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Chemokines, a large family of highly versatile small soluble proteins, play crucial roles in defining innate and adaptive immune responses by regulating the trafficking of leukocytes, and also play a key role in various aspects of human physiology. Chemokines share the characteristic feature of reversibly existing as monomers and dimers, and their functional response is intimately coupled to interaction with glycosaminoglycans (GAGs). Currently, nothing is known regarding the structural basis or molecular mechanisms underlying CXCL5-GAG interactions. To address this missing knowledge, we characterized the interaction of a panel of heparin oligosaccharides to CXCL5 using solution NMR, isothermal titration calorimetry, and molecular dynamics simulations. NMR studies indicated that the dimer is the high-affinity GAG binding ligand and that lysine residues from the N-loop, 40s turn, β3 strand, and C-terminal helix mediate binding. Isothermal titration calorimetry indicated a stoichiometry of two oligosaccharides per CXCL5 dimer. NMR-based structural models reveal that these residues form a contiguous surface within a monomer and, interestingly, that the GAG-binding domain overlaps with the receptor-binding domain, indicating that a GAG-bound chemokine cannot activate the receptor. Molecular dynamics simulations indicate that the roles of the individual lysines are not equivalent and that helical lysines play a more prominent role in determining binding geometry and affinity. Further, binding interactions and GAG geometry in CXCL5 are novel and distinctly different compared with the related chemokines CXCL1 and CXCL8. We conclude that a finely tuned balance between the GAG-bound dimer and free soluble monomer regulates CXCL5-mediated receptor signaling and function., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: In this study, we developed horseradish peroxidase (HRP)-catalyzed sprayable gelatin hydrogels (GH) as a bioactive wound dressing that can deliver cell-attracting chemotactic cytokines to the injured tissues for diabetic wound healing. We hypothesized that topical administration of chemokines using GH hydrogels might improve wound healing by inducing recruitment of the endogenous cells. Two types of chemokines (interleukin-8; IL-8, macrophage inflammatory protein-3α; MIP-3α) were simply loaded into GH hydrogels during in situ cross-linking, and then their wound-healing effects were evaluated in streptozotocin-induced diabetic mice. The incorporation of chemokines did not affect hydrogels properties including swelling ratio and mechanical stiffness, and the bioactivities of IL-8 and MIP-3α released from hydrogel matrices were stably maintained. In vivo transplantation of chemokine-loaded GH hydrogels facilitated cell infiltration into the wound area, and promoted wound healing with enhanced re-epithelialization/neovascularization and increased collagen deposition, compared with no treatment or the GH hydrogel alone. Based on our results, we suggest that cell-recruiting chemokine-loaded GH hydrogel dressing can serve as a delivery platform of various therapeutic proteins for wound healing applications., Statement of Significance: Despite development of materials combined with therapeutic agents for diabetic wound treatment, impaired wound healing by insufficient chemotactic responses still remain as a significant problem. In this study, we have developed enzyme-catalyzed gelatin (GH) hydrogels as a sprayable dressing material that can deliver cell-attracting chemokines for diabetic wound healing. The chemotactic cytokines (IL-8 and MIP-3α) were simply loaded within hydrogel during in situ gelling, and wound healing efficacy of chemokine-loaded GH hydrogels was investigated in STZ-induced diabetic mouse model. These hydrogels significantly promoted wound-healing efficacy with faster wound closure, neovascularization, and thicker granulation. Therefore, we expect that HRP-catalyzed in situ forming GH hydrogels can serve as an injectable/sprayable carrier of various therapeutic agents for wound healing applications., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The attachment of neutrophils to the endothelial surface and their migration towards the site of inflammation following chemokine gradients play an essential role in the innate immune response. Chemokines adhere to glycosaminoglycans on the endothelial surface to be detected by leukocytes and trigger their movement along surface- bound gradients in a process called haptotaxis. In assays to systematically study the response of leukocytes to surface-bound compounds both the spatial arrangement of the compound as well as the mode of immobilization need to be controlled. In this study microcontact printing was employed to create patterns of hydrophobic or functionalized thiols on gold-coated glass slides and CXCL8 was immobilized on the thiol coated areas using three different strategies. Human neutrophils adhered to the CXCL8-coated lines but not to the PEG-coated background. We could show that more cells adhered to CXCL8 adsorbed to hydrophobic octadecanethiol than on CXCL8 covalently bound to amino undecanethiol or CXCL8 specifically bound to immobilized heparin on aminothiol. Likewise general cell activity such as lamellipodia formation and random migration were most pronounced for CXCL8 adsorbed on a hydrophobic surface which may be attributed to the larger amounts of protein immobilized on this type of surface., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Macromolecules such as proteins are attracting increasing interest for molecular imaging. We previously proposed a novel strategy for preparing macromolecules labeled with a PET radionuclide, (11)C, using a cell-free translation system with (11)C-methionine. However, macromolecules tend to exhibit slower kinetics, thus requiring a longer scanning time. Here, we expand our strategy using (18)F, which has a longer half-life, with the cell-free translation system with 4-(18)F-fluoro-L-proline ((18)F-FPro). We evaluated (18)F-interleukin-8 ((18)F-IL-8) produced by this method in vitro and in vivo to provide a proof of concept of our strategy., Methods: We tested some fluorinated amino acids to be incorporated into a protein. Trans-(18)F-FPro was radiolabeled from the corresponding precursor. (18)F-IL-8 was produced using the cell-free translation system with trans-(18)F-FPro instead of natural L-proline with incubation at 37°C for 120 min. An in vitro binding assay of (18)F-IL-8 was performed using IL-8 receptor-expressing cells. After intravenous administration of (18)F-IL-8, in vivo PET imaging of IL-8 receptor-expressing xenograft-bearing mice was performed using a small-animal PET system., Results: FPro was identified as an amino acid incorporated into the protein. (18)F-IL-8 was successfully prepared using the cell-free translation system and trans-(18)F-FPro with the radiochemical yield of 1.5% (decay-corrected) based on trans-(18)F-FPro. In vitro binding assays of (18)F-IL-8 demonstrated its binding to IL-8 receptor. In vivo PET imaging demonstrated that (18)F-IL-8 clearly accumulated in IL-8 receptor-expressing xenografts in mice, unlike trans-(18)F-FPro., Conclusion: (18)F-IL-8 produced by this method binds to IL-8 receptors in vitro, and (18)F-IL-8 PET clearly visualizes its target receptor-expressing xenograft in vivo. Therefore, this technique might be useful for labeling macromolecules and performing preclinical evaluations of proteins of interest in vitro and in vivo., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Chronic ulcers represent a major health burden in our society. Despite many available therapies, a large number of ulcers do not heal. Protein based therapies fail in part due to proteolytic activity in the chronic wound bed. The aim of this in vitro study was to determine whether typical inflammatory cytokines and human salivary histatins remain stable when incubated with chronic wound extracts. Furthermore we determined whether a short exposure of histatins or cytokines was sufficient to exert long term effects on fibroblast migration. Stability of human recombinant cytokines IL-6 and CXCL8, and histatin variants (Hst1, Hst2, cyclic Hst1, minimal active domain of Hst1) in the presence of chronic wound extracts isolated from non-healing ulcers, was monitored by capillary zone electrophoresis. Migration-stimulating activity was assessed using a dermal fibroblast wound healing scratch assay. Histatins and cytokines stayed stable in saline for > 24 h at 37°C, making them ideal as an off-the-shelf product. However, incubation with chronic wound extracts resulted in serious breakdown of Hst1 and Hst2 (~50% in 8 h) and to lesser extent cyclic Hst1 and the minimal active domain of Hst1 (~20% in 8 h). The cytokines IL-6 and CXCL8 were more stable in chronic wound extracts (~40% degradation in 96 h). An initial 8-hour pulse of histatins or cytokines during a 96-hour study period was sufficient to stimulate fibroblast migration equally well as a continuous 96-hour exposure, indicating that they may possibly be used as novel bioactive therapeutics, exerting their activity for up to four days after a single exposure.

