Human IL-18 Binding Protein Research Articles

Acquisition of different transcriptional shear mRNA and biological function of porcine interleukin 18 binding protein in PRRSV infection.

Interleukin-18 binding protein (IL-18BP), a natural regulator molecule of the pro-inflammatory cytokine interleukin-18 (IL-18), plays an important role in regulating the expression of the cellular immunity factor interferon-γ (IFN-γ). In a previous RNA-seq analysis of porcine alveolar macrophages (PAM) infected with the TIM and TJ strains of porcine reproductive and respiratory syndrome virus (PRRSV), we unexpectedly found that the mRNA expression of porcine interleukin 18-binding protein (pIL-18BP) in PAM cells infected with the TJM strain was significantly higher than that infected with the TJ strain. Studies have shown that human interleukin-18 binding protein (hIL-18bp) plays an important role in regulating cellular immunity in the course of the disease. However, there is a research gap on pIL-18BP. At the same time, PRRSV infection in pigs triggers weak cellular immune response problems. To explore the expression and the role of pIL-18BP in the cellular immune response induced by PRRSV, we strived to acquire the pIL-18BP gene from PAM or peripheral blood mononuclear cell (PBMC) with RT-PCR and sequencing. Furthermore, pIL-18BP and pIL-18 were both expressed prokaryotically and eukaryotically. The colocalization and interaction based on recombinant pIL-18BP and pIL-18 on cells were confirmed in vitro. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in pigs with different PRRSV infection states to interpret the biological function of pIL-18BP in vivo. The results showed there were five shear mutants of pIL-18BP. The mutant with the longest coding region was selected for subsequent functional validation. First, it was demonstrated that TJM-induced pIL-18BP mRNA expression was higher than that of TJ. A direct interaction between pIL-18BP and pIL-18 was confirmed through fluorescence colocalization, bimolecular fluorescent complimentary (BIFC), and co-immunoprecipitation (CO-IP). pIL-18BP also can regulate pIFN-γ mRNA expression. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in different PRRSV infection states. Surprisingly, both mRNA and protein expression of pIL-18 were suppressed. These findings fill the gap in understanding the roles played by pIL-18BP in PRRSV infection and provide a foundation for further research.IMPORTANCEPRRSV-infected pigs elicit a weak cellular immune response and the mechanisms of cellular immune regulation induced by PRRSV have not yet been fully elucidated. In this study, we investigated the role of pIL-18BP in PRRSV-induced immune response referring to the regulation of human IL-18BP to human interferon-gamma (hIFN-γ). This is expected to be used as a method to enhance the cellular immune response induced by the PRRSV vaccine. Here, we mined five transcripts of the pIL-18BP gene and demonstrated that it interacts with pIL-18 and regulates pIFN-γ mRNA expression. Surprisingly, we also found that both mRNA and protein expression of pIL-18 were suppressed under different PRRSV strains of infection status. These results have led to a renewed understanding of the roles of pIL-18BP and pIL-18 in cellular immunity induced by PRRSV infection, which has important implications for the prevention and control of PRRS.

Pharmacokinetic Study of rhIL-18BP and Its Effect on Radiation-Induced Cytokine Changes in Mouse Serum and Intestine

Administration of recombinant human IL-18 binding protein (rhIL-18BP), a natural antagonist of IL-18, significantly increased mouse survival after lethal doses of irradiation. To further understand the roles of IL-18BP in radiation mitigation, we studied the pharmacokinetic (PK) parameters of rhIL-18BP, and the serum and intestinal cytokine changes in CD2F1 mice treated with vehicle or rhIL-18BP after 9.0 Gy total body irradiation (TBI). For the PK study, non-compartmental pharmacokinetic analysis was performed using PKsolver. Serum and intestine specimens were collected to measure 44-cytokine levels. Principal component analysis showed a clear separation of the non-irradiated samples from the irradiated samples; and partial separation with or without rhIL-18BP treatment. Cytokine clusters that were significantly correlated in the serum or intestine, respectively were identified. On the individual cytokine levels, serum and intestinal cytokines that were significantly changed by irradiation and rhIL-18BP treatment were identified. Finally, cytokines that were significantly correlated between their serum and intestinal levels were identified. The current study established the PK parameters of rhIL-18BP in mice, identified significantly changed cytokines in mouse serum and intestine after radiation exposure and rhIL-18BP treatment. Current data provide critical insights into IL-18BP's mechanism of action as a radiation mitigator.

