Lack of nuclear translocation of cytoplasmic domains of IL-2/IL-15 receptor subunits

Some sensors of extracellular signaling molecules such as Notch and sterol response element binding protein (SREBP) receive ligand-induced intra-membrane proteolysis followed by nuclear translocation of their cytoplasmic domains to regulate gene expression programs in the nucleus. It has not been extensively examined whether ligand-induced intra-membrane proteolysis of type I cytokine receptors and nuclear translocation of cytoplasmic domains occur. Here, by using a sensitive reporter system, we examined this possibility for the interleukin-2 (IL-2) receptor (IL-2R) β-chain (IL-2Rβ) and the IL-15 receptor (IL-15R) α-chain (IL-15Rα). Flowcytometric analysis revealed that ligand stimulation does not induce nuclear translocation of their cytoplasmic domains. In addition, overexpression of the cytoplasmic domain of the common cytokine receptor γ-chain (γc) in an IL-2R-reconstituted Ba/F3-derived cell line did not affect any biological responses including cell survival, disproving potential roles of the cleaved cytoplasmic domain of γc as a signal transducer. Collectively, these results indicated that potential nuclear function of cleaved type I cytokine receptor subunits is not plausible.     

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.     

Selective Expansion of Chimeric Antigen Receptor-targeted T-cells with Potent Effector Function using Interleukin-4

Polyclonal T-cells can be directed against cancer using transmembrane fusion molecules known as chimeric antigen receptors (CARs). Although preclinical studies have provided encouragement, pioneering clinical trials using CAR-based immunotherapy have been disappointing. Key obstacles are the need for robust expansion ex vivo followed by sustained survival of infused T-cells in patients. To address this, we have developed a system to achieve selective proliferation of CAR⁺ T-cells using IL-4, a cytokine with several pathophysiologic and therapeutic links to cancer. A chimeric cytokine receptor (4αβ) was engineered by fusion of the IL-4 receptor α (IL-4Rα) ectodomain to the β_c subunit, used by IL-2 and IL-15. Addition of IL-4 to T-cells that express 4αβ resulted in STAT3/STAT5/ERK phosphorylation and exponential proliferation, mimicking the actions of IL-2. Using receptor-selective IL-4 muteins, partnering of 4αβ with γ_c was implicated in signal delivery. Next, human T-cells were engineered to co-express 4αβ with a CAR specific for tumor-associated MUC1. These T-cells exhibited an unprecedented capacity to elicit repeated destruction of MUC1-expressing tumor cultures and expanded through several logs in vitro. Despite prolonged culture in IL-4, T-cells retained specificity for target antigen, type 1 polarity, and cytokine dependence. Similar findings were observed using CARs directed against two additional tumor-associated targets, demonstrating generality of application. Furthermore, this system allows rapid ex vivo expansion and enrichment of engineered T-cells from small blood volumes, under GMP-compliant conditions. Together, these findings provide proof of principle for the development of IL-4-enhanced T-cell immunotherapy of cancer.     

Molecular Dissection of Human Interleukin-31-mediated Signal Transduction through Site-directed Mutagenesis

Interleukin (IL)-31 is a recently described cytokine, preferentially produced by T helper 2 lymphocytes and associated with skin diseases, such as atopic dermatitis. IL-31 is a member of the four α-helix bundle cytokine family and is related to the IL-6 subgroup. Its heterodimeric membrane receptor is composed of the gp130-like receptor (GPL) subunit associated to the oncostatin M receptor subunit. We identified critical amino acids implicated in the ligand receptor interaction by computational analysis combined with site-directed mutagenesis. Six IL-31 residues selected for their putative involvement in cytokine receptor contact sites were alanine-substituted, and the corresponding proteins were expressed in mammalian and bacterial systems. Biochemical, membrane binding, cell signaling, and cell proliferation analyses showed that mutation E44A, E106A, or H110A abolished IL-31 binding to GPL and the subsequent signaling events. A second ligand receptor-binding site involved Lys¹³⁴, with alanine substitution leading to a protein that still binds GPL, but is unable to recruit the second receptor subunit and the subsequent signaling pathways. The results indicate that IL-31 recognizes its receptor complex through two different binding sites, and we propose a three-dimensional model for IL-31.     