In humans, the chemokine CXCL1/MGSA (hCXCL1) plays fundamental and diverse roles in pathophysiology, from microbial killing to cancer progression, by orchestrating the directed migration of immune and non-immune cells. Cellular trafficking is highly regulated and requires concentration gradients that are achieved by interactions with sulfated glycosaminoglycans (GAGs). However, very little is known regarding the structural basis underlying hCXCL1-GAG interactions. We addressed this by characterizing the binding of GAG heparin oligosaccharides to hCXCL1 using NMR spectroscopy. Binding experiments under conditions at which hCXCL1 exists as monomers and dimers indicate that the dimer is the high-affinity GAG ligand. NMR experiments and modeling studies indicate that lysine and arginine residues mediate binding and that they are located in two non-overlapping domains. One domain, consisting of N-loop and C-helical residues (defined as α-domain) has also been identified previously as the GAG-binding domain for the related chemokine CXCL8/IL-8. The second domain, consisting of residues from the N terminus, 40s turn, and third β-strand (defined as β-domain) is novel. Eliminating β-domain binding by mutagenesis does not perturb α-domain binding, indicating two independent GAG-binding sites. It is known that N-loop and N-terminal residues mediate receptor activation, and we show that these residues are also involved in extensive GAG interactions. We also show that the GAG-bound hCXCL1 completely occlude receptor binding. We conclude that hCXCL1-GAG interactions provide stringent control over regulating chemokine levels and receptor accessibility and activation, and that chemotactic gradients mediate cellular trafficking to the target site., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Interleukin 1β (IL-1β) and interleukin 8 (IL-8) are two major pro-inflammatory cytokines which play a central role in initiation of inflammatory responses against bacterial- and viral-infections. IL-1β is a member of the interleukin 1 family proteins and IL-8 is classified as a CXC-chemokine. In the current study, putative IL-1β and IL-8 counterparts were identified from a black rockfish transcriptomic database and designated as RfIL-1β and RfIL-8. The RfIL-1β cDNA sequence consists of 1140 nucleotides with a 759bp open reading frame (ORF) which encodes a 252 amino acid (aa) protein, whereas the RfIL-8 cDNA sequence (898bp) harbors a 300bp ORF encoding a 99 aa protein. Furthermore, the RfIL-1β aa sequence contains an IL-1 super family-like domain and an N-terminal IL-1 super family propeptide, while the amino acid sequence of RfIL-8 consists of a typical chemokine-CXC domain. Analysis of sequenced BAC clones containing RfIL-1β and RfIL-8 showed each gene to contain 4 exons interrupted by 3 introns. Pairwise comparison and phylogeny analysis of these cytokine sequences clearly revealed their closer relationship with other corresponding members of teleosts compared to birds and mammals. Constitutive differences in RfIL-1β and RfIL-8 mRNA expression were detected in a tissue-specific manner with the highest expression of each mRNA in spleen tissue. Two immune challenge experiments were conducted with Streptococcus iniae and polyinosinic:polycytidylic acid (poly I:C; a viral double stranded RNA mimic), and transcripts were quantified in spleen and peripheral blood cells. Significantly increased RfIL-1β and RfIL8 transcript levels were detected with almost similar profile patterns, further suggesting a putative involvement of these pro-inflammatory cytokines in the rockfish immunity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: To investigate the correlation of the levels of interleukin-8 (IL-8) and IL-6 in the prostatic fluid with serum levels of serum prostate-specific antigen (PSA) in patients with benign prostatic hyperplasia (BPH) complicated by prostatitis., Methods: A series of 211 patients undergoing surgery of BPH were divided into BPH group (n=75) and BPH with prostatitis group (n=136) according to the white blood cell count in the prostatic fluid. The clinical and laboratory findings were compared between the two groups, and stepwise regression analysis was used to assess the association of IL-8 and IL-6 with serum PSA level., Results: No significant differences were found in age, BMI, blood pressure, blood glucose, blood lipids, IPSS score, PSA-Ratio, or prostate volume between the two groups (P<0.05). The patients with prostatitis had significantly increased serum PSA and prostate fluid IL-8 and IL-6 levels compared with those without prostatitis (P<0.001). Multiple linear regression analysis showed that IL-8 and IL-6 levels and white blood cell count in the prostatic fluid were all positively correlated with serum PSA level., Conclusion: Prostatitis is an important risk factor for elevated serum PSA level in patients with BPH, and both IL-8 and IL-6 levels in the prostatic fluid are correlated with serum PSA level.

Interleukin (IL)-1β and IL-8 are pro-inflammatory cytokines produced primarily by monocytes and macrophages in response to a variety of microbial and nonmicrobial agents. As yet, no molecular data have been reported for IL-1β and IL-8 of the Asian elephant. In the present study, we have cloned and sequenced the cDNA encoding IL-1β and IL-8 of the Asian elephant. The open reading frame (ORF) of Asian elephant IL-1β is 789 bp in length, encoded a propeptide of 263 amino acid polypeptide. The predicted protein revealed the presence of IL-1 family signature motif and an ICE cut site. Whereas, IL-8 contained 321 bp of open reading frame. Interestingly, the predicted protein sequence of 106 aa, contains an ELR motif immediately upstream of the CQC residues, common in all vertebrate IL-8 molecules. Identity levels of the nucleic acid and deduced amino acid sequences of Asian elephant IL-1β ranged from 68.48 (Squirrel monkey) to 98.57% (African elephant), and 57.78 (Sheep) to 98.47% (African elephant), respectively, whereas that of IL-8 ranged from 72.9% (Human) to 87.8% (African elephant), and 63.2 (human, gorilla, chimpanzee) to 74.5% (African elephant, buffalo), respectively. The phylogenetic analysis based on deduced amino acid sequenced showed that the Asian elephant IL-1β and IL-8 were most closely related to African elephant. Molecular characterization of these two cytokines, IL-1β and IL-8, in Asian elephant provides fundamental information necessary to progress the study of functional immune responses in this animal and gives the potential to use them to manipulate the immune response as recombinant proteins.

Chemokine CXCL8/interleukin-8 (IL-8) plays a crucial role in directing neutrophils and oligodendrocytes to combat infection/injury and tumour cells in metastasis development. CXCL8 exists as monomers and dimers and interaction of both forms with glycosaminoglycans (GAGs) mediate these diverse cellular processes. However, very little is known regarding the structural basis underlying CXCL8-GAG interactions. There are conflicting reports on the affinities, geometry and whether the monomer or dimer is the high-affinity GAG ligand. To resolve these issues, we characterized the binding of a series of heparin-derived oligosaccharides [heparin disaccharide (dp2), heparin tetrasaccharide (dp4), heparin octasaccharide (dp8) and heparin 14-mer (dp14)] to the wild-type (WT) dimer and a designed monomer using solution NMR spectroscopy. The pattern and extent of binding-induced chemical shift perturbation (CSP) varied between dimer and monomer and between longer and shorter oligosaccharides. NMR-based structural models show that different interaction modes coexist and that the nature of interactions varied between monomer and dimer and oligosaccharide length. MD simulations indicate that the binding interface is structurally plastic and provided residue-specific details of the dynamic nature of the binding interface. Binding studies carried out under conditions at which WT CXCL8 exists as monomers and dimers provide unambiguous evidence that the dimer is the high-affinity GAG ligand. Together, our data indicate that a set of core residues function as the major recognition/binding site, a set of peripheral residues define the various binding geometries and that the structural plasticity of the binding interface allows multiplicity of binding interactions. We conclude that structural plasticity most probably regulates in vivo CXCL8 monomer/dimer-GAG interactions and function., (© 2015 Authors; published by Portland Press Limited.)

