexameric, IL-6/IL-6Rα/gp130 signaling complex and strategies for therapeutic targeting. We propose that the signaling complex originally involved specific interactions between IL-6 and IL-6Rα (site I) and between the D1 domain of gp130 and IL-6/IL-6Rα (site III), with the later inclusion of interactions between the D2 and D3 domains of gp130 and IL-6/IL-6Rα (site II) through serendipity. It seems likely that IL-6 signaling benefited from the evolution of a multipurpose, nonspecific protein interaction surface on gp130, now known as the cytokine binding homology region (site II contact surface), which fortuitously contributes to stabilization of the IL-6/IL-6Rα/gp130 signaling complex.     

The Amino Acid Exchange R28E in Ciliary Neurotrophic Factor (CNTF) Abrogates Interleukin-6 Receptor-dependent but Retains CNTF Receptor-dependent Signaling via Glycoprotein 130 (gp130)/Leukemia Inhibitory Factor Receptor (LIFR)

Ciliary neurotrophic factor (CNTF) is a neurotrophic factor with therapeutic potential for neurodegenerative diseases. Moreover, therapeutic application of CNTF reduced body weight in mice and humans. CNTF binds to high or low affinity receptor complexes consisting of CNTFR·gp130·LIFR or IL-6R·gp130·LIFR, respectively. Clinical studies of the CNTF derivative Axokine revealed intolerance at higher concentrations, which may rely on the low-affinity binding of CNTF to the IL-6R. Here, we aimed to generate a CNTFR-selective CNTF variant (CV). CV-1 contained the single amino acid exchange R28E. Arg²⁸ is in close proximity to the CNTFR binding site. Using molecular modeling, we hypothesized that Arg²⁸ might contribute to IL-6R/CNTFR plasticity of CNTF. CV-2 to CV-5 were generated by transferring parts of the CNTFR-binding site from cardiotrophin-like cytokine to CNTF. Cardiotrophin-like cytokine selectively signals via the CNTFR·gp130·LIFR complex, albeit with a much lower affinity compared with CNTF. As shown by immunoprecipitation, all CNTF variants retained the ability to bind to CNTFR. CV-1, CV-2, and CV-5, however, lost the ability to bind to IL-6R. Although all variants induced cytokine-dependent cellular proliferation and STAT3 phosphorylation via CNTFR·gp130·LIFR, only CV-3 induced STAT3 phosphorylation via IL-6R·gp130·LIFR. Quantification of CNTF-dependent proliferation of CNTFR·gp130·LIFR expressing cells indicated that only CV-1 was as biologically active as CNTF. Thus, the CNTFR-selective CV-1 will allow discriminating between CNTFR- and IL-6R-mediated effects in vivo.     

Recombinant p35 from Bacteria Can Form Interleukin (IL-)12, but Not IL-35

The Interleukin (IL)-12 family contains several heterodimeric composite cytokines which share subunits among each other. IL-12 consists of the subunits p40 (shared with IL-23) and p35. p35 is shared with the composite cytokine IL-35 which comprises of the p35/EBI3 heterodimer (EBI3 shared with IL-27). IL-35 signals via homo- or heterodimers of IL-12Rβ2, gp130 and WSX-1, which are shared with IL-12 and IL-27 receptor complexes, respectively. p35 was efficiently secreted in complex with p40 as IL-12 but not with EBI3 as IL-35 in several transfected cell lines tested which complicates the analysis of IL-35 signal transduction. p35 and p40 but not p35 and EBI3 form an inter-chain disulfide bridge. Mutation of the responsible cysteine residue (p40_C197A) reduced IL-12 formation and activity only slightly. Importantly, the p40_C197A mutation prevented the formation of antagonistic p40 homodimers which enabled the in vitro reconstitution of biologically active IL-12 with p35 produced in bacteria (p35_bac). Reconstitution of IL-35 with p35_bac and EBI3 did, however, fail to induce signal transduction in Ba/F3 cells expressing IL-12Rβ2 and gp130. In summary, we describe the in vitro reconstitution of IL-12, but fail to produce recombinant IL-35 by this novel approach.     