Interleukin-21 Is a Critical Regulator of CD4 and CD8 T Cell Survival during Priming under Interleukin-2 Deprivation Conditions

Optimal T cell activation and expansion require binding of the common gamma-chain (γc) cytokine Interleukin-2 (IL-2) to its cognate receptor that in turn engages a γc/Janus tyrosine kinase (Jak)3 signaling pathway. Because of its restricted expression by antigen-activated T cells and its obligatory role in promoting their survival and proliferation, IL-2 has been considered as a selective therapeutic target for preventing T cell mediated diseases. However, in order to further explore IL-2 targeted therapy, it is critical to precisely understand its role during early events of T cell activation. In this study, we delineate the role of IL-2 and other γc cytokines in promoting the survival of CD4 and CD8 T cells during early phases of priming. Under IL-2 inhibitory conditions (by neutralizing anti-IL-2 mAbs), the survival of activated CD8⁺ T cells was reduced, whereas CD4⁺ T cells remained much more resistant. These results correlated with reduced Bcl-2 expression, and mitochondrial membrane potential in CD8⁺ T cells in comparison to CD4⁺ T cells. However, using transwell co-culture assays we have found that CD4⁺ T cells could rescue the survival of CD8⁺ T cells even under IL-2 deprived conditions via secretion of soluble factors. A cytokine screen performed on CD8⁺ T cells cultured alone revealed that IL-21, another γc cytokine, was capable of rescuing their survival under IL-2 deprivation. Indeed, blocking the IL-21 signaling pathway along with IL-2 neutralization resulted in significantly reduced survival of both CD4⁺ and CD8⁺ T cells. Taken together, we have shown that under IL-2 deprivation conditions, IL-21 may act as the major survival factor promoting T cell immune responses. Thus, investigation of IL-2 targeted therapies may need to be revisited to consider blockade of the IL-21 signaling pathways as an adjunct to provide more effective control of T cell immune responses.     

Kinetic Approach to Pathway Attenuation Using XOMA 052, a Regulatory Therapeutic Antibody That Modulates Interleukin-1β Activity

Many therapeutic antibodies act as antagonists to competitively block cellular signaling pathways. We describe here an approach for the therapeutic use of monoclonal antibodies based on context-dependent attenuation to reduce pathologically high activity while allowing homeostatic signaling in biologically important pathways. Such attenuation is achieved by modulating the kinetics of a ligand binding to its various receptors and regulatory proteins rather than by complete blockade of signaling pathways. The anti-interleukin-1β (IL-1β) antibody XOMA 052 is a potent inhibitor of IL-1β activity that reduces the affinity of IL-1β for its signaling receptor and co-receptor but not for its decoy and soluble inhibitory receptors. This mechanism shifts the effective dose response of the cytokine so that the potency of IL-1β bound by XOMA 052 is 20–100-fold lower than that of IL-1β in the absence of antibody in a variety of in vitro cell-based assays. We propose that by decreasing potency of IL-1β while allowing binding to its clearance and inhibitory receptors, XOMA 052 treatment will attenuate IL-1β activity in concert with endogenous regulatory mechanisms. Furthermore, the ability to bind the decoy receptor may reduce the potential for accumulation of antibody·target complexes. Regulatory antibodies like XOMA 052, which selectively modulate signaling pathways, may represent a new mechanistic class of therapeutic antibodies.     

The Role of Interchain Heterodisulfide Formation in Activation of the Human Common β and Mouse βIL-3 Receptors

The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common β (βc) receptor is shared by the three cytokines and functions together with cytokine-specific α subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human βc (hβc) and unidentified cysteine residues in the N-terminal domains of the α receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hβc is part of the cytokine binding epitope of this receptor and that an IL-3Rα isoform lacking the N-terminal Ig-like domain (the “SP2” isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hβc and the related mouse receptor, β_IL-3, could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hβc led to both a complete loss of high affinity binding with the human IL-3Rα SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, β_IL-3, not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Rα SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hβc and β_IL-3 in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.     

Blockade of IL-36 Receptor Signaling Does Not Prevent from TNF-Induced Arthritis

Introduction

Interleukin (IL)-36α is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36α and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36α induces MAP-kinase and NFκB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines.