Structural basis of human IL-18 sequestration by the decoy receptor IL-18 binding protein in inflammation and tumor immunity

Human Interleukin-18 (IL-18) is an omnipresent proinflammatory cytokine of the IL-1 family with central roles in autoimmune and inflammatory diseases and serves as a staple biomarker in the evaluation of inflammation in physiology and disease, including the inflammatory phase of COVID-19. The sequestration of IL-18 by its soluble decoy receptor IL-18-Binding Protein (IL-18BP) is critical to the regulation of IL-18 activity. Since an imbalance in expression and circulating levels of IL-18 is associated with disease, structural insights into how IL-18BP outcompetes binding of IL-18 by its cognate cell-surface receptors are highly desirable; however, the structure of human IL-18BP in complex with IL-18 has been elusive. Here, we elucidate the sequestration mechanism of human IL-18 mediated by IL-18BP based on the crystal structure of the IL-18:IL-18BP complex. These detailed structural snapshots reveal the interaction landscape leading to the ultra-high affinity of IL-18BP toward IL-18 and identify substantial differences with respect to previously characterized complexes of IL-18 with IL-18BP of viral origin. Furthermore, our structure captured a fortuitous higher-order assembly between IL-18 and IL-18BP coordinated by a disulfide-bond distal to the binding surface connecting IL-18 and IL-18BP molecules from different complexes, resulting in a novel tetramer with 2:2 stoichiometry. This tetrapartite assembly was found to restrain IL-18 activity more effectively than the canonical 1:1 complex. Collectively, our findings provide a framework for innovative, structure-driven therapeutic strategies and further functional interrogation of IL-18 in physiology and disease.

Balance between Interleukin-18 and Interleukin-18 binding protein in auto-inflammatory diseases.

Interleukin (IL)-18 is a member of the IL-1 family of cytokines with pleiotropic and potent pro-inflammatory activities that are tightly controlled at the level of production and in the extracellular space. Indeed, IL-18 is translated as a leaderless biologically inert pro-peptide that is cleaved by caspase-1 in its N-terminus domain to become active. Mature Il-18 is then released out of the cells via a phenomenon of inflammatory cell death termed pyroptosis. The biological activity of IL-18 is also regulated by a naturally-occurring soluble inhibitor, IL-18 binding protein (IL-18BP) that binds IL-18 and forms high affinity complexes, thus preventing IL-18 to signal through its cell surface receptors. IL-18BP is present in high amount in the circulation, thus unbound free Il-18 is virtually absent in normal and most pathological conditions. Recent findings showed that IL-18 is present in remarkably high concentrations in some autoinflammatory diseases, including adult-onset Still's disease, systemic juvenile idiopathic arthritis and in various conditions associated with hemophagocytic lymphohistiocytosis/macrophage activation syndrome. Furthermore, elevated levels of free IL-18 are present in correlation with clinical and biological signs of disease activity. Most importantly, some patients with these diseases responded remarkably well to the administration of recombinant human IL-18BP, further indicating the pathogenic role of Il-18 and providing a strong rational for the use of IL-18 inhibitors in some of these difficult to treat auto-inflammatory diseases.