Background: Different strategies have been proposed to target neoangiogenesis in gliomas, besides those targeting Vascular Endothelial Growth Factor (VEGF). The chemokine Interleukin-8 (IL-8) has been shown to possess both tumorigenic and proangiogenic properties. Although different pathways of induction of IL-8 gene expression have been already elucidated, few data are available on its post-transcriptional regulation in gliomas., Methods: Here we investigated the role of the microRNA miR-93 on the expression levels of IL-8 and other pro-inflammatory genes by RT-qPCR and Bio-Plex analysis. We used different disease model systems, including clinical samples from glioma patients and two glioma cell lines, U251 and T98G., Results: IL-8 and VEGF transcripts are highly expressed in low and high grade gliomas in respect to reference healthy brain; miR-93 expression is also increased and inversely correlated with transcription of IL-8 and VEGF genes. Computational analysis showed the presence of miR-93 consensus sequences in the 3'UTR region of both VEGF and IL-8 mRNAs, predicting possible interaction with miR-93 and suggesting a potential regulatory role of this microRNA. In vitro transfection with pre-miR-93 and antagomiR-93 inversely modulated VEGF and IL-8 gene expression and protein release when the glioma cell line U251 was considered. Similar data were obtained on IL-8 gene regulation in the other glioma cell line analyzed, T98G. The effect of pre-miR-93 and antagomiR-93 in U251 cells has been extended to the secretion of a panel of cytokines, chemokines and growth factors, which consolidated the concept of a role of miR-93 in IL-8 and VEGF gene expression and evidenced a potential regulatory role also for MCP-1 and PDGF (also involved in angiogenesis)., Conclusion: In conclusion, our results suggest an increasing role of miR-93 in regulating the level of expression of several genes involved in the angiogenesis of gliomas.

The interaction of leukocytes with surface bound ligands can be limited by the location of the molecules relative to the surface topology of the cell. In this report, we examine the dynamic response of neutrophils to IL-8-fractalkine chimera immobilized on bead surfaces, taking into account changes in receptor occupancy resulting from changes in surface topography. As a readout for receptor signaling, we observe the dynamics of calcium release in neutrophils following contact with the IL-8 coated surface. After a delay that depended on the initial area of contact and the surface density of IL-8, the cell began to phagocytose the IL-8 coated bead. This appeared to be a pre-requisite for release of calcium, which typically followed shortly after the initiation of phagocytosis. In separate experiments, effective kinetic coefficients for the formation of bonds between immobilized IL-8 and receptors on the cell surface were determined. Using these coefficients, we were able to estimate the number of bound receptors in the nascent contact zone. Kinetic modeling of the signaling response predicted that cell spreading and a concomitant increase in the density of occupied receptors would be required for the experimentally observed calcium dynamics. Postulating that there is an increase in receptor occupancy resulting from smoothing of the cell surface as it is stretched over the bead enabled us to obtain model predictions consistent with experimental observations. This study reveals the likely importance of membrane microtopology as a rate-limiting property and potential means of regulation of cell responses stimulated by two-dimensional surface interactions.

The use of paramagnetic constraints in protein NMR is an active area of research because of the benefits of long-range distance measurements (>10 Å). One of the main issues in successful execution is the incorporation of a paramagnetic metal ion into diamagnetic proteins. The most common metal ion tags are relatively long aliphatic chains attached to the side chain of a selected cysteine residue with a chelating group at the end where it can undergo substantial internal motions, decreasing the accuracy of the method. An attractive alternative approach is to incorporate an unnatural amino acid that binds metal ions at a specific site on the protein using the methods of molecular biology. Here we describe the successful incorporation of the unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) into two different membrane proteins by heterologous expression in E. coli. Fluorescence and NMR experiments demonstrate complete replacement of the natural amino acid with HQA and stable metal chelation by the mutated proteins. Evidence of site-specific intra- and inter-molecular PREs by NMR in micelle solutions sets the stage for the use of HQA incorporation in solid-state NMR structure determinations of membrane proteins in phospholipid bilayers.

We report the design and synthesis of an aquacarbonyl Ru(II) dication cis-[Ru(CO)2(H2O)4](2+) reagent for histidine (His)-selective metallation of interleukin (IL)-8 at site 33. The artificial, non-toxic interleukin (IL)-8-Ru(II)(CO)2 metalloprotein retained IL-8-dependent neutrophil chemotactic activity and was shown to spontaneously release CO in live cells.

Chemokine CXCL8 and its receptor CXCR1 are key mediators in combating infection and have also been implicated in the pathophysiology of various diseases including chronic obstructive pulmonary disease (COPD) and cancer. CXCL8 exists as monomers and dimers but monomer alone binds CXCR1 with high affinity. CXCL8 function involves binding two distinct CXCR1 sites - the N-terminal domain (Site-I) and the extracellular/transmembrane domain (Site-II). Therefore, higher monomer affinity could be due to stronger binding at Site-I or Site-II or both. We have now characterized the binding of a human CXCR1 N-terminal domain peptide (hCXCR1Ndp) to WT CXCL8 under conditions where it exists as both monomers and dimers. We show that the WT monomer binds the CXCR1 N-domain with much higher affinity and that binding is coupled to dimer dissociation. We also characterized the binding of two CXCL8 monomer variants and a trapped dimer to two different hCXCR1Ndp constructs, and observe that the monomer binds with ∼10- to 100-fold higher affinity than the dimer. Our studies also show that the binding constants of monomer and dimer to the receptor peptides, and the dimer dissociation constant, can vary significantly as a function of pH and buffer, and so the ability to observe WT monomer peaks is critically dependent on NMR experimental conditions. We conclude that the monomer is the high affinity CXCR1 agonist, that Site-I interactions play a dominant role in determining monomer vs. dimer affinity, and that the dimer plays an indirect role in regulating monomer function., (© 2014 The Protein Society.)

The evaluation of the potential of nanoparticles (NP) for adsorbing biomolecules and use of control approaches are important for accurate presentation of in vitro analytical data. In this study, seven types of NP including carbon black (CB), cerium dioxide (CeO2), copper oxide (CuO), indium trioxide (In2O3), nickel oxide (NiO), silicon dioxide (SiO2), and titanium dioxide (TiO2) were used for determining the adsorption ability of interleukin-8 (IL-8) under either (1) a cell-free condition where NP were incubated with supernatant of A549 cells, or (2) a cell-based condition, where cells were treated with NP. Under the cell-free condition, CB and TiO2 NP showed a high adsorption affinity for IL-8 in supernatants of both lipopolysaccharide (LPS)-stimulated and unstimulated A549 cells. In contrast, SiO2 and In2O3 NP displayed a relatively low adsorption affinity. Further, IL-8 adsorption was markedly reduced when NP were predispersed in fetal bovine serum. The results obtained under cell-based conditions using both stimulated and unstimulated cells were consistent with those of the cell-free condition. Data indicate that adsorption of IL-8 onto NP surface is variable depending on type of NP, preparation method of NP, and cellular inflammatory state. Thus, the cell-free adsorption assay may be utilized for reliable interpretation of data produced by in vitro cell-based methodology.