Characterization and Favorable in Vivo Properties of Heterodimeric Soluble IL-15·IL-15Rα Cytokine Compared to IL-15 Monomer

Interleukin-15 (IL-15), a 114-amino acid cytokine related to IL-2, regulates immune homeostasis and the fate of many lymphocyte subsets. We reported that, in the blood of mice and humans, IL-15 is present as a heterodimer associated with soluble IL-15 receptor α (sIL-15Rα). Here, we show efficient production of this noncovalently linked but stable heterodimer in clonal human HEK293 cells and release of the processed IL-15·sIL-15Rα heterodimer in the medium. Purification of the IL-15 and sIL-15Rα polypeptides allowed identification of the proteolytic cleavage site of IL-15Rα and characterization of multiple glycosylation sites. Administration of the IL-15·sIL-15Rα heterodimer reconstituted from purified subunits resulted in sustained plasma IL-15 levels and in robust expansion of NK and T cells in mice, demonstrating pharmacokinetics and in vivo bioactivity superior to single chain IL-15. These identified properties of heterodimeric IL-15 provide a strong rationale for the evaluation of this molecule for clinical applications.     

Humanin Inhibits Neuronal Cell Death by Interacting with a Cytokine Receptor Complex or Complexes Involving CNTF Receptor α/WSX-1/gp130

Humanin (HN) inhibits neuronal death induced by various Alzheimer''s disease (AD)-related insults via an unknown receptor on cell membranes. Our earlier study indicated that the activation of STAT3 was essential for HN-induced neuroprotection, suggesting that the HN receptor may belong to the cytokine receptor family. In this study, a series of loss-of-function tests indicated that gp130, the common subunit of receptors belonging to the IL-6 receptor family, was essential for HN-induced neuroprotection. Overexpression of ciliary neurotrophic factor receptor α (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it. These results suggest that both CNTFR and WSX-1 may be also involved in HN binding to cells. Consistent with these results, loss-of-functions of CNTFR or WSX-1 in neuronal cells nullified their responsiveness to HN-mediated protection. In vitro–reconstituted binding assays showed that HN, but not the other control peptide, induced the hetero-oligomerization of CNTFR, WSX-1, and gp130. Together, these results indicate that HN protects neurons by binding to a complex or complexes involving CNTFR/WSX-1/gp130.     

Novel IL27p28/IL12p40 Cytokine Suppressed Experimental Autoimmune Uveitis by Inhibiting Autoreactive Th1/Th17 Cells and Promoting Expansion of Regulatory T Cells

IL-12 family cytokines are important in host immunity. Whereas some members (IL-12, IL-23) play crucial roles in pathogenesis of organ-specific autoimmune diseases by inducing the differentiation of Th1 and Th17 lymphocytes, others (IL-27 and IL-35) suppress inflammatory responses and limit tissue injury induced by these T cell subsets. In this study, we have genetically engineered a novel IL27p28/IL12p40 heterodimeric cytokine (p28/p40) that antagonizes signaling downstream of the gp130 receptor. We investigated whether p28/p40 can be used to ameliorate uveitis, a CNS inflammatory disease. Experimental autoimmune uveitis (EAU) is the mouse model of human uveitis and is mediated by Th1 and Th17 cells. We show here that p28/p40 suppressed EAU by inhibiting the differentiation and inflammatory responses of Th1 and Th17 cells while promoting expansion of IL-10⁺- and Foxp3⁺-expressing regulatory T cells. Lymph node cells from mice treated with p28/p40 blocked adoptive transfer of EAU to naïve syngeneic mice by immunopathogenic T cells and suppressive effects of p28/p40 derived in part from antagonizing STAT1 and STAT3 pathways induced by IL-27 and IL-6. Interestingly, IL27p28 also suppressed EAU, but to a lesser extent than p28/p40. The inhibition of uveitogenic lymphocyte proliferation and suppression of EAU by p28/p40 and IL27p28 establish efficacy of single chain and heterodimeric IL-12 family cytokines in treatment of a CNS autoimmune disease. Creation of the biologically active p28/p40 heterodimeric cytokine represents an important proof-of-concept experiment, suggesting that cytokines comprising unique IL-12 α- and β-subunit pairing may exist in nature and may constitute a new class of therapeutic cytokines.     

