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The three-dimensional structure of a novel four amino acid truncated form of the CXC chemokine GRObeta [5-73] isolated from bone marrow stromal cells with potent hematopoietic and anti-infective activities has been determined by two-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy in solution. On the basis of 1878 upper distance constraints derived from nuclear Overhauser effects (NOE) and 314 dihedral angle constraints, a group of 20 conformers representing the solution structure of the human GRObeta [5-73] was computed with the program DYANA. At the concentrations used for NMR study, GRObeta [5-73] forms a dimer in solution that is architectured by a six-stranded antiparallel beta-sheet (residues 25 to 29, 39 to 44, 49 to 52) and a pair of helices (residues 58 to 68) with 2-fold symmetry, while the C terminus of the protein is disordered. The average of the pairwise root-mean-square deviations of individual NMR conformers relative to the mean coordinates for the backbone atoms N, C(alpha) and C' of residues 5 to 68 is 0.47 A. Overall, the global fold of GRObeta [5-73] is similar to that of the previously reported NMR structure of GROalpha and the NMR and X-ray structures of interleukin-8. Among these three CXC chemokines, GRObeta [5-73] is most similar in structure to GROalpha. Significant differences between GRObeta [5-73], GROalpha and interleukin-8 are in the N-terminal loop comprising residues 12 to 19. The N-terminal arm containing the conserved ELR motif and the loop of residues 30 to 38 containing the GPH motif are different among these three CXC chemokines. The structural differences in these two regions may be responsible for the specificity of the receptor binding and biological activity of different chemokines., (Copyright 1999 Academic Press.)

Cytokine-induced neutrophil chemoattractant (CINC/Gro) is a chemotactic factor for neutrophils in the rat and a member of the CXC chemokine family. The refined three-dimensional structure of CINC/Gro was derived with the aid of heteronuclear magnetic resonance spectroscopy and a hybrid method of distance geometry and simulated annealing. The backbone atomic r.m.s. differences for 19 structures about the mean coordinates for residues 7 to 70 are 0.69+/-0.15 A for the dimer and 0.56+/-0.13 A for the monomer. The N terminal region containing an ELR sequence, an essential motif for chemotactic activity, is disordered in solution, as in other CXC chemokines. The dimer structure consists of a six-stranded anti-parallel beta sheet with two C-terminal alpha helices on the beta sheet. The overall dimer structure of CINC/Gro is similar to that of human MGSA, though the backbone r.m.s.d.s between CINC/Gro and the two MGSA structures were high (1.81 and 2.34 A for the monomer) in spite of the high sequence homology (67%). The major difference resides in the relative position of the C-terminal alpha helix with respect to the beta sheet. This results in a difference of interhelical distances in the dimer: wider (15 A) in CINC/Gro, as in IL-8, than in MGSA (11.7 and 10 A).

T-cell infiltration into synovium is a crucial process for rheumatoid arthritis (RA). To investigate the mechanism of T-cell infiltration, we studied T-cell attracting activity in synovial tissue extracts of RA or osteoarthritis (OA) whose synovium lacks T-cell accumulation. RA extracts attracted twofold more T cells than OA extracts. By gel filtration column chromatography the activity of RA extracts was separated into two peaks; one was eluted at the 67-kDa region and the other was at the 12-kDa region, while the latter was absent in OA extracts. The activity eluted at the 12-kDa region was absorbed mostly by an antibody against IL-8/NAP-1, a potent T-cell chemotactic factor. IL-8/NAP-1 concentrations in RA extracts were much higher than those in OA extracts and correlated to T-cell attracting activity eluted at the 12-kDa region. The checkerboard analysis revealed that the 67-kDa activity was chemokinetic but not chemotactic. These results suggest that IL-8/NAP-1 is the major T-cell chemoattractant in RA-synovium.

Interleukin-8 (IL-8), a CXC chemokine, is known to bring about chemotaxis and activation of neutrophils through high affinity binding to at least two distinct receptors, receptor-A and receptor-B. The IL-8 homolog melanoma growth stimulating activity (MGSA) is also active toward neutrophils. In contrast to IL-8, MGSA binds receptor-B with high affinity and binds receptor-A with approximately 400-fold lower affinity. Using the structure of IL-8 (Clore et al.(1990) Biochemistry, 29, 1689-1696; Baldwin et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 502-506) and the NMR-determined structure of MGSA (Fairbrother et al. (1994) J. Mol. Biol. 242, 252-270), we designed variants of both IL-8 and MGSA to investigate the basis of specificity for binding of these chemokines to the IL-8 receptors. The most outstanding structural difference between IL-8 and MGSA lies in the loop preceding the first beta-strand. When the corresponding (shorter) loop from MGSA was swapped into IL-8, both receptor-A and receptor-B binding affinities were significantly (>300-fold) reduced. However, with additional mutations that affect packing interactions, an IL-8 variant specific for receptor-B binding was produced. Conversely, when the same loop from IL-8 was swapped into MGSA, receptor-B binding was maintained with only a approximately 30-fold reduction in receptor-A affinity. Again, mutations affecting packing of the loop yielded a MGSA variant with high affinity for both receptors, like IL-8. Finally, we show, through point mutations in a monomeric IL-8 framework, that individual side chain substitutions can affect receptor specificity.