IL-18 binding protein (IL-18BP) as a novel radiation countermeasure after radiation exposure in mice

Recent studies suggested that radiation exposure causes local and systemic inflammatory responses and induces cell and tissue damage. We have reported that IL-18 plays an important role in radiation-induced injury. Here, we demonstrate that IL-18 binding protein (IL-18BP), a natural antagonist of IL-18, was significantly increased (1.7–63 fold) in mouse serum on day 1 after 0.5–10 Gy TBI. However, this high level of IL-18BP was not sufficient to neutralize the active IL-18 in irradiated mice, resulting in a radiation dose-dependent free IL-18 increase in these mice’s serum which led to pathological alterations to the irradiated cells and tissues and finally caused animal death. Administration of recombinant human (rh) IL-18BP (1.5 mg/kg) with single (24, 48 or 72 h post-TBI) or double doses (48 h and 5 days post-TBI) subcutaneous (SC) injection increased 30-day survival of CD2F1 mice after 9 Gy TBI 12.5–25% compared with the vehicle control treated group, respectively. Furthermore, the mitigative effects of rhIL-18BP included balancing the ratio of IL-18/IL-18BP and decreasing the free IL-18 levels in irradiated mouse serum and significantly increasing blood cell counts, BM hematopoietic cellularity and stem and progenitor cell clonogenicity in mouse BM. Furthermore, IL-18BP treatment inhibited the IL-18 downstream target interferon (IFN)-γ expression in mouse BM, decreased reactive oxygen species (ROS) level in the irradiated mouse heart tissues, attenuated the stress responsive factor GDF-15 (growth differentiation factor-15) and increased the intestine protector citrulline level in total body irradiated mouse serum, implicating that IL-18BP may protect multiple organs from radiation-induced inflammation and oxidative stress. Our data suggest that IL-18 plays a key role in radiation-induced cell and tissue damage and dysfunction; and for the first time demonstrated that IL-18BP counters IL-18 activation and therefore may mitigate/treat radiation-induced multiple organ injuries and increase animal survival with a wider therapeutic window from 24 h and beyond after lethal doses of radiation exposure.

Molecular cloning and characterisation of chicken IL-18 binding protein

The human IL-1 receptor family is comprised of 11 membrane bound or soluble receptors and the IL-18 binding protein (IL-18BP). These receptors are dispersed across seven genomic loci, with the majority at a single locus. Direct orthologues were identified in the chicken at conserved genomic loci; however, the IL-18BP remained absent from the first four builds of the chicken genome sequence. Subsequent assemblies identified the gene at a locus syntenic with mammals; however, these predicted sequences differed between genome builds and contained multiple errors. A partial IL-18BP-like sequence in the NCBI EST database was used to clone the full-length cDNA. A splice variant, which lacks the exon that encodes part of the signal peptide, was also cloned. Human IL-18BP is differentially spliced to produce a number of variants, which are all secreted. By contrast, the spliced chicken isoform was predicted to be intracellular, and we identified similar variants with the same exon missing in a limited number of divergent vertebrate species. Mammalian and viral IL-18BPs inhibit IL-18 activity by directly binding to this cytokine. Full-length and intracellular chicken IL-18BPs were equally effective at inhibiting IL-18-mediated IFN-γ release from an avian B-cell line. Analysis of the predicted chIL-18BP protein sequence revealed two crucial residues, which account for 50% of the binding affinity between human IL-18 and IL-18BP, are conserved in the chicken and a fowlpox-encoded homologue, fpv214. This suggests specific fowlpox viruses used in humans as a vaccine vector have the potential to dampen anti-viral host immune responses.

Open-label, multicentre, dose-escalating phase II clinical trial on the safety and efficacy of tadekinig alfa (IL-18BP) in adult-onset Still’s disease

ObjectivesAdult-onset Still’s disease (AOSD) is a rare systemic autoinflammatory disease; its management is largely empirical. This is the first clinical study to determine if interleukin (IL)-18 inhibition, using the recombinant...

IL1R9 Is Evolutionarily Related to IL18BP and May Function as an IL-18 Receptor.