Methods

To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36α signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36α was tested in murine and human osteoclast assays.

Results

Diseased mice showed an increased expression of IL-36R and IL-36α in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays.

Conclusion

Thus we conclude that IL-36α does not affect the development of inflammatory arthritis.     

An abundant chick gizzard integrin is the avian α1β1 integrin heterodimer and functions as a divalent cation-dependent collagen IV receptor

The α-subunit of an abundant chick gizzard integrin was isolated ([12.], J. Biol. Chem. 262, 17,189–17,199) and fragmented by proteolytic digestion. The N-terminal sequences of the intact polypeptide and of several internal peptides were determined and were found to be highly homologous to the mammalian integrin α₁-subunit. Monoclonal antibodies to the chick integrin β₁-chain react on immunoblots with the gizzard integrin β-subunit ([28.], J. Biol. Chem. 265, 14,561–14,565). The chain composition of the abundant chick gizzard integrin is therefore α₁β₁. Polyclonal antibodies to the avian integrin α₁-subunit block attachment of embryonic gizzard cells to human and chick collagen IV completely and inhibit attachment to mouse Engelbreth-Holm-Swarm (EHS) tumor laminin partially. In ELISA-style receptor assays, the isolated α₁β₁ integrin bound to human and chick collagen IV and to mouse EHS tumor and chick heart laminin. While the binding to collagen IV was abolished by removal of divalent cations, the binding to laminin was not sensitive to EDTA under the conditions used. Collagen I bound the isolated avian α₁β₁ integrin only weakly. As collagen IV was the only extracellular matrix protein for which a consistent, divalent cation-dependent, binding to the avian α₁β₁ integrin could be demonstrated in both cellular and molecular assays we suggest that it is a preferred ligand for this integrin.     

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (K_D ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC₅₀ typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.     

The cytokine interleukin-5 (IL-5) effects cotransport of its receptor subunits to the nucleus in vitro

Interleukin (IL)-5 is central in regulating eosinophilia in allergic disease and parasitic infections. We have recently shown that human (h) IL-5 both possesses a functional nuclear localization signal capable of targeting a heterologous protein to the nucleus and localises to the nucleus of intact receptor-expressing cells. In this study, the extracellular domains of the hIL-5 α- and β-receptor subunits were expressed in baculovirus, fluorescently labelled and assayed for nuclear targeting in vitro in the absence and presence of IL-5. The β-subunit, which lacks IL-5 binding activity, only accumulated in the nucleus in the presence of both the hIL-5 binding α-subunit and hIL-5. The IL-5-binding α-subunit showed similar results. IL-5 thus effected nuclear transport of its α- and β-receptor subunits apparently through a ‘piggy back' mechanism, raising the possibility that IL-5's nuclear signalling role may be to cotarget its receptor subunits to the nucleus. This is the first demonstration of nuclear protein piggy back transport in vitro.     

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.     

Lack of Expression of Gp-130 Makes Pancreatic Beta Cell Lines Unresponsive to the IL-6 Family of Cytokines

Cytokine receptors from the IL-6 receptor family are comprised of ligand specific α chains and a common signalling chain, gp-130, which is also required for high affinity binding. A cDNA library generated from the β-TC3 SV40 T-antigen transformed insulinoma cell line was screened for members of this receptor family potentially relevant to both beta cell development and autoimmunity. Degenerate oligonucleotide primers to a consensus region of these receptors were used and the IL-11 receptor (α chain was identified. Despite confirmation of IL-11 receptor mRNA expression, iodinated bioactive IL-11 did not bind specifically to β-TC3 cells and gp-130-dependent cytokines did not elicit signalling events in beta cell lines. This was explained by absence of gp-130 protein or mRNA in the beta cell lines tested and in primary islets. We conclude from these resuits that the previously recognised effects of IL-6 family member cytokines on pancreatic islets must be indirect via other non-beta cells within the islet, rather than due to direct effects on beta cells themselves.     

