1.9 A crystal structure of interleukin 6: implications for a novel mode of receptor dimerization and signaling.

Interleukin 6 (IL-6) has many biological activities in vivo, and deregulation has been implicated in many disease processes. IL-6, a 185 amino acid polypeptide was refolded, purified and crystallized. The crystals diffracted to beyond 1.9 A and the structure was solved using single isomorphous replacement. The X-ray structure of IL-6 is composed of a four helix bundle linked by loops and an additional mini-helix. 157 out of 185 residues are well defined in the final structure, with 18 N-terminal and 8 A-B loop amino acids displaying no interpretable electron density. The three-dimensional structure has been used to construct a model of IL-6 interacting with the IL-6 receptor (alpha-chain) and gp130 (beta-chain) that gives new insight into the process of molecular recognition and signaling. Based on this model, we predict a fourth binding site on IL-6, a low affinity IL-6-IL-6 interaction, which may be necessary for the sequential assembly of a functional hexameric IL-6 receptor complex.     

Crystal structure of interleukin-19 defines a new subfamily of helical cytokines

Interleukin-19 (IL-19) is a novel cytokine that was initially identified during a sequence data base search aimed at finding potential IL-10 homologs. IL-19 shares a receptor complex with IL-20, indicating that the biological activities of these two cytokines overlap and that both may play an important role in regulating development and proper functioning of the skin. We determined the crystal structure of human recombinant IL-19 and refined it at 1.95-A resolution to an R-factor of 0.157. Unlike IL-10, which forms an intercalated dimer, the molecule of IL-19 is a monomer made of seven amphipathic helices, A-G, creating a unique helical bundle. On the basis of the observed structure, we propose that IL-19, IL-20, and other putative members of the proposed IL-10 family together form a distinct subfamily of helical cytokines.     

The immunosuppressive activity of peptide fragments of vaccinia virus C10L protein and a hypothesis on the role of this protein in the viral invasion

Our previous studies revealed that the 143-148 fragment of interleukin-1 receptor antagonist (IL-1 Ra) molecule with a Val-Thr-Lys-Phe-Tyr-Phe (VTKFYF) sequence inhibits the interleukin-1 (IL-1) interaction with its cellular receptor. The Val-Thr-Arg-Phe-Tyr-Phe (VTRFYF) sequence of the 322-327 fragment of the C-terminal domain of vaccinia virus protein related to the C10L vaccinia gene shows a very high homology to the 143-148 IL-1 Ra fragment, suggesting a similar inhibitory activity. To test this suggestion, we investigated the inhibitory activity of a series of synthetic peptides derived from 316 to 327 fragment of C10L on the interaction of IL-1 with its receptor. We also tested the peptides for their influence on the humoral and cellular immune response. The results indicate that biological activities of the C10L fragments are similar to those obtained for respective fragments of IL-1 Ra. The C-terminal domain of C10L protein can be easily folded into spatial structure similar to the crystallographic one of IL-1 Ra. Based on the crystallographic structure of IL-1 Ra, we constructed a 3-D model of the C10L protein. According to the model, the Val(322)-Asn(328) sequence is localized on the surface of the molecule and, therefore, it may be involved in the interactions with receptors. Our results indicate that the C10L viral protein can play an important role in vaccinia virus evasion of the host immune system. It may consist in the blockade of IL-1 receptors by the C10L protein, a homologue of the IL-1 Ra.     

Crystal structure of the IL-22/IL-22R1 complex and its implications for the IL-22 signaling mechanism

Interleukin-22 (IL-22) is a member of the interleukin-10 cytokine family, which is involved in anti-microbial defenses, tissue damage protection and repair, and acute phase responses. Its signaling mechanism involves the sequential binding of IL-22 to interleukin-22 receptor 1 (IL-22R1), and of this dimer to interleukin-10 receptor 2 (IL-10R2) extracellular domain. We report a 1.9A crystal structure of the IL-22/IL-22R1 complex, revealing crucial interacting residues at the IL-22/IL-22R1 interface. Functional importance of key residues was confirmed by site-directed mutagenesis and functional studies. Based on the X-ray structure of the binary complex, we discuss a molecular basis of the IL-22/IL-22R1 recognition by IL-10R2. STRUCTURED SUMMARY:     