The objective of this study was to locate the functional region responsible for the chemotaxis-inducing activity of flounder interleukin 8 (IL-8), which lacks the glutamic acid-leucine-arginine (ELR) motif essential for the induction of neutrophil migration by mammalian IL-8. Using a human cell line, we produced a secretory recombinant protein of flounder IL-8, and analyzed its chemotaxis-inducing activity on leukocytes collected from the flounder kidney. The recombinant IL-8 induced significant migration in neutrophils, which were morphologically and functionally characterized. Using the Edman degradation method, the N-terminal amino acid sequence of rIL-8 was identified as VSLRSLGV. To examine the significance of the N-terminal region for the bioactivity of flounder IL-8, we prepared several recombinant proteins that containing mutations at the N-terminus. Modification of three residues (residues 9-11: serine-leucine-histidine) corresponding in position to the ELR motif in mammalian IL-8 did not reduce its chemotaxis-inducing activity. However, deletion of the first six or more residues significantly reduced its chemotaxis-inducing activity. We propose that residue 6 (leucine) at the N-terminus is important for the chemotaxis-inducing activity of flounder IL-8., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Peptide based inhibitors of protein-protein interactions are of great interest in proteomics, structural biology and medicinal chemistry. Optimized inhibitors can be designed by experimental approaches or by computational prediction. Ideally, computational models are adjusted to the peptide-protein complex of interest according to experimental data obtained in specific binding experiments. The chemokine CXCL8 (interleukin-8) is an interesting target for drug discovery due to its role in inflammatory diseases. Given the available structural data and information on its receptor interactions it constitutes a basis for the rational design of inhibitor peptides. Starting from the reported structure of CXCL8 in complex with a peptide derived from its receptor CXCR1 we developed a computational docking procedure to estimate the changes in binding energy as a function of individual amino acid exchanges. This indicates whether the respective amino acid residue must be preserved or can be substituted to maintain or improve affinity, respectively. To validate and improve the assumptions made in this docking simulation we established a fluorescence polarization assay for receptor-derived peptides binding to CXCL8. A peptide library was tested comprising selected mutants characterized by docking simulations. A number of predictions regarding electrostatic interactions were confirmed by these experiments and it was revealed that the model needed to be corrected for backbone flexibility. Therefore, the assay presented here is a promising tool to systematically improve the computational model by iterative cycles of modeling, experimental validation and refinement of the algorithm, leading to a more reliable model and peptides with improved affinity.

Chemokine CXCL-8 plays a central role in human immune response by binding to and activate its cognate receptor CXCR1, a member of the G-protein coupled receptor (GPCR) family. The full-length structure of CXCR1 is modeled by combining the structures of previous NMR experiments with those from homology modeling. Molecular docking is performed to search favorable binding sites of monomeric and dimeric CXCL-8 with CXCR1 and a mutated form of it. The receptor-ligand complex is embedded into a lipid bilayer and used in multi ns molecular dynamics (MD) simulations. A multi-steps binding mode is proposed: (i) the N-loop of CXCL-8 initially binds to the N-terminal domain of receptor CXCR1 driven predominantly by electrostatic interactions; (ii) hydrophobic interactions allow the N-terminal Glu-Leu-Arg (ELR) motif of CXCL-8 to move closer to the extracellular loops of CXCR1; (iii) electrostatic interactions finally dominate the interaction between the N-terminal ELR motif of CXCL-8 and the EC-loops of CXCR1. Mutation of CXCR1 abrogates this mode of binding. The detailed binding process may help to facilitate the discovery of agonists and antagonists for rational drug design.

Interleukin-8 (CXCL8, IL-8) is a proinflammatory chemokine important for the regulation of inflammatory and immune responses via its interaction with G-protein coupled receptors, including CXC receptor 1 (CXCR1). CXCL8 exists as both a monomer and as a dimer at physiological concentrations, yet the molecular basis of CXCL8 interaction with its receptor as well as the importance of CXCL8 dimer formation remain poorly characterized. Although several biological studies have indicated that both the CXCL8 monomer and dimer are active, biophysical studies have reported conflicting results regarding the binding of CXCL8 to CXCR1. To clarify this problem, we expressed and purified a peptide (hCXCR1pep) corresponding to the N-terminal region of human CXCR1 (hCXCR1) and utilized nuclear magnetic resonance (NMR) spectroscopy to interrogate the binding of wild-type CXCL8 and a previously reported mutant (CXCL8M) that stabilizes the monomeric form. Our data reveal that the CXCL8 monomer engages hCXCR1pep with a slightly higher affinity than the CXCL8 dimer, but that the CXCL8 dimer does not dissociate upon binding hCXCR1pep. These investigations also showed that CXCL8 is dynamic on multiple timescales, which may help explain the versatility in this interleukin for engaging its target receptors., (© 2014 The Protein Society.)

Dendritic cells (DCs) play a crucial role in the development of cell-mediated immunotherapy due to their ability to induce and maintain strong immune responses. In our study, we evaluated a biocompatible Ni(II)-NTA-modified poly(ethylene imine) dendritic glycopolymer (Ni(II)-NTA-DG) as new carrier system to increase the antigen uptake into iDCs for future DC-based immunotherapy. Ni(II)-NTA-DG led to an increase in His6-Gp160 uptake in monocytes and iDCs, where His6-Gp160 is localized in the early endosomal and lysosomal compartments. Ni(II)-NTA-DG and the formed polyplexes induced an activation of iDCs, showing an increasing expression of costimulatory molecules CD86, CD80, and proinflammatory cytokines IL-6 and IL-8. Beside no influencing effect of Ni(II)-NTA-DG and polyplexes on the maturation of antigen-bearing DCs, the mature peptide bearing DCs remained their ability to migrate along a gradient of CCR7 ligands. Thus, Ni(II)-NTA-DG with advancing biological properties is a promising carrier system for the future application in DC-based immunotherapy.

Interleukin-8 (IL-8) is a proinflammatory CXC chemokine that has been associated with the promotion of neutrophil chemotaxis, degranulation, and the pathogenesis of several neutrophil-infiltrating chronic inflammatory diseases. In the current study, we generated and characterized a 2'-fluoro-pyrimidine modified RNA aptamer (8A-35) against human IL-8. The 8A-35 aptamer binds to IL-8 with high specificity and affinity, yielding an estimated K(D) of 1.72 pM. NMR data revealed that the residues of Lys8, Leu10, Val63, Val66, Lys69 and Ala74 of IL-8 interact with aptamer. Moreover, the 8A-35 aptamer has a potent IL-8-neutralizing activity that can modulate multiple biological activities of IL-8 in human neutrophils, including migration, intracellular signaling, and intracellular Ca(2+) mobilization. Our results suggest that the 8A-35 aptamer has great potential to be a lead structure in the development of effective therapeutic agents against inflammatory diseases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Having unique architectural features, cationic polymeric nanocapsules (NCs) with well-defined covalently stabilized biodegradable structures were generated as potentially universal and safe therapeutic nanocarriers. These NCs were synthesized from allyl-functionalized cationic polylactide (CPLA) by highly efficient UV-induced thiol-ene interfacial cross-linking in transparent miniemulsions. With tunable nanoscopic sizes, negligible cytotoxicity and remarkable degradability, they are able to encapsulate doxorubicin (Dox) with inner cavities and bind interleukin-8 (IL-8) small interfering RNA (siRNA) with cationic shells. The Dox-encapsulated NCs can effectively bypass the P-glycoprotein (Pgp)-mediated multidrug resistance of MCF7/ADR cancer cells, thereby resulting in increased intracellular drug concentration and reduced cell viability. In vitro studies also showed that the NCs loaded with Dox, IL-8 siRNA and both agents can be readily taken up by PC3 prostate cancer cells, resulting in a significant chemotherapeutic effect and/or IL-8 gene silencing.