A 72-amino-acid chimeric protein, Chi1, was constructed from the N-terminal part of interleukin 8, IL-8-(1-53), and the C-terminal part of melanoma growth stimulatory activity, MGSA-(54-72). Chi1 protein showed receptor-binding specificity and biological activity similar, but not identical to IL-8 and decidedly different from MGSA. The structure of Chi1 was determined in solution by two-dimensional NMR and molecular-dynamics calculations. The structure resembled the structures of MGSA and IL-8 closely, containing a triple-stranded beta-sheet in the IL-8 part and an amphipathic alpha-helix in the MGSA part. Chi1 formed dimers at millimolar concentrations via the first strand from the N-terminus, analogous to IL-8 and MGSA. In contrast to the latter molecules, however, the alpha-helix of Chi1 did not pack against the beta-sheet part, but was an independent structural element. This structural difference could be explained mainly by the modulation of hydrophobic interactions between the helix and the rest of the protein in Chi1 as compared to IL-8 and MGSA. It is concluded that tight helix packing is not required for receptor binding and biological activity of Chi1.

In the present investigation, we evaluated the potential of bradykinin (BK), histamine, and serotonin to induce the release of neutrophil and monocyte chemotactic activity (NCA and MCA) from bronchial epithelial cells (BEC). BK significantly stimulated BEC to release NCA and MCA in a dose- and time-dependent manner. Histamine weakly but significantly induced the release of both NCA and MCA in a similar fashion. Serotonin did not stimulate BEC. Checkerboard analysis showed that the NCA and MCA released in response to BK were chemotactic. Molecular-sieve column chromatography by Sephadex G-75 revealed that BK induced a single low-molecular-weight peak (approximately 400 Da) for both NCA and MCA. The releases of NCA and MCA in response to BK and histamine were inhibited by lipoxygenase inhibitors (P < 0.01). The released NCA was inhibited by leukotriene B4 (LTB4) receptor antagonist (P < 0.01) and was slightly inhibited by platelet-activating factor receptor antagonist. LTB4 was increased in BK-stimulated BEC supernatant (P < 0.01). BK B2-receptor antagonist attenuated the release of NCA and MCA. These data suggest that BK and histamine may stimulate BEC to release NCA and MCA and may modulate neutrophil and monocyte recruitment into the airways in patients with asthma.

Structural analysis of chemokines has revealed that the alpha/beta structural-fold is highly conserved among both the CXC and CC chemokine classes. Although dimerization and aggregation is often observed, the chemokines function as monomers. The critical receptor binding regions are in the NH2-terminal 20 residues of the protein and are the least ordered in solution. The flexible NH2-terminal region is the most critical receptor binding site and a second site also exists in the loop that follows the two disulfides. The well-ordered regions are not directly involved in receptor binding but, along with the disulfides, they provide a scaffold that determines the conformation of the sites that are critical for receptor binding. These general requirements for function are common to all the chemokines. For the CC chemokines, receptor activation and receptor binding regions are separate within the 10 residue NH2-terminal region. This has allowed identification of high affinity analogs that do not activate the receptor and are potent antagonists.

The solution conformation of rat cytokine-induced neutrophil chemoattractant (CINC/Gro), a small protein consisting of 72 amino acid residues with proinflammatory activities, and a member of the interleukin 8 family corresponding to a counterpart of human Gro, was investigated with homonuclear 2D and 3D NMR spectroscopy. At each phase of the structural analysis, the homonuclear 3D NOESY-HOHAHA and HOHAHA-NOESY spectra afforded valuable data, removing ambiguities intractable by conventional 2D NMR techniques. CINC/Gro exists as a dimer in solution and contains a triple stranded anti-parallel beta-sheet and C-terminal alpha-helix in the monomer structure, as observed in human IL-8, but non-trivial differences are also observed.

The solution structure of melanoma growth stimulating activity (MGSA), a dimeric chemokine consisting of 73 residues per monomer, has been determined using two-dimensional homonuclear and three-dimensional heteronuclear NMR spectroscopy. Structure calculations were carried out using a hybrid distance geometry-simulated annealing approach with the programs DGII and X-PLOR. The structure is based on a total of 2362 experimental restraints, comprising 2150 NOE-derived distance restraints (2076 unambiguous intrasubunit restraints, 60 unambiguous intersubunit restraints, and 14 ambiguous restraints with potential contributions from both intra- and intersubunit NOEs), 84 distance restraints for 42 backbone hydrogen bonds, and 128 torsion angle restraints. The ambiguous distance restraints were treated using a target function which accounts for both intra- and intermolecular contributions to the NOE intensity. A total of 25 structures were calculated, with the backbone (N, C alpha, C) atomic r.m.s. distribution about the mean coordinates for residues 8 to