Structure of interleukin 1 alpha at 2.7-A resolution

The interleukin 1 (IL-1) family of proteins has a central role in modulating immune and inflammatory responses. Two major IL-1 proteins, designated alpha (IL-1 alpha) and beta (IL-1 beta), are produced by activated macrophages and other cell types. In an effort to understand the similarities and differences in the physicochemical and functional properties of these two proteins, a program was initiated to determine their structures. Crystals of IL-1 alpha were grown, and the three-dimensional structure at 2.7-A resolution was solved. The technique of multiple-wavelength anomalous dispersion (MAD) with the selenomethionine form of IL-1 alpha was utilized in combination with a single mercury derivative to provide the starting phases. Partial refinement of the IL-1 alpha model has been performed as well. The overall structure is composed of 14 beta-strands and a 3(10) helix. The core of this structure is a capped beta-barrell that possesses 3-fold symmetry and displays a topology similar to that observed for IL-1 beta [Priestle, J. P., et al. (1988) EMBO J. 7, 339-343] and soybean trypsin inhibitor (STI) [McLachlan, A. D. (1979) J. Mol. Biol. 133, 557-563]. In this paper, the overall structure of IL-1 alpha and the nature and fidelity of the internal 3-fold symmetry are discussed. Comparisons with IL-1 beta and STI are made within these contexts.     

Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding

In order to identify residues required for the binding of interleukin-8 (IL-8) to its receptor, mutants were constructed in which clusters of charged amino acids were systematically replaced with alanine along the entire IL-8 sequence. The mutants were tested for their ability to induce a receptor-mediated rise in cytosolic free Ca2+, a property of wild-type IL-8 which can readily be detected by flow cytometry using neutrophils loaded with the calcium probe Indo-1. Eleven of the 12 mutants caused neutrophil calcium mobilization at 5 nM; the exception being a triple alanine mutant at positions K3, E4, and R6, which was inactive at all concentrations tested (150 nM maximum). A second set of mutants was generated in which residues 1-15 were individually mutated to alanine. Mutants E4A, L5A, or R6A were all inactive in the Ca2+ assay at 5 nM and competed poorly with 125I-IL-8 for neutrophil receptor binding; I10A, E4A, L5A, and R6A had approximately 30-, 100-, 100-, and 1000-fold reduced affinity, as compared with control IL-8, respectively. The nuclear magnetic resonance structure of IL-8 indicates that, in solution, the side chains of E4, L5, R6, and I10 point away from the core of the protein and do not participate in any intramolecular hydrogen bonds or salt bridges (Clore, G. M., and Gronenborn, A. M. (1991) J. Mol. Biol. 217, 611-620).     

Structure of IL-17A in Complex with a Potent, Fully Human Neutralizing Antibody

IL-17A is a pro-inflammatory cytokine produced by the newly identified Th17 subset of T-cells. We have isolated a human monoclonal antibody to IL-17A (CAT-2200) that can potently neutralize the effects of recombinant and native human IL-17A. We determined the crystal structure of IL-17A in complex with the CAT-2200 Fab at 2.6 Å resolution in order to provide a definitive characterization of the epitope and paratope regions. Approximately a third of the IL-17A dimer is disordered in this crystal structure. The disorder occurs in both independent copies of the complex in the asymmetric unit and does not appear to be influenced by crystal packing. The complex contains one IL-17A dimer sandwiched between two CAT-2200 Fab fragments. The IL-17A is a disulfide-linked homodimer that is similar in structure to IL-17F, adopting a cystine-knot fold. The structure is not inconsistent with the previous prediction of a receptor binding cavity on IL-17 family members. The epitope recognized by CAT-2200 is shown to involve 12 amino acid residues from the quaternary structure of IL-17A, with each Fab contacting both monomers in the dimer. All complementarity-determining regions (CDRs) in the Fab contribute to a total of 16 amino acid residues in the antibody paratope. In vitro affinity optimization was used to generate CAT-2200 from a parental lead antibody using random mutagenesis of CDR3 loops. This resulted in seven amino acid changes (three in VL-CDR3 and four in VH-CDR3) and gave an approximate 30-fold increase in potency in a cell-based neutralization assay. Two of the seven amino acids form part of the CAT-2200 paratope. The observed interaction site between CAT-2200 and IL-17A is consistent with data from hydrogen/deuterium exchange mass spectrometry and mutagenesis approaches.     