Chemokines mediate diverse functions from organogenesis to mobilizing leucocytes, and are unusual agonists for class-A GPCRs (G-protein-coupled receptors) because of their large size and multi-domain structure. The current model for receptor activation, which involves interactions between chemokine N-loop and receptor N-terminal residues (Site-I) and between chemokine N-terminal and receptor extracellular loop/transmembrane residues (Site-II), fails to describe differences in ligand/receptor selectivity and the activation of multiple signalling pathways. In the present study, we show in neutrophil-activating chemokine CXCL8 that the highly conserved GP (glycine-proline) motif located distal to both N-terminal and N-loop residues couples Site-I and Site-II interactions. GP mutants showed large differences from native-like to complete loss of function that could not be correlated with the specific mutation, receptor affinity or subtype, or a specific signalling pathway. NMR studies indicated that the GP motif does not influence Site-I interactions, but molecular dynamics simulations suggested that this motif dictates substates of the CXCL8 conformational ensemble. We conclude that the GP motif enables diverse receptor functions by controlling cross-talk between Site-I and Site-II, and further propose that the repertoire of chemokine functions is best described by a conformational ensemble model in which a network of long-range coupled indirect interactions mediate receptor activity.

Interleukin-8 (IL-8) is a CXC chemokine that plays key regulatory roles in the immune and inflammatory responses implicated in many human diseases. In this study, we identified and characterized an IL-8 homologue from the grass carp, Ctenopharyngodon idellus. A sequence alignment of the full-length cDNA and genomic DNA showed that the exon/intron organization of grass carp IL-8 (gcIL-8) is identical to those of other known CXC chemokine genes. A multiple alignment analysis showed that gcIL-8 is an ELR(-)CXC chemokine, and its deduced amino acid sequence shares 81% and 36% identity with common carp IL-8s L1 (GenBank ID: ABE47600) and L2 (GenBank ID: AB470924), respectively, suggesting that it belongs to the lineage 1 group of fish IL-8 proteins. On a phylogenetic tree, gcIL-8 clustered with other teleost IL-8 proteins to form a fish-specific clade, clearly distinct from those of bird, mammal, and amphibian proteins. Real-time quantitative PCR analysis indicated that gcIL-8 is differentially expressed in various tissues under normal conditions and that the expression of gcIL-8 mRNA in immune-related tissues is clearly upregulated by Aeromonas hydrophila infection. To explore the biological effects of gcIL-8, we produced a recombinant protein, rgcIL-8, in a prokaryotic expression system. Purified rgcIL-8 was confirmed to be chemoattractive for head kidney neutrophils and mononuclear leukocytes in vitro. Our histopathological study also revealed that rgcIL-8 exerts proinflammatory effects by inducing neutrophil infiltration and erythrocyte extravasation. Overall, these results suggest that IL-8 is crucially involved in the inflammatory responses of fish., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Although the interaction between interleukin-8 (IL-8) and glycosaminoglycans (GAGs) is crucial for the mediation of inflammatory effects, little is known about the site specificity of this interaction. Therefore, we studied complexes of IL-8 and heparin (HEP) as well as other GAGs in a multidisciplinary approach, involving site-directed mutagenesis, mass spectrometry, fluorescence and solution NMR spectroscopy as well as computer modeling. The interaction between GAG and IL-8 is largely driven by the amine groups of the lysine and the guanidinium groups of arginine side chains. However, due to fast exchange with the solvent, it is typically not possible to detect NMR signals of those groups. Here, we applied reductive (13)C-methylation of the lysine side chains providing sensitive NMR probes for monitoring directly the sites of GAG interaction in (1)H-(13)C correlation experiments. We focused on the lysine side chains K25, K28, K59, K69 and K72 of IL-8 (1-77), which were reported to be involved in the binding to GAGs. The NMR signals of these residues were assigned in (1)H-(13)C HSQC spectra through the help of site-directed mutagenesis. NMR and fluorescence titration experiments in combination with molecular docking and molecular dynamics simulations were applied to investigate the involvement of each lysine in the binding with HEP and various GAG hexasaccharides. We identified K25, K69 and K72 to be the most relevant binding anchors of IL-8(1-77) for the analyzed GAGs.

Rationale: Intestinal epithelial cells (IEC) secrete many chemokines in response to proinflammatory stimuli. We investigated their role in the mucosal inflammatory response in the intestine, by developing a non-targeted approach for analyzing the profile of peptides secreted by stimulated IEC, based on differential mass spectrometry analysis., Methods: Lipopolysaccharide (LPS) was incubated with IEC as a proinflammatory stimulus. Differential peptidomic analysis was then carried out, comparing the profiles of IEC with and without LPS stimulation. A mass spectrometry procedure was developed, based on a liquid chromatography/tandem mass spectrometry (LC/MS/MS) approach without enzymatic pretreatment of the peptides. Partial de novo sequencing was carried out by Fourier transform ion cyclotron resonance (FTICR), and the native peptides in the culture media were identified., Results: A major ion (m/z 7862.51) detected after stimulation was identified as GRO alpha and a minor ion (m/z 8918.17) was identified as IL-8. ELISA-based comparisons gave results consistent with those obtained by MS. Surprisingly, GRO alpha was secreted in amounts 5 to 15 times higher than those for IL-8 in our cellular model. The truncated form of IL-8, resulting from activation, was detected and distinguished from the native peptide by MS, whereas this was not possible with enzyme-linked immunosorbent assay (ELISA)., Conclusions: Mass spectrometric analysis of culture media can be used to identify the principal peptides produced in response to the stimulation of IEC, and their metabolites. Mass spectrometry provides a comprehensive view of the chemokines and peptides potentially involved in gut inflammation, making it possible to identify the most appropriate peptides for further quantification., (Copyright © 2013 John Wiley & Sons, Ltd.)

Microcystin-LR (MCLR) is a widespread cyanotoxin and has immunotoxicity to animals, including fish. Chemokines are considered to play important roles in inflammatory response induced by MCLR. In this study, we cloned the full-length cDNA of interleukin-8 (IL-8) from bighead carp (Aristichthys nobilis) for the first time. The full-length IL-8 cDNA was 552 bp and contained a 297-bp open-reading frame that encoded for a 98-amino acid protein. The deduced IL-8 protein had a typical aspartic acid (D)-leucine (L)-arginine (R) and a CXC motif at the N-terminal, which were conserved in most fish species. Phylogenetic analysis showed that bighead carp IL-8 protein was grouped in the teleost IL-8 lineage 2. Under normal conditions, the expression of IL-8 is constitutive and weak in all tested tissues. However, MCLR treatment could significantly increase the transcription of IL-8 in bighead carp in a temporal- and dose-dependent pattern. The present study will help us to understand more about the evolution of IL-8 and its function in the MCLR induced proinflammatory response in bighead carp.