Interleukin-33 Primes Mast Cells for Activation by IgG Immune Complexes

Mast cells (MCs) are heterogeneous cells whose phenotype is modulated by signals received from the local microenvironment. Recent studies have identified the mesenchymal-derived cytokine IL-33 as a potent direct activator of MCs, as well as regulator of their effector phenotype, and have implicated this activity in the ability of mast cells to contribute to murine experimental arthritis. We explored the hypothesis that IL-33 enables participation of synovial MCs in murine K/BxN arthritis by promoting their activation by IgG immune complexes. Compared to wild-type (WT) control mice, transgenic animals lacking the IL-33 receptor ST2 exhibited impaired MC-dependent immune complex-induced vascular permeability (flare) and attenuated K/BxN arthritis. Whereas participation of MCs in this model is mediated by the activating IgG receptor FcγRIII, we pre-incubated bone marrow-derived MCs with IL-33 and found not only direct induction of cytokine release but also a marked increase in FcγRIII-driven production of critical arthritogenic mediators including IL-1β and CXCL2. This “priming” effect was associated with mRNA accumulation rather than altered expression of Fcγ receptors, could be mimicked by co-culture of WT but not ST2^−/− MCs with synovial fibroblasts, and was blocked by antibodies against IL-33. In turn, WT but not ST2^−/− MCs augmented fibroblast expression of IL-33, forming a positive feedback circuit. Together, these findings confirm a novel role for IL-33 as an amplifier of IgG immune complex-mediated inflammation and identify a potential MC-fibroblast amplification loop dependent on IL-33 and ST2.     

Two Modes of β-Receptor Recognition Are Mediated by Distinct Epitopes on Mouse and Human Interleukin-3

The cytokine interleukin-3 (IL-3) is a critical regulator of inflammation and immune responses in mammals. IL-3 exerts its effects on target cells via receptors comprising an IL-3-specific α-subunit and common β-subunit (βc; shared with IL-5 and granulocyte-macrophage colony-stimulating factor) or a β-subunit that specifically binds IL-3 (β_IL-3; present in mice but not humans). We recently identified two splice variants of the α-subunit of the IL-3 receptor (IL-3Rα) that are relevant to hematopoietic progenitor cell differentiation or proliferation: the full length (“SP1” isoform) and a novel isoform (denoted “SP2”) lacking the N-terminal Ig-like domain. Although our studies demonstrated that each mouse IL-3 (mIL-3) Rα isoform can direct mIL-3 binding to two distinct sites on the β_IL-3 subunit, it has remained unclear which residues in mIL-3 itself are critical to the two modes of β_IL-3 recognition and whether the human IL-3Rα SP1 and SP2 orthologs similarly instruct human IL-3 binding to two distinct sites on the human βc subunit. Herein, we describe the identification of residues clustering around the highly conserved A-helix residue, Glu²³, in the mIL-3 A- and C-helices as critical for receptor binding and growth stimulation via the β_IL-3 and mIL-3Rα SP2 subunits, whereas an overlapping cluster was required for binding and activation of β_IL-3 in the presence of mIL-3Rα SP1. Similarly, our studies of human IL-3 indicate that two different modes of βc binding are utilized in the presence of the hIL-3Rα SP1 or SP2 isoforms, suggesting a possible conserved mechanism by which the relative orientations of receptor subunits are modulated to achieve distinct signaling outcomes.     

Molecular modeling of the interleukin-19 receptor complex

The interleukin-10 (IL-10) cytokine family consists of several viral and human homologs that exhibit distinct receptor binding specificities. In the present study, the complex between interleukin-19 (IL-19) and its physiological receptor—the interleukin-20 receptor -chain (IL-20R1)—was modeled.The most prominent feature of this complex is an extended binding interface formed by a long loop of IL-20R1 and a bulge region of IL-19. The two regions exhibit complementary charges and have no structural counterparts in the IL-10/IL-10R1 complex but show some resemblance to the complex between interferon- (IFN-) and its receptor.Sequence comparison of the three cytokines (IL-19, IL-20, IL-24) that bind the IL-20R1 reveals a considerable conservation of the length of the interacting loops. One residue suggested to play a key role in receptor binding specificity is a conserved glutamate. The binding interface of IL-20R1 is rich in aromatic residues while the interfaces of its cytokine ligands are mainly formed by more flexible aliphatic amino acids. This structural feature might play an important role for the specific recognition of a single receptor chain by three different cytokines. Figure Comparison of the ligand/receptor interfaces in the a IL-10/IL-10R1, b IL-19/IL-20R1 and c IFN-/receptor complexes. The translucent Connolly surfaces of the receptor and the ligand are shown in yellow and white, respectively. The backbone of the receptor and ligand are highlighted by a red and blue/green tube, respectively. For clarity, only one domain of the intertwined dimes is shown for IL-10 and IFN-. Arrows denote the location of helix B and the corresponding structural elements in IL-19 and IFN- as well as the location of receptor loop L2. As evident from b and c an extended interaction surface is created in the IL-19/IL-20R1 and IFN-/receptor complexes by the interaction of this structural element with a long loop of the respective receptor     