Inhibition of interleukin 1 synthesis by tenidap: a new drug for arthritis

Tenidap is a new antiarthritic drug of novel chemical structure. This study shows the effects of tenidap on the in vitro synthesis of interleukin 1 (IL-1). IL-1 production by murine peritoneal macrophages was induced either by stimulation with lipopolysaccharide (LPS) or by phagocytosis of zymosan. With either stimulus, tenidap inhibited IL-1 production as measured by a quantitative competitive IL-1 receptor binding assay. Approximately 20 ng/mL of IL-1 was produced by 10(6) macrophages in response to LPS and about half that amount was produced in response to zymosan. Fifty percent inhibition of IL-1 production by tenidap was found at 3 microM for both stimuli. Using goat anti-IL-1 alpha and Western blot analysis, the appearance of intracellular 34 kDa pro-IL-1 alpha was inhibited by tenidap down to 3 microM. Tenidap decreased [35S]Met incorporation into cellular protein at 30 microM but not at 10 or 3 microM, indicating selectivity for IL-1 inhibition relative to total protein synthesis. Because tenidap inhibited IL-1 induction by both zymosan and LPS, it must act subsequently to receptor triggering. As the appearance of IL-1 was inhibited both intracellularly and extracellularly, the primary drug effect cannot be on secretion.     

Noncompetitive antibody neutralization of IL-10 revealed by protein engineering and x-ray crystallography

IL-10 is a dimeric cytokine that must engage its high-affinity cell surface receptor, IL-10R1, to induce multiple cellular activities. Here we report the 1.9 A crystal structure of an engineered IL-10 monomer (IL-10M1) in complex with a neutralizing Fab fragment (9D7Fab). 9D7Fab and IL-10R1 bind distinct nonoverlapping surfaces on IL-10M1. Antagonism of the IL-10M1/IL-10R1 interaction is the result of 9D7Fab-induced conformational changes in the CD loop of IL-10M1 that indirectly alter the structure of the IL-10R1 binding site. A single mutation (Ile87Ala) in the same CD loop region of the Epstein-Barr virus IL-10 (ebvIL-10) also reduces IL-10R1 binding affinity, suggesting that ebvIL-10 and 9D7Fab use similar allosteric mechanisms to modulate IL-10R1 affinity and biological activity.     

Evaluation of the anti-inflammatory effect of milk fermented by a strain of IL-10-producing Lactococcus lactis using a murine model of Crohn's disease

Interleukin-10 (IL-10) is the most important anti-inflammatory cytokine at intestinal level, and its absence is involved in inflammatory bowel diseases. However, oral treatment with IL-10 is difficult because of its low survival in the gastrointestinal tract and systemic treatments lead to undesirable side effects. The aim of this paper was to evaluate the anti-inflammatory effect of the administration of milks fermented by Lactococcus lactis strains that produce IL-10 under the control of the xylose-inducible expression system using a trinitrobenzenesulfonic acid-induced colitis murine model. Mice that received milks fermented by L. lactis strains producing IL-10 in the cytoplasm (Cyt strain) or secreted to the product (Sec strain) showed lower damage scores in their large intestines, decreased IFN-γ levels in their intestinal fluids and lower microbial translocation to liver, compared to mice receiving milk fermented by the wild-type strain or those not receiving any treatment. The results obtained in this study show that the employment of fermented milks as a new form of administration of IL-10-producing L. lactisis effective in the prevention of inflammatory bowel disease in a murine model.     

Association of the interleukin-10 1082G/A, 819C/T and 3575T/A gene polymorphisms with systemic sclerosis: a meta-analysis

Many environmental and genetic factors have been contributed to the development of systemic sclerosis (SSc). To determine whether IL-10 gene polymorphisms are associated with SSc, we conducted a meta-analysis approach. A total of eight studies involving 1,034 SSc cases and 1,815 controls were obtained by electronic database, i.e. Embase, Blackwell, Scopus, China National Knowledge Infrastructure database, Chinese Biomedical database, Google searching. We analyzed three gene polymorphisms, including IL-10 −1082G/A (rs1800896), IL-10 −819C/T (rs1800871), IL-10 −3575T/A (rs1800890). The combined odds ratio (OR) with its 95% confidence interval (95% CI) was calculated using fixed or random effect models. We found that IL-10 819C allele might contribute to SSc susceptibility by fixed effect model and IL-10 3575A allele could be an important risk factor for SSc, especially in European descent. No significant heterogeneity were observed. Under random effect model, there was no evidence of statistically significant association between IL-10 1082G/A polymorphism and SSc. Publication bias was absent in all analyses. However, larger scale primary studies are required to further evaluate the IL-10 polymorphism and SSc.     