Prerequisites for Functional Interleukin 31 Signaling and Its Feedback Regulation by Suppressor of Cytokine Signaling 3 (SOCS3)

Interleukin-31 (IL-31) is a T helper type 2 cell-derived cytokine tightly associated with inflammatory skin disorders. IL-31-induced signaling is mediated by a receptor complex composed of oncostatin M receptor β and the cytokine-specific receptor subunit IL-31Rα, of which there are several isoforms. The latter can be classified as long or short isoforms with respect to their intracellular domain. At present, the signaling capabilities of the different isoforms remain inchoately understood, and potential mechanisms involved in negative regulation of IL-31Rα signaling have so far not been studied in detail. Here, we show that both the long and short isoforms of IL-31Rα are capable of inducing STAT signaling. However, the presence of a functional JAK-binding box within IL-31Rα is an essential prerequisite for functional IL-31-mediated STAT3 signaling. Moreover, both the long and short isoforms require oncostatin M receptor β for their activity. We also show that IL-31 induces expression of four suppressor of cytokine signaling family members and provide evidence that SOCS3 acts as a potent feedback inhibitor of IL-31-induced signaling. Taken together, this study identifies crucial requirements for IL-31 signaling and shows its counter-regulation by SOCS3.     

Paracrine and Transpresentation Functions of IL-15 Are Mediated by Diverse Splice Versions of IL-15Rα in Human Monocytes and Dendritic Cells

Species-specific differences of post-translational modifications suggested the existence of human IL-15Rα isoforms. We identified eight new isoforms that are predicted to modify the intracellular C termini of IL-15Rα, and another N-terminal exon “Ex2A” that was consistently present in all but one of the C-terminal isoforms. Ex2A encodes a 49-amino acid domain that allowed the transfer of IL-15/IL-15Rα complex to the cell surface but prevented its cleavage from cell membranes and its secretion thus facilitating the transpresentation of IL-15 as part of the immunological synapse. The Ex2A domain also affected the O-glycosylation of IL-15Rα that explained the species-specific differences. The Ex2A domain appeared to be removed from major IL-15Rα species during protein maturation, but both Ex2A and IL-15Rα appeared on the surface of monocytic cells upon activation. The membrane-associated form of the only C-terminal isoform that lacked Ex2A (IC3) was retained inside the cell, but soluble IL-15/IL-15Rα complexes were readily released from cells that expressed IL-15/IL-15Rα-IC3 thus limiting this IL-15/IL-15Rα isoform to act as a secreted molecule. These data suggest that splice versions of IL-15Rα determine the range of IL-15 activities.     

Critical and Independent Role for SOCS3 in Either Myeloid or T Cells in Resistance to Mycobacterium tuberculosis

Suppressor of cytokine signalling 3 (SOCS3) negatively regulates STAT3 activation in response to several cytokines such as those in the gp130-containing IL-6 receptor family. Thus, SOCS3 may play a major role in immune responses to pathogens. In the present study, the role of SOCS3 in M. tuberculosis infection was examined. All Socs3^fl/fl LysM cre, Socs3^fl/fl lck cre (with SOCS3-deficient myeloid and lymphoid cells, respectively) and gp130^F/F mice, with a mutation in gp130 that impedes binding to SOCS3, showed increased susceptibility to infection with M. tuberculosis. SOCS3 binding to gp130 in myeloid cells conveyed resistance to M. tuberculosis infection via the regulation of IL-6/STAT3 signalling. SOCS3 was redundant for mycobacterial control by macrophages in vitro. Instead, SOCS3 expression in infected macrophages and DCs prevented the IL-6-mediated inhibition of TNF and IL-12 secretion and contributed to a timely CD4+ cell-dependent IFN-γ expression in vivo. In T cells, SOCS3 expression was essential for a gp130-independent control of infection with M. tuberculosis, but was neither required for the control of infection with attenuated M. bovis BCG nor for M. tuberculosis in BCG-vaccinated mice. Socs3^fl/fl lck cre mice showed an increased frequency of γδ+ T cells in different organs and an enhanced secretion of IL-17 by γδ+ T cells in response to infection. Socs3^fl/fl lck cre γδ+ T cells impaired the control of infection with M. tuberculosis. Thus, SOCS3 expression in either lymphoid or myeloid cells is essential for resistance against M. tuberculosis via discrete mechanisms.     

How a cytokine is chaperoned through the secretory pathway by complexing with its own receptor: lessons from interleukin-15 (IL-15)/IL-15 receptor alpha

While it is well appreciated that receptors for secreted cytokines transmit ligand-induced signals, little is known about additional roles for cytokine receptor components in the control of ligand transport and secretion. Here, we show that interleukin-15 (IL-15) translocation into the endoplasmic reticulum occurs independently of the presence of IL-15 receptor α (IL-15Rα). Subsequently, however, IL-15 is transported through the Golgi apparatus only in association with IL-15Rα and then is secreted. This intracellular IL-15/IL-15Rα complex already is formed in the endoplasmic reticulum and, thus, enables the further trafficking of complexed IL-15 through the secretory pathway. Just transfecting IL-15Rα in cells, which transcribe but normally do not secrete IL-15, suffices to induce IL-15 secretion. Thus, we provide the first evidence of how a cytokine is chaperoned through the secretory pathway by complexing with its own high-affinity receptor and show that IL-15/IL-15Rα offers an excellent model system for the further exploration of this novel mechanism for the control of cytokine secretion.     