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline's unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

IL (interleukin)-8 [CXCL8 (CXC chemokine ligand 8)] exerts its role in inflammation by triggering neutrophils via its specific GPCRs (G-protein-coupled receptors), CXCR1 (CXC chemokine receptor 1) and CXCR2, for which additional binding to endothelial HS-GAGs (heparan sulphate-glycosaminoglycans) is required. We present here a novel approach for blocking the CXCL8-related inflammatory cascade by generating dominant-negative CXCL8 mutants with improved GAG-binding affinity and knocked-out CXCR1/CXCR2 activity. These non-signalling CXCL8 decoy proteins are able to displace WT (wild-type) CXCL8 and to prevent CXCR1/CXCR2 signalling thereby interfering with the inflammatory response. We have designed 14 CXCL8 mutants that we subdivided into three classes according to number and site of mutations. The decoys were characterized by IFTs (isothermal fluorescence titrations) and SPR (surface plasmon resonance) to determine GAG affinity. Protein stability and structural changes were evaluated by far-UV CD spectroscopy and knocked-out GPCR response was shown by Boyden chamber and Ca2+ release assays. From these experiments, CXCL8(Δ6F17KF21KE70KN71K) emerged with the most promising in vitro characteristics. This mutant was therefore further investigated in a murine model of mBSA (methylated BSA)-induced arthritis in mice where it showed strong anti-inflammatory activity. Based on these results, we propose that dominant-negative CXCL8 decoy proteins are a promising class of novel biopharmaceuticals with high therapeutic potential in inflammatory diseases.

Recently, sortase A (SrtA) from Staphyloccus aureus moved into the focus of bioscience because of its ability to incorporate site specific modifications into proteins. The enzyme was mostly used to modify target proteins in an analytical scale, to study biomolecules in their cellular context. In this study, we show the applicability of SrtA mediated ligation for site specific modification of proteins in a large scale. Therefore, the reaction was first optimized using peptides and subsequently new reaction conditions were applied for the large scale biotinylation of interleukin-8. Furthermore, we established C-terminal immobilization of the SrtA on a PEG based resin and could demonstrate maintaining enzymatic activity. Immobilized SrtA significantly facilitates previous ligation protocols as the enzyme can be easily recycled. Also, the removal of excess reaction solution and the whole washing process is significantly accelerated, as centrifugation or filtration techniques can be applied instead of time-consuming chromatography steps., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Macrophage migration inhibitory factor (MIF) is a proinflammatory molecule in mammals that, unusually for a cytokine, exhibits tautomerase and oxidoreductase enzymatic activities. Homologues of this well conserved protein are found within diverse phyla including a number of parasitic organisms. Herein, we produced recombinant histidine-tagged Toxoplasma gondii MIF (TgMIF), a 12-kDa protein that lacks oxidoreductase activity but exhibits tautomerase activity with a specific activity of 19.3 μmol/min/mg that cannot be inhibited by the human MIF inhibitor ISO-1. The crystal structure of the TgMIF homotrimer has been determined to 1.82 Å, and although it has close structural homology with mammalian MIFs, it has critical differences in the tautomerase active site that account for the different inhibitor sensitivity. We also demonstrate that TgMIF can elicit IL-8 production from human peripheral blood mononuclear cells while also activating ERK MAPK pathways in murine bone marrow-derived macrophages. TgMIF may therefore play an immunomodulatory role during T. gondii infection in mammals.

Chemokines, 8 kDa proteins implicated in leukocyte migration via oligomerization, bind to glycosaminoglycans (GAGs) during the inflammation response as a means to regulate chemokine migration. Structural characterization of chemokines non-covalently bound to GAGs provides physiologically meaningful data in regard to routine inmmunosurveillance and disease response. In order to analyze the structures resulting from the GAG:chemokine interaction, we employed ion mobility mass spectrometry (IMMS) to analyze monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, and interleukin-8 (IL-8), a CXC chemokine, along with their individual interactions with GAG heparin octasaccharides. We show that MCP-1 and IL-8 are physiologically present as a dimer, with MCP-1 having two variants of its dimeric form and IL-8 having only one. We also show that the MCP-1 dimer adopts two conformations, one extended and one compact, when bound to a dodecasulfated heparin octasaccharide. Binding of MCP-1 to heparin octasaccharide isomers of varying sulfation patterns results in similar arrival time distribution values, which suggests minimal distinguishing features among the resultant complexes. Additionally, tandem mass spectrometry (MS/MS) showed that the binding of MCP-1 to a heparin octasaccharide has different dissociation patterns when compared with the corresponding IL-8 bound dimer. Overall, IMMS and MS/MS were used to better define the structural tendencies and differences associated with CC and CXC dimers when associated with GAG octasaccharides.

IL-8 plays a crucial role in acute inflammation by recruiting and mediating neutrophils and other cells and in initiating the oxidative burst in neutrophils and inducing wound healing by promoting angiogenesis. In the present study, the full-length cDNA and genome sequence of interleukin-8 (LcIL-8) were cloned from large yellow croaker Larimichthys crocea. The LcIL-8 cDNA sequence was 931 bp, containing a 118-bp 5'-untranslated region (UTR), a 528-bp 3'-UTR and a 285-bp open reading frame (ORF) which encoded 94 amino acids. A putative signal peptide including 20 amino acid residues was found at N-terminal in LcIL-8 protein. And a small cytokine (SCY) domain showing a typical CXC chemokine gene organization was predicted in LcIL-8. The genome sequence of LcIL-8 gene was composed of 1930 nucleotides, including four exons and three introns. Quantitative real-time PCR analysis indicated a broad expression of LcIL-8 in most detected tissues, with the most predominant expression in liver. After injection with LPS, Vibrio parahaemolyticus and poly I:C, LcIL-8 expression levels showed up-regulation in head-kidney and spleen. The peak value was in the spleen with 6 times (at 6 h) greater expression than in the control after LPS injection (p < 0.05). However, LcIL-8 transcripts showed down-regulation in the liver after all the three stimulants injection. Recombinant LcIL-8 mature peptide was produced by Escherichia coli, which enhanced the production of superoxide anion in PCK cells. In addition, 5 single-nucleotide polymorphisms (SNPs) were identified in LcIL-8 gene. The results suggested that LcIL-8 might play an important role in fish's immune response, and the SNPs might be used as potential candidate molecular markers for selection for disease-resistant large yellow croaker., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Atherosclerosis is a chronic inflammatory disease with multiple contributing factors. Hyperlipidemia is one of the major independent risks, and interleukin-8 (IL-8), as an inflammatory factor, plays an important role in the development of atherosclerosis. The aims of the study were to examine the therapeutic efficacy of G31P, an antagonist of IL-8 receptor, with a mouse model of hyperlipidemia and the potential mechanisms of G31P through the vascular smooth muscle cell (VSMC) proliferation and migration in a cell line. In vivo study: Male BALB/c mice were fed a high-fat diet for 6 months. G31P was injected subcutaneously. Blood keratinocyte chemoattractant, lipid profile, and aorta expression of inflammatory factors, matrix metalloproteinases, MMP2 and MMP9 were investigated. In vitro study: A7R5 cells were treated with IL-8 with/without G31P. Cell proliferation and migration were investigated. G31P significantly suppressed the hyperlipidermia-induced abnormal lipid profile and increased IL-8, proinflammatory factor, MMP2 and MMP9 expression. G31P also inhibited VSMC proliferation and migration both in vitro and in vivo. These findings indicate the potential therapeutic effects of G31P in preventing the development of atherosclerosis by antagonizing IL-8 receptor and decreasing the biologic activity of IL-8., (Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.)

CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation. CXCR1 is a class A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences, as well as addition of stabilizing antibodies and small molecules that facilitate crystallization in cubic phase monoolein mixtures. The intracellular loops of GPCRs are crucial for G-protein interactions, and activation of CXCR1 involves both amino-terminal residues and extracellular loops. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.