LPS／D—GalN诱发ⅣF—KB转基因小鼠急性致死性肝损伤模型的建立

目的以NF—KB转基因BALB／c小鼠建立一个LPS／D—GaIN诱发的急性致死性肝损伤模型。方法采取腹腔注射高剂量的LPS／D-GalN建立急性致死性肝损伤小鼠模型,观察模型小鼠的促炎症细胞因子水平和NF—KB的活性改变,以及肝脏功能和病理改变情况。结果模型组小鼠生存时间为8—10h,模型建立后小鼠血清TNF—a、IL-6和MCP-1水平显著升高,在2—4h达到高峰;肝脏外观出现瘀血和出血,肝脏小叶被严重破坏,肝细胞严重坏死和出血;血清ALT／AST水平在模型诱发后持续迅速上升;整体成像显示胛-KB的活性在4～6h达到高峰。正常对照组小鼠以上指标无显著变化。结论成功建立LPS／D-GalN诱发的M-船转基因小鼠的急性致死性肝损伤模型。     

Immunomodulatory role of C10 chemokine in a murine model of allergic bronchopulmonary aspergillosis

The immunomodulatory role of the chemokine C10 was explored in allergic airway responses during experimental allergic bronchopulmonary aspergillosis (ABPA). The intratracheal delivery of Asperigillus fumigatus Ag into A. fumigatus-sensitized mice resulted in significantly increased levels of C10 within the bronchoalveolar lavage, and these levels peaked at 48 h after A. fumigatus challenge. In addition, C10 levels in BAL samples were greater than 5-fold higher than levels of other chemokines such as monocyte-chemoattractant protein-1, eotaxin, and macrophage-inflammatory protein-1alpha. From in vitro studies, it was evident that major pulmonary sources of C10 may have included alveolar macrophages, lung fibroblasts, and vascular smooth muscle cells. Experimental ABPA was associated with severe peribronchial eosinophilia, bronchial hyperresponsiveness, and augmented IL-13 and IgE levels. The immunoneutralization of C10 with polyclonal anti-C10 antiserum 2 h before the intratracheal A. fumigatus challenge significantly reduced the airway inflammation and hyperresponsiveness in this model of ABPA, but had no effect on IL-10 nor IgE levels. Taken together, these data suggest that C10 has a unique role in the progression of experimental ABPA.     

3D modeling and molecular dynamics simulation of an immune-regulatory cytokine,interleukin-10, from the Indian major carp, <Emphasis Type="Italic">Catla catla</Emphasis>

Interleukin-10 (IL-10) is a pleiotropic immune-regulatory cytokine that is expressed in various species of fish and higher vertebrates, and is activated during infection. In spite of its important role, IL-10 has not been well characterized either functionally or structurally in fish. To analyze its properties and function, we constructed a 3D model of IL-10 in the Indian major carp, the catla (Catla catla), which is a highly preferred fish species and the most commercially important one in the Indian subcontinent. The catla IL-10 model was constructed by comparative modeling using human IL-10 (2ILK) as the template, and a 5 ns molecular dynamics (MD) simulation was carried out to characterize its structural and dynamical features, which was validated by the SAVES, WHAT IF and MolProbity servers. Analysis using the VAST server revealed a comparatively low level of homology between catla and human IL-10 amino acids at the N-terminal (22.7%) compared to the C-terminal (38.29%). Six conserved domains (A–F) were predicted in catla that threaded well with human IL-10, but their putative interaction sites varied significantly. The amino acid residues in helices A and F differed in length between catla and human IL-10, which may lead to the differences in the IL-10/IL-10R complexes of these two species. The existence of two highly conserved amino acid residues (Cys5 and Cys10) in fish IL-10 but not in higher vertebrate (including human) IL-10 was analyzed in this 3D model. CastP, cons-PPISP and InterProSurf server identified several binding pockets with various probe radii, but Cys5 and Cys10 did not form any significant bonds relating to structural stabilization or protein–protein interactions.     

Identification of the Glycosaminoglycan Binding Site of Interleukin-10 by NMR Spectroscopy