Interleukin-1α Activity in Necrotic Endothelial Cells Is Controlled by Caspase-1 Cleavage of Interleukin-1 Receptor-2: IMPLICATIONS FOR ALLOGRAFT REJECTION*

Inflammation is a key instigator of the immune responses that drive atherosclerosis and allograft rejection. IL-1α, a powerful cytokine that activates both innate and adaptive immunity, induces vessel inflammation after release from necrotic vascular smooth muscle cells (VSMCs). Similarly, IL-1α released from endothelial cells (ECs) damaged during transplant drives allograft rejection. However, IL-1α requires cleavage for full cytokine activity, and what controls cleavage in necrotic ECs is currently unknown. We find that ECs have very low levels of IL-1α activity upon necrosis. However, TNFα or IL-1 induces significant levels of active IL-1α in EC necrotic lysates without alteration in protein levels. Increased activity requires cleavage of IL-1α by calpain to the more active mature form. Immunofluorescence and proximity ligation assays show that IL-1α associates with interleukin-1 receptor-2, and this association is decreased by TNFα or IL-1 and requires caspase activity. Thus, TNFα or IL-1 treatment of ECs leads to caspase proteolytic activity that cleaves interleukin-1 receptor-2, allowing IL-1α dissociation and subsequent processing by calpain. Importantly, ECs could be primed by IL-1α from adjacent damaged VSMCs, and necrotic ECs could activate neighboring normal ECs and VSMCs, causing them to release inflammatory cytokines and up-regulate adhesion molecules, thus amplifying inflammation. These data unravel the molecular mechanisms and interplay between damaged ECs and VSMCs that lead to activation of IL-1α and, thus, initiation of adaptive responses that cause graft rejection.     

Structural Basis for the Binding Specificity of Human Recepteur d'Origine Nantais (RON) Receptor Tyrosine Kinase to Macrophage-stimulating Protein

Recepteur d''origine nantais (RON) receptor tyrosine kinase and its ligand, serum macrophage-stimulating protein (MSP), play important roles in inflammation, cell growth, migration, and epithelial to mesenchymal transition during tumor development. The binding of mature MSPαβ (disulfide-linked α- and β-chains) to RON ectodomain modulates receptor dimerization, followed by autophosphorylation of tyrosines in the cytoplasmic receptor kinase domains. Receptor recognition is mediated by binding of MSP β-chain (MSPβ) to the RON Sema. Here we report the structure of RON Sema-PSI-IPT₁ (SPI₁) domains in complex with MSPβ at 3.0 Å resolution. The MSPβ serine protease-like β-barrel uses the degenerate serine protease active site to recognize blades 2, 3, and 4 of the β-propeller fold of RON Sema. Despite the sequence homology between RON and MET receptor tyrosine kinase and between MSP and hepatocyte growth factor, it is well established that there is no cross-reactivity between the two receptor-ligand systems. Comparison of the structure of RON SPI₁ in complex with MSPβ and that of MET receptor tyrosine kinase Sema-PSI in complex with hepatocyte growth factor β-chain reveals the receptor-ligand selectivity determinants. Analytical ultracentrifugation studies of the SPI₁-MSPβ interaction confirm the formation of a 1:1 complex. SPI₁ and MSPαβ also associate primarily as a 1:1 complex with a binding affinity similar to that of SPI₁-MSPβ. In addition, the SPI₁-MSPαβ ultracentrifuge studies reveal a low abundance 2:2 complex with ∼10-fold lower binding affinity compared with the 1:1 species. These results support the hypothesis that the α-chain of MSPαβ mediates RON dimerization.