The interactions between regulatory proteins such as interleukin-8 (IL-8) and glycosaminoglycans are of great interest both for the general understanding of regulatory processes in biology and for the development of implant coatings and innovative materials that suppress undesired immune responses and improve wound healing. In previous work, a number of residues of IL-8 that interact strongly with several glycosaminoglycans (GAGs) have been identified. In particular, the negatively charged Glu75 was reported to be involved in interactions with charged GAGs. To improve understanding of the role of this residue, we generated a selectively (15)N-labeled E75K variant of IL-8(1-77) by expressed protein ligation. NMR and fluorescence spectroscopy in combination with molecular modeling were applied to evaluate the particular role of residue 75 in interactions with GAGs. Remarkably, more residues in the variant responded to GAG binding than in the wild-type. For the first time, we identified amino acids 34 to 36 as additional residues in the loop region of IL-8(1-77) that participate in the interactions with GAGs. These findings indicate that the N terminus of the E75K variant is more important as a second binding site for GAGs than that of the wild-type IL-8(1-77)., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An efficient aqueous Wittig reaction was enabled on protein bioconjugation for the first time. By investigating the reaction on small molecules, peptides, and proteins, a site-specific reaction targeting "aldehyde tag" was presented. A variety of functional groups could be introduced into the protein of interest.

Glycosaminoglycans (GAGs) of the extracellular matrix (ECM) contribute to the regulation of physiological processes by binding various immune-competent proteins. Due to their large structural diversity, the analysis of the binding properties and their functional consequences is challenging. The cytokine interleukin-8 (IL-8) is involved in the recruitment of neutrophils to inflammatory sites. Here, we investigated the interaction of heparin hexasaccharides and recombinant human IL-8, consisting of 77 amino acids using fluorescence and NMR spectroscopy. A dissociation constant of 2.0±0.4 μM was determined for the heparin-IL-8 complex, which is slightly higher than what has been found for chondroitin-6-sulfate (K(D)=1.4±0.4 μM) [Pichert, A.; Samsonov, S. A.; Theisgen, S.; Thomas, L.; Baumann, L.; Schiller, J.; Beck-Sickinger, A. G.; Huster, D.; Pisabarro, M. T. Glycobiology2012, 22, 134-145], suggesting an important role of the sulfate group at position 6 of the second ring in the disaccharide unit of the GAGs in this interaction. In addition, the influence of long-chain hyaluronan, chondroitin sulfate, and heparin on IL-8-induced chemotaxis and oxidative activity of neutrophils was examined. Only the incubation of heparin with IL-8 affected the IL-8-mediated chemotaxis of neutrophils. However, all investigated GAGs enhanced the IL-8-induced formation of reactive oxygen species in neutrophils, which is an entirely new finding. This work provides a representative example of how protein functions can be regulated by different GAGs of the ECM., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

During the immune response, the cytokine interleukin 8 (IL-8, CXCL8) functions as a strong chemoattractant for polymorphonuclear leukocytes helping to direct these cells to infected/injured sites. This review focuses on the interaction of IL-8 with sulfated glycosaminoglycans expressed on cell surfaces and the extracellular matrix. This interaction contributes to the recruitment of polymorphonuclear cells from blood, penetration of these cells through the vessel wall, and their directed migration to inflammatory sites. Regulatory aspects of the interplay between IL-8 and heparan sulfate, the most abundant glycosaminoglycan, are highlighted. In this field, the large natural heterogeneity of glycosaminoglycans represents a great challenge that impedes the modeling of IL-8 functions. The interaction of IL-8 with newly developed artificial sulfated hyaluronan derivatives is also considered as these artificial substrates are an important tool for development of new materials in regenerative medicine.

Oligomerization plays an important role in the function of many proteins. Thus, understanding, predicting, and, ultimately, engineering oligomerization presents a long-standing interest. From the perspective of structural biology, protein-protein interactions have mainly been analyzed in terms of the biophysical nature and evolution of protein interfaces. Here, our aim is to quantify the importance of the larger structural context of protein interfaces in protein interaction evolution. Specifically, we ask to what extent intersubunit geometry affects oligomerization state. We define a set of structural parameters describing the overall geometry and relative positions of interfaces of homomeric complexes with different oligomeric states. This allows us to quantify the contribution of direct sequence changes in interfaces versus indirect changes outside the interface that affect intersubunit geometry. We find that such indirect, or allosteric mutations affecting intersubunit geometry via indirect mechanisms are as important as interface sequence changes for evolution of oligomeric states.

Interleukin-8 (IL-8 or CXCL8), the archetypal member of the CXC chemokine subfamily, stimulates neutrophil chemotaxis by activating receptors CXCR1/IL8RA and CXCR2/IL8RB. Previous mutational studies have implicated both the N-terminal and third extracellular loop (E3) regions of these receptors in binding to IL-8. To investigate the interactions of these receptor elements with IL-8, we have constructed soluble proteins in which the N-terminal and E3 elements of either CXCR1 or CXCR2 are juxtaposed on a soluble scaffold protein; these are termed CROSS-N(X1)E3(X1) and CROSS-N(X2)E3(X2), respectively. Isothermal titration calorimetry and nuclear magnetic resonance spectroscopy were used to compare the IL-8 binding properties of the receptor mimics to those of control proteins containing only the N-terminal or E3 receptor element. CROSS-N(X2)E3(X2) bound to monomeric IL-8 with the same affinity and induced the same chemical shift changes as the control protein containing only the N-terminal element of CXCR2, indicating that the E3 element of CXCR2 did not contribute to IL-8 binding. In contrast, CROSS-N(X1)E3(X1) bound to IL-8 with ~10-fold increased affinity and induced different chemical shift changes compared to the control protein containing only the N-terminal element of CXCR1, suggesting that the E3 region of CXCR1 was interacting with IL-8. However, a chimeric protein containing the N-terminal region of CXCR1 and the E3 region of CXCR2 (CROSS-N(X1)E3(X2)) bound to IL-8 with thermodynamic properties and induced chemical shift changes indistinguishable from those of CROSS-N(X1)E3(X1) and substantially different from those of CROSS-N(X2)E3(X2). These results indicate that the N-terminal and E3 regions of CXCR1 interact synergistically to achieve optimal binding interactions with IL-8.

Chemokines exert their function by binding the GPCR class of receptors on leukocytes and cell surface GAGs in target tissues. Most chemokines reversibly exist as monomers and dimers, but very little is known regarding the molecular mechanisms by which the monomer-dimer equilibrium modulates in vivo function. For the chemokine CXCL8, we recently showed in a mouse lung model that monomers and dimers are active and that the monomer-dimer equilibrium of the WT plays a crucial role in regulating neutrophil recruitment. In this study, we show that monomers and dimers are also active in the mouse peritoneum but that the role of monomer-dimer equilibrium is distinctly different between these tissues and that mutations in GAG-binding residues render CXCL8 less active in the peritoneum but more active in the lung. We propose that tissue-specific differences in chemokine gradient formation, resulting from tissue-specific differences in GAG interactions, are responsible for the observed differences in neutrophil recruitment. Our observation of differential roles played by the CXCL8 monomer-dimer equilibrium and GAG interactions in different tissues is novel and reveals an additional level of complexity of how chemokine dimerization regulates in vivo recruitment.