The biological function of interleukin-10 (IL-10), a pleiotropic cytokine with an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are highly negatively charged polysaccharides and integral components of the extracellular matrix with important functions in the biology of many growth factors and cytokines. The molecular mechanism of the IL-10/GAG interaction is unclear. In particular, experimental evidence about IL-10/GAG binding sites is lacking, despite its importance for understanding the biological role of the interaction. Here, we report the experimental determination of a GAG binding site of IL-10. Although no co-crystal structure of the IL-10·GAG complex could be obtained, its structural characterization was possible by NMR spectroscopy. Chemical shift perturbations of IL-10 induced by GAG binding were used to narrow down the location of the binding site and to assess the affinity for different GAG molecules. Subsequent observation of NMR pseudocontact shifts of IL-10 and its heparin ligand, as induced by a protein-attached lanthanide spin label, provided structural restraints for the protein·ligand complex. Using these restraints, pseudocontact shift-based rigid body docking together with molecular dynamics simulations yielded a GAG binding model. The heparin binding site is located at the C-terminal end of helix D and the adjacent DE loop and coincides with a patch of positively charged residues involving arginines 102, 104, 106, and 107 and lysines 117 and 119. This study represents the first experimental characterization of the IL-10·GAG complex structure and provides the starting point for revealing the biological significance of the interaction of IL-10 with GAGs.     

Mapping of the interleukin-10/interleukin-10 receptor combining site.

The discontinuous interleukin-10(IL-10)/interleukin-10 receptor (IL-10R) combining site was mapped using sets of overlapping peptides derived from both binding partners bound to continuous cellulose membranes. Low affinity binding of single regions of the discontinuous contact sites on IL-10 and IL-10R could be identified due to (1) high peptide density on the membrane support, (2) incubation with high protein concentrations, (3) indirect immunodetection of the ligates after electrotransfer onto polyvinylene difluoride membranes, and (4) use of highly overlapping peptide scans of different length (6-mers and 15-mers). The single binding regions identified for each protein species are separated in the protein sequences, but form continuous areas on the surface of IL-10 (X-ray structure) and IL-10R (computer model). Furthermore, four epitopes of neutralizing anti-IL-10 and anti-IL-10R antibodies were mapped and overlap with these binding regions. Soluble peptides (15- to 19-mers) each spanning one of the three identified IL-10-derived receptor binding regions displayed no significant affinity to IL-10R as expected, whereas a peptide (35-mer) comprising two of these regions had considerably higher binding activity. The data are consistent with a previously published computer model of the IL-10/IL-10R complex. This approach should be generally applicable for the mapping of non-linear protein-protein contact sites.     

Cloning and characterization of IL-22 binding protein, a natural antagonist of IL-10-related T cell-derived inducible factor/IL-22

The class II cytokine receptor family includes the receptors for IFN-alphabeta, IFN-gamma, IL-10, and IL-10-related T cell-derived inducible factor/IL-22. By screening genomic DNA databases, we identified a gene encoding a protein of 231 aa, showing 33 and 34% amino acid identity with the extracellular domains of the IL-22 receptor and of the IL-20R/cytokine receptor family 2-8, respectively, but lacking the transmembrane and cytoplasmic domains. A lower but significant sequence identity was found with other members of this family such as the IL-10R (29%), cytokine receptor family 2-4/IL-10Rbeta (30%), tissue factor (26%), and the four IFN receptor chains (23-25%). This gene is located on chromosome 6q24, at 35 kb from the IFNGR1 gene, and is expressed in various tissues with maximal expression in breast, lungs, and colon. The recombinant protein was found to bind IL-10-related T cell-derived inducible factor/IL-22, and to inhibit the activity of this cytokine on hepatocytes and intestinal epithelial cells. We propose to name this natural cytokine antagonist IL-22BP for IL-22 binding protein.     

Impact of bile duct obstruction on hepatic E. coli infection: role of IL-10

Biliary obstruction in the setting of hepatic bacterial infection has great morbidity and mortality. We developed a novel murine model to examine the effect of biliary obstruction on the clearance of hepatic Escherichia coli infection. This model may allow us to test the hypothesis that biliary obstruction itself adversely affects clearance of hepatic infections even if the bacteria are introduced into the liver by a nonbiliary route. We ligated the bile ducts of C57BL/6 mice on days -1, 0, or +1, relative to a day 0 portal venous injection of E. coli. We monitored survival, hepatic bacterial growth, pathology, and IL-10 protein levels. The role of IL-10 in this model was further examined using IL-10 knockout mice. Mice with bile duct ligation at day +1 or 0, relative to portal venous infection at day 0, had decreased survival compared with mice with only portal venous infection. The impaired survival was associated with greater hepatic bacterial growth, hepatic necrosis, and increased production of IL-10. Interestingly, the transgenic knockout of IL-10 resulted in impaired survival in mice with bile duct ligation and portal venous infection. Biliary obstruction had a dramatic detrimental effect on hepatic clearance of portal venous E. coli infection. This impaired clearance is associated with increased IL-10 production. However, transgenic knockout of IL-10 increased mortality after hepatic infection.