Prognostic Relevance of Cytokine Receptor Expression in Acute Myeloid Leukemia: Interleukin-2 Receptor α-Chain (CD25) Expression Predicts a Poor Prognosis

A variety of cytokine/cytokine receptor systems affect the biological behavior of acute leukemia cells. However, little is known about the clinical relevance of cytokine receptor expression in acute myeloid leukemia (AML). We quantitatively examined the expression of interleukin-2 receptor α-chain (IL-2Rα, also known as CD25), IL-2Rβ, IL-3Rα, IL-4Rα, IL-5Rα, IL-6Rα, IL-7Rα, the common β-chain (βc), γc, granulocyte-macrophage colony-stimulating factor (GM-CSF)Rα, G-CSFR, c-fms, c-mpl, c-kit, FLT3, and GP130 in leukemia cells from 767 adult patients with AML by flow cytometry and determined their prevalence and clinical significance. All cytokine receptors examined were expressed at varying levels, whereas the levels of IL-3Rα, GM-CSFRα, IL-2Rα, γc, c-kit, and G-CSFR exhibited a wide spectrum of ≥10,000 sites/cell. In terms of their French-American-British classification types, GM-CSFRα and c-fms were preferentially expressed in M4/M5 patients, G-CSF in M3 patients, and IL-2Rα in non-M3 patients. Elevated levels of IL-3Rα, GM-CSFRα, and IL-2Rα correlated with leukocytosis. In patients ≤60 years old, higher levels of these 3 receptors correlated with poor responses to conventional chemotherapy, but only IL-2Rα was associated with a shorter overall survival. By incorporating IL-2Rα status into cytogenetic risk stratification, we could sort out a significantly adverse-risk cohort from the cytogenetically intermediate-risk group. Analyses with various phenotypical risk markers revealed the expression of IL-2Rα as an independent prognostic indicator in patients with intermediate-risk cytogenetics. These findings were not observed in patients >60 years old. Our results indicate that several cytokine receptors were associated with certain cellular and clinical features, but IL-2Rα alone had prognostic value that provides an additional marker to improve current risk evaluation in AML patients ≤60 years old.     

In vitro reconstitution of recognition and activation complexes between interleukin-6 and gp130.

Gp130 is a shared signal-transducing receptor for a family of four-helix cytokines, of which interleukin-6 is a prototypic member. IL-6-type cytokines activate gp130 to elicit downstream intracellular JAK/STAT signaling cascades through formation of hetero-oligomeric receptor complexes. Interleukin-6 must first complex with its specific alpha-receptor (Ralpha) in order to bind and activate gp130. We have dissected the extracellular activation pathway of human gp130 by human IL-6 through reconstitution of soluble complexes representing intermediate and final states in the hierarchical assembly of the IL-6/IL-6Ralpha/gp130 signaling complex. To isolate these hetero-complexes, we have applied a protein engineering strategy of covalently linking IL-6 to its Ralpha, which results in a "hyperactive" single-chain complex (hyper-IL-6) which we express in both Escherichia coli and insect cells. We have determined that IL-6/IL-Ralpha and the cytokine-binding homology region (CHR) of gp130 (D2D3) form a stable trimolecular "recognition" complex (trimer) consisting of 1IL-6,1 IL-6Ralpha, and 1 gp130-CHR. Addition of the N-terminal (D1) Ig-like domain (IGD) of gp130 to the CHR results in a transition to a hexameric "activation" complex containing 2 IL-6, 2IL-6Ralpha, and 2 gp130. These results clearly demonstrate that the recognition and activation complexes are disparate hetero-oligomeric molecular species linked by the recruitment of the gp130 IGD by the unique site III epitope present on all gp130-class cytokines. The results of these studies are relevant to other members of the IL-6 family of gp130-cytokines and address a longstanding question concerning the respective roles of the gp130 CHR and IGD in assembly of the active signaling oligomer.