The IL-1 families of ligands and receptors exhibit similarity of coding sequences, protein structures, and chromosomal positions, suggesting that they have arisen via duplication of ancestral genes. Within these families there is selectivity in ligand-receptor interactions as well as promiscuity. IL-18 and its receptor are members of these families. IL-18 is recognized as binding to the protein products of the IL18R1 and IL18RAP genes, and with high affinity to a separate IL-18 binding protein (IL-18BP). However, IL-18BP is anomalous, as it exhibits little resemblance to IL-18R proteins. Additionally, IL-18 is produced in the brain in medial habenula neurons, which project IL-18-containing axons to the interpeduncular nucleus. However, there is a lack of focal IL-18R expression in their terminal field. Given these anomalies, we hypothesized that another receptor for IL-18 may exist, and that IL18BP is evolutionarily related to this receptor. We examined Ensembl and National Center for Biotechnology Information databases to identify available IL18BP records (n = 86 species) and show through bioinformatics approaches that across mammalian species with IL18BP genes, IL-18BP is consistently most similar to IL-1R9 (IL-1R accessory protein-like 2), another member of the IL-1R family. IL-1R9 and the related IL-1R8, but not other IL-1R family members, exhibit an amino acid sequence similar to binding site A of human and viral IL-18BPs. Conserved intron/exon boundaries, protein structure, and key binding site amino acids suggest that IL18BP and IL1R9 are evolutionarily related, and that IL-1R9 and IL-1R8 may bind IL-18.

Interleukin 18–binding protein ameliorates liver ischemia–reperfusion injury

BackgroundThe aim of this study was to investigate the possible protective effect of interleukin 18–binding protein (IL-18BP) on ischemia–reperfusion (I/R)–induced liver injury in experimental rat models. Liver is one of the most affected organs from I/R process. IL-18 is an important proinflammatory cytokine, which may induce some events such as production of reactive oxygen substances and release of various cytokines. IL-18BP acts as an inhibitor of IL-18. The relationship between IL-18 and IL-18BP has an important place in inflammatory process. Materials and methodsRats were equally divided into three groups as follows: sham: Hepatic pedicle dissection was done, but hepatic pedicle clamping was not used. I/R: Sixty minutes of ischemia and 2 h of reperfusion were applied. IR + IL-18BP: Recombinant human IL-18BP (100 μg/kg) was administered 30 min before the surgery. Hepatic pedicle was clamped during 60 min of ischemia and 2 h of reperfusion was achieved. ResultsLiver enzyme levels were significantly lower in the IR + IL-18BP group, when compared with the I/R group. Serum and tissue levels of tumor necrosis factor-α, IL-6, and IL-18 were considerably lower in the IR + IL-18BP group, when compared with the I/R group, but hepatic interferon-γ and IL1β levels were not significant. Serum oxidative stress index level was significantly higher in the I/R group, when compared with the IR + IL-18BP group. In immunostaining, it was observed that pathologic changes were lower in IR + IL-18BP group than the I/R group. ConclusionsIL-18BP exhibited anti-inflammatory, antioxidant, and protective effects in I/R-mediated hepatic injury via regulating some liver enzyme activities and cytokine levels. Additionally, these effects have been verified by histomorphologic examination and oxidative stress markers.

Interleukin-18 as a therapeutic target in acute myocardial infarction and heart failure.

Interleukin 18 (IL-18) is a proinflammatory cytokine in the IL-1 family that has been implicated in a number of disease states. In animal models of acute myocardial infarction (AMI), pressure overload, and LPS-induced dysfunction, IL-18 regulates cardiomyocyte hypertrophy and induces cardiac contractile dysfunction and extracellular matrix remodeling. In patients, high IL-18 levels correlate with increased risk of developing cardiovascular disease (CVD) and with a worse prognosis in patients with established CVD. Two strategies have been used to counter the effects of IL-18:IL-18 binding protein (IL-18BP), a naturally occurring protein, and a neutralizing IL-18 antibody. Recombinant human IL-18BP (r-hIL-18BP) has been investigated in animal studies and in phase I/II clinical trials for psoriasis and rheumatoid arthritis. A phase II clinical trial using a humanized monoclonal IL-18 antibody for type 2 diabetes is ongoing. Here we review the literature regarding the role of IL-18 in AMI and heart failure and the evidence and challenges of using IL-18BP and blocking IL-18 antibodies as a therapeutic strategy in patients with heart disease.