The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix, and proteins such as growth factors and chemokines play critical roles in cellular regulation processes. Therefore, the design of GAG derivatives for the development of innovative materials with bio-like properties in terms of their interaction with regulatory proteins is of great interest for tissue engineering and regenerative medicine. Previous work on the chemokine interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. However, the extracellular matrix contains other GAGs, such as hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), which have so far not been characterized in terms of their distinct molecular recognition properties towards IL-8 in relation to their length and sulfation patterns. NMR and molecular modeling have been in great part the methods of choice to study the structural and recognition properties of GAGs and their protein complexes. However, separately these methods have challenges to cope with the high degree of similarity and flexibility that GAGs exhibit. In this work, we combine fluorescence spectroscopy, NMR experiments, docking and molecular dynamics simulations to study the configurational and recognition properties of IL-8 towards a series of HA and CS derivatives and DS. We analyze the effects of GAG length and sulfation patterns in binding strength and specificity, and the influence of GAG binding on IL-8 dimer formation. Our results highlight the importance of combining experimental and theoretical approaches to obtain a better understanding of the molecular recognition properties of GAG-protein systems.

Introduction: This review presents the current in vitro and in vivo animal and human research on the roles of IL-8 in ocular inflammatory diseases., Materials and Methods: Data sources were a literature review using Pub Med, Medline, and ISI databases (from 1990 to 2011). Search items included interleukine-8 (IL-8), CXCL8, chemokines, cytokines, alone or in combination with the, serum, aqueous, vitreous, eye, ocular, ocular tissues, ophthalmic, and review., Results: IL-8 may be involved in primary or secondary ocular inflammations. Ocular effects of IL-8 differ based on the source of the secretion and site of the action. The most important effects of IL-8 in the eyes are angiogenic activities and induction of ocular inflammation., Conclusion: IL-8 plays important roles in ocular inflammation and angiogenesis in conjunctiva, cornea, iris, retina, and orbit. Anti-IL-8 targeted immunotherapy has been introduced as an important treatment modality, provided that IL-8 signal blocking takes place in desired areas and tissues.

In this study, an IL-8 homologue has been cloned and identified from South African clawed frog Xenopus laevis (designated XlIL-8). The open reading frame (ORF) of XlIL-8 consists of 312 bases encoding a protein of 103 amino acids. The chemokine CXC domain, which contained Glu-Leu-Arg (ELR) motif and four cysteine residues, was well conserved in South African clawed frog IL-8. By quantitative real-time PCR, mRNA transcript of XlIL-8 was detectable in all the examined tissues with higher level in spleen and kidney. The temporal expression of XlIL-8 mRNA in the monocytes was up-regulated by lipopolysaccharide (LPS) stimulation and reached the maximum level at about 6h post-stimulation. Recombinant soluble XlIL-8 (XlsIL-8) was fused with a small ubiquitin-related modifier gene (SUMO) to enhance the soluble expression level in Escherichia coli BL21 (DE3). The fusing protein SUMO-XlsIL-8 was purified using metal chellate affinity chromatography (Ni-NTA) and cleaved by a SUMO-specific protease, then confirmed by SDS-PAGE and Western blotting analysis. Chemotaxis assays showed that lymphocytes but not monocytes could be recruited toward SUMO-XlsIL-8 or XlsIL-8 protein in a dose-dependent manner in vitro. The present study may be useful for understanding the anti-bacteria immunity in amphibian and gives the potential to use the recombinant proteins to manipulate the immune response., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The biopolymer matrigel loaded with cytokine can be used for the recruitment in vivo of specific cell populations and as a vector for the preparation of cell cultures. Data demonstrate that the injection of the matrigel biopolymer supplemented with interleukin-8 (IL-8) in the leech Hirudo medicinalis can be used to purify cell populations showing the same morphofunctional and molecular mechanisms of specific populations of vertebrate hematopoietic precursor cells involved in tissue repair. These cells spontaneously differentiated into myofibroblasts. This approach highlights how the innovative use of a cytokine-loaded biopolymer for an in vivo cell sorting method, applied to a simple invertebrate model, can be a tool for studying myofibroblast cell biology and its regulation, step by step., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chemokines and their receptors control cell migration associated with routine immune surveillance, inflammation and development. They are also implicated in a large number of inflammatory diseases, cancer and HIV. Here we describe a rapid and efficient way to express and purify milligram quantities of multiple chemokine ligands (CCL7/MCP-3, CCL14/HCC-1, CCL3/MIP-1α and CXCL8/IL-8) containing C-terminal modifications to enable coupling to fluorescent dyes or small molecules such as biotin, in vitro. These labeled chemokines display wild-type behavior in both receptor binding and calcium mobilization assays. The ability to rapidly and inexpensively produce labeled chemokines opens the way for their use in many applications, including non-traditional chemokine-receptor interaction studies, both on intact cells and with purified receptor reconstituted in artificial membranes in vitro. Furthermore, the ability to immobilize chemokines to obtain ligand affinity columns aids in efforts to purify chemokine receptors for structural and biophysical studies, by facilitating the separation of functional proteins from their non-functional counterparts., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Neutrophil infiltration caused by IL-8 production is a central mechanism in alcohol-induced hepatitis. This study was performed to examine if an epigenetic mechanism is involved in alcohol-induced IL-8 production. Mice were pair-fed an alcohol-containing liquid diet for 4 weeks. Alcohol exposure induced hepatitis as indicated by increased expression of keratinocyte-derived cytokine (mouse IL-8) and neutrophil infiltration. Alcohol exposure induced histone 3 hyperacetylation owing to inhibition of histone deacetylase (HDAC) in association with NF-κB activation. Cell culture studies showed that alcohol exposure induced IL-8 and cytokine-induced neutrophil chemoattractant-1 (CINC-1, rat IL-8) production in human VL-17A cells and rat H4IIEC3 cells, respectively, dependent on acetaldehyde production, oxidative stress, and zinc release. Zinc deprivation alone induced CINC-1 production and acted synergistically with lipopolysaccharide or tumor necrosis factor-α on CINC-1 production. Zinc deprivation induced histone 3 hyperacetylation at lysine 9 through suppression of HDAC activity. Zinc deprivation caused nuclear translocation of NF-κB, and reduced HDAC binding to NF-κB. Chromatin immunoprecipitation (ChIP) showed that zinc deprivation caused histone 3 hyperacetylation as well as increased NF-κB binding to the CINC-1 promoter. In conclusion, inactivation of HDAC caused by zinc deprivation is a novel mechanism underlying IL-8 up-regulation in alcoholic hepatitis., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Methyl thiazolyl tetrazolium (MTT) and interleukin-8 (IL-8) assays are common colorimetric methods to measure mitochondrial activity and drug induced pro-inflammatory factors. However, many reports have described how MTT absorbance and cytokine adsorption could limit their applicability in evaluating the cytotoxicity of nanomaterials. In this study, we used an acid-containing isopropanol complex as a substitute for dimethyl sulfoxide (DMSO) solvent to dissolve MTT formazan, which was expected to diminish the absorbance of nano-ZnO at 570 nm where maximum absorbance for the MTT formazan was detected. In addition, we used a serum-containing medium to prevent the possible effects of IL-8 protein adsorption in the nano-ZnO and nano-TiO2. The results showed that the modified method by using acid-containing isopropanol step in MTT assay, nano-ZnO exposed to human lung epithelial cells had the lowest cell viability (from 12.5 to 50 microg mL(-1)) and EC50 value (8.4 microg mL(-1)) comparing with the conventional MTT protocol or adding phosphate buffered saline (PBS) to wash cells. The reason for this was the acid-containing isopropanol completely dissolved nano-ZnO with no additional absorbance when compared to the background solvent at 570 nm. On the other hand, the IL-8 protein had a marked influence on the adsorption of nano-TiO2 in the serum-free medium. While only at 100 microg mL(-1) of nano-ZnO, an influence on the adsorption of IL-8 was observed. This could be attributed to the different charges on the surface of nanomaterials. This problem could be overcome through the addition of fetal bovine serum (FBS) to the medium.