Aberrant Regulation of Interleukin 18 Binding Protein A (IL-18BPa) by IL-18BPa Autoantibodies in Rheumatoid Arthritis

Objective: This study aimed to identify the role of IL-18BPa in the regulation of immune responses associated with the pathogenesis of Rheumatoid Arthritis. Methods: 65 Rheumatoid Arthritis (RA) patients, 22 Osteoarthritis (OA) patients, and 40 sex and age matched healthy donors were enrolled in this study. Synovial fluids mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) were prepared by using Ficoll-Hypaque separation procedure. Super Array analysis was used to measure the expression profile of immune-related genes in RA synovial tissues (RA-ST) and in normal PBMC treated with recombinant human IL-18 binding protein a (IL-18BPa). The mRNA levels of T-helper 1 (TH1) and T-helper 2 (TH2) cytokines were measured by real-time PCR, and the protein levels of IFN-γ, IL-4 and IL-18BPa autoantibodies were detected by ELISA. Results: High expression of IL-18BPa protein and messenger RNA (mRNA) are found in RA-SF and RA-ST. SuperArray analysis of immune related gene expression profile in normal PBMC treated with IL-18BPa indicated decreases in the gene expression of IFN-γ. IL-18BPa downregulated the mRNA expression of IFN-γ and IL-12, as well as, upregulated the mRNA expression of IL-4 and IL-10 in RA and normal subjects. IFN-γ and IL-12 upregulated the mRNA and protein expression of IL-18BPa in RA subjects. Autoantibodies against IL-18BPa were detected in plasma of RA patients (41.7%), in healthy subjects (4.0%), and none of OA patients, and also detected in SF of RA patients (37.9%) and OA patients (9%). Conclusion: This study emphasizes the anti-inflammatory properties of IL-18BPa on cytokines milieu and the IL-18BPa auto-antibodies may play a role in aberrant regulation of IL-18BPa in RA patients.

Development of Isoform-specific Monoclonal Antibodies Against Human IL-18 Binding Protein

Interleukin-18 binding protein (IL-18BP) is a soluble antagonist of IL-18 originally discovered while attempting to isolate a soluble receptor by using IL-18-ligand affinity column. IL-18BP has four isoforms (a, b, c, and d) in humans and two isoforms (c and d) in mice. The human isoforms IL-18BPa and IL-18BPc neutralize IL-18 activity sufficiently at an equimolar ratio; however IL-18BPb and IL-18BPd isoforms lack a complete Ig domain at C-terminus and lose the ability to neutralize IL-18 activity. Mouse IL-18BPc and IL-18BPd isoforms, possessing a similar complete Ig domain, also neutralize the biological activity of mouse IL-18 at an equimolar ratio. Here we expressed recombinant proteins of the active human IL-18BP isoforms and developed monoclonal antibodies (MAbs) against human IL-18BP a and c isoforms. We obtained two MAbs (78-4 and 38-3) of human IL-18BPa and two MAbs (18-7 and 29-6) of human IL-18BPc. The MAb clones 18-7 and 29-6 specifically recognized recombinant IL-18BPc in Western blot analyses and ELISA, whereas the MAb clone 78-4 recognized both isoforms in Western blot analyses, but only human IL-18BPa isoform in ELISA. We developed a sandwich ELISA by using the monoclonal antibody specific to human IL-18BPa isoform. The isoform-specific anti-human IL-18BP MAb may be a useful tool in categorizing a distinct group of patients from various autoimmune diseases related to IL-18BP.

Cloning and characterization of rhesus IL-18 binding protein, a natural antagonist to IL-18

IL-18 is a proinflammatory cytokine that is important for host defense, but is also involved in the pathogenesis of a number of disease processes, ranging from autoimmune disorders to atherosclerosis. IL-18 binding protein (IL-18BP) is a constitutively expressed glycoprotein that specifically neutralizes the effects of IL-18, resulting in decreased production of IFN-γ and reduction in Th1 immune responses. In this study we cloned and sequenced a full-length cDNA of the rhesus IL-18BP (RhIL-18BP) from the spleen of rhesus macaques (Macaca mulatta) and compared its nucleotide and amino acid sequences to the functional murine and human IL-18BP orthologues. In addition, we fused RhIL-18BP to the Fc portion of human IgG1 to make recombinant RhIL-18BP·Fcγ1 in order to facilitate its detection by Western blot analysis and determined the approximate molecular weight of RhIL-18BP·Fcγ1 to be 66 kD. With this fusion protein, we showed that RhIL-18BP was functional and could significantly reduce murine IL-18 and LPS-induced IFN-γ production by murine splenocytes. Furthermore, we demonstrated the expression of IL-18BP in atherosclerotic lesions in a rhesus model of atherosclerosis, underscoring the need to fully understand the role of this protein as a primary negative regulator of IL-18 in multiple disease processes.

IL-18 binding protein-expressing mesenchymal stem cells improve myocardial protection after ischemia or infarction

IL-18 is a proinflammatory cytokine known to cause tissue injury by inducing inflammation and cell death. Increased levels of IL-18 are associated with myocardial injury after ischemia or infarction. IL-18-binding protein (IL-18BP), the naturally occurring inhibitor of IL-18 activity, decreases the severity of inflammation in response to injury. In the present study, mesenchymal stem cells (MSCs) derived from mice transgenic for over expression of human IL-18BP were tested in rat models of global myocardial ischemia and acute myocardial infarction. Improved myocardial function is associated with production of VEGF, and in vitro, IL-18BP MSCs secreted higher levels of constitutive VEGF compared to wild-type MSCs. Whereas IL-18 increased cell death and reduced VEGF in wild-type MSCs, IL-18BP MSCs were protected. In an isolated heart model, intracoronary infusion of IL-18BP MSCs before ischemia increased postischemic left ventricular (LV) developed pressure to 79.5 + or - 9.47 mmHg compared to 59.3 + or - 7.8 mmHg in wild-type MSCs and 37.8 + or - 5 mmHg in the vehicle group. Similarly, using a coronary artery ligation model, intramyocardial injection of IL-18BP MSCs improved LV ejection fraction to 67.8 + or - 1.76% versus wild-type MSCs (57.4 + or - 1.33%) and vehicle (39.2 + or - 2.07%), increased LV fractional shortening 1.25-fold over wild-type MSCs and 1.95-fold over vehicle, decreased infarct size to 38.8 + or - 2.16% compared to 46.4 + or - 1.92% in wild-type MSCs and 60.7 + or - 2.2% in vehicle, reduced adverse ventricular remodeling, increased myocardial VEGF production, and decreased IL-6 levels. This study provides the concept that IL-18BP genetically modified stem cells improve cardioprotection over that observed with unmodified stem cells.

Structural basis for antagonism of human interleukin 18 by poxvirus interleukin 18-binding protein

Human interleukin-18 (hIL-18) is a cytokine that plays an important role in inflammation and host defense against microbes. Its activity is regulated in vivo by a naturally occurring antagonist, the human IL-18-binding protein (IL-18BP). Functional homologs of human IL-18BP are encoded by all orthopoxviruses, including variola virus, the causative agent of smallpox. They contribute to virulence by suppressing IL-18-mediated immune responses. Here, we describe the 2.0-A resolution crystal structure of an orthopoxvirus IL-18BP, ectromelia virus IL-18BP (ectvIL-18BP), in complex with hIL-18. The hIL-18 structure in the complex shows significant conformational change at the binding interface compared with the structure of ligand-free hIL-18, indicating that the binding is mediated by an induced-fit mechanism. EctvIL-18BP adopts a canonical Ig fold and interacts via one edge of its beta-sandwich with 3 cavities on the hIL-18 surface through extensive hydrophobic and hydrogen bonding interactions. Most of the ectvIL-18BP residues that participate in these interactions are conserved in both human and viral homologs, explaining their functional equivalence despite limited sequence homology. EctvIL-18BP blocks a putative receptor-binding site on IL-18, thus preventing IL-18 from engaging its receptor. Our structure provides insights into how IL-18BPs modulate hIL-18 activity. The revealed binding interface provides the basis for rational design of inhibitors against orthopoxvirus IL-18BP (for treating orthopoxvirus infection) or hIL-18 (for treating certain inflammatory and autoimmune diseases).

Glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of glycan-binding proteins

The extensive involvement of glycan-binding proteins (GBPs) as regulators in diverse biological phenomena provides a fundamental reason to investigate their glycan-binding specificities. Here, we developed a glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of GBPs. Eighty-nine selected multivalent glycoconjugates comprising natural glycoproteins, neo-glycoproteins, and polyacrylamide (PAA)-conjugated glycan epitopes were immobilized on an epoxy-activated glass slide. The GBP binding was monitored by an evanescent-field fluorescence-assisted scanner at equilibrium without washing steps. The detection principle also allows direct application of unpurified GBPs with the aid of specific antibodies. Model experiments using plant lectins (RCA120, ConA, and SNA), galectins (3 and 8), a C-type lectin (DC-SIGN) and a siglec (CD22) provided data consistent with previous work within 4 h using less than 40 ng of GBPs per analysis. As an application, serum profiling of antiglycan antibodies (IgG and IgM) was performed with Cy3-labeled secondary antibodies. Moreover, novel carbohydrate-binding ability was demonstrated for a human IL-18 binding protein. Thus, the developed glycan array is useful for investigation of various types of GBPs, with the added advantage of wash-free analysis.

Expression, Purification and Structural Analysis of Human IL-18 Binding Protein: A Potent Therapeutic Molecule for Allergy

While interleukin-18 (IL-18) plays an important role in the innate and adaptive immune responses, it can also cause severe allergic inflammatory reactions. Thus it is a molecule currently being targeted for therapy. The natural intrinsic inhibitor of IL-18 receptor activation, IL-18 binding protein (IL-18BP), shows a great potential for the treatment of allergy. Expression and purification of recombinant human IL-18BP (rhIL-18BP) were performed using the baculovirus system to develop a therapeutic molecule for the treatment of IL-18-related diseases and to investigate the structural basis of its inhibitory mechanism. Purified rhIL-18BP potently inhibited the production of interferon-gamma by peripheral blood mononuclear cells in the presence of lipopolysaccharide and by human myelomonocytic KG-1 cells in the presence of IL-18 (IC50 = 0.4 nM). Surface plasmon resonance showed a high affinity (Kd = 0.46 nM) for rhIL-18BP in binding hIL-18. Structural analysis indicated that the stoichiometry between IL-18 and IL-18BP is 1 : 1 in solution and the model structure of the complex suggests that the key residues on IL-18 (L5, K53, S55) and the estimated key residues on IL-18BP (F93,Y97, F104) could have interactions. The structural mechanism of IL-18BP inhibition might be a competition for Site 2 on rIL-18 so that IL-18BP can prevent IL-18 receptor alpha from binding to Site 2 and inhibit IL-18 receptor activation. IL-18BP has unique features with respect to its structure, binding mode and inhibitory mechanism. It is a molecule that has a great potential for the therapy of allergy.

Pharmacokinetics of IL-18 binding protein in healthy volunteers and subjects with rheumatoid arthritis or plaque psoriasis

IL-18 binding protein (BP) neutralizes the activity of IL-18, a cytokine implicated in psoriasis and rheumatoid arthritis (RA). We investigated the pharmacokinetics, pharmacodynamics and safety of recombinant human IL-18 BP (r-hIL-18 BP) in healthy volunteers and subjects with psoriasis or RA in four phase I studies. A) Healthy volunteers (n = 24) were randomised to receive a single subcutaneous (sc) injection of r-hIL-18 BP (20, 70, 210 or 350 mg) or placebo. B) Healthy volunteers (n = 10) were randomised to receive six sc injections of r-hIL-18 BP (35 or 175 mg, 48 h between injections) or placebo. C) Subjects with moderate-to-severe plaque psoriasis (n = 35) were randomised to receive r-hIL-18 BP (20, 160 or 320 mg, sc tiw) or placebo for 6 weeks. D) Subjects with active, moderate-to-severe RA (n = 36) were randomised to receive r-hIL-18 BP (20, 80, 160 mg, sc tiw) or placebo for 6 weeks. Pharmacokinetics, pharmacodynamics and safety were assessed in all four studies. r-hIL-18 BP showed a dose-dependent pharmacokinetic profile, with a peak serum concentration of 6-48 hours. With repeated sc injections tiw, a steady state was achieved in 1-2 weeks among subjects with psoriasis or RA. The majority of adverse events were mild or moderate in severity. Injection site reactions were the most frequently reported event in subjects with psoriasis or RA. r-hIL-18 BP displays dose-dependent pharmacokinetics, has a favourable safety profile and is well-tolerated in healthy volunteers and in subjects with moderate-to-severe plaque psoriasis or active, moderate-to-severe RA.

Generation and characterization of mice transgenic for human IL-18-binding protein isoform a.

Interleukin (IL)-18 binding protein (IL-18BP) is a natural inhibitor of the pleiotropic cytokine IL-18. To study the role of IL-18BP in modulating inflammatory responses in vivo, mice transgenic for human IL-18BP isoform a (IL-18BP-Tg) were generated. The transgene was expressed at high levels in each organ examined. High levels of bioactive human IL-18BPa were detectable in the circulation of IL-18BP-Tg mice, which were viable, fertile, and had no tissue or organ abnormality. The high levels of IL-18BP in the transgenic mice were able to completely neutralize the interferon-gamma (IFN-gamma)-inducing activity of exogenously administered IL-18. Following administration of endotoxin, with or without prior sensitization with heat-inactivated Propionibacterium acnes, IL-18BP-Tg mice produced significantly lower serum levels of IFN-gamma and macrophage-inflammatory protein-2 compared with nontransgenic littermates. Significantly reduced production of IFN-gamma in response to endotoxin was also observed in cultures of IL-18BP-Tg splenocytes. Finally, IL-18BP-Tg mice were completely protected in a model of hepatotoxicity induced by administration of concanavalin A. These results indicate that high endogenous levels of IL-18BP in trangenic mice effectively neutralize IL-18 and are protective in response to different inflammatory stimuli.

Mutation scan of a type 1 diabetes candidate gene: the human interleukin-18 binding protein gene.

Type 1 (insulin-dependent) diabetes, T1DM, is the result of an immune-mediated destruction of the pancreatic beta cells dependent mainly on T helper cells and macrophages. Interleukin-18 (IL-18) is a proinflammatory cytokine produced mainly by macrophages. IL-18 is capable of inducing T lymphocyte synthesis of IFNgamma, thereby skewing the T helper response toward a T helper type 1 (Th1) profile. IL-18 binding protein (IL18BP) neutralizes IL-18 and leads to a reduced Th1 response. Polymorphisms in IL18BP may affect the activity of IL-18 and the magnitude of the Th1 response and may play a role in the pathogenesis of T1DM. The aim of the study was therefore to identify polymorphisms in IL18BP and to test these for association with T1DM. We evaluated the human IL18BP gene on chromosome 11q13 as a candidate susceptibility gene for T1DM and scanned the entire IL18BP (promoter, exons 1-6, and 3'UTR) for polymorphisms using single-strand conformational polymorphism analysis and direct sequencing. We identified a total of 11 polymorphisms, all having allele frequencies ranging between 0.05 and 0.10. Four were in the 5'UTR: -257G-->T, -78C-->T, -65G-->A, and -59A-->G. Three were in intron 3: IVS3+140A-->C, IVS4-147G-->T, and IVS4-59G-->T. The last four, 38*A-->T, 48*T-->A, 388*C-->G, and 440*_441*insG, were in the 3'UTR of IL18BP. However, none of these were frequent enough to permit association studies in T1DM and we conclude that IL18BP does not contribute to the overall genetic susceptibility to type 1 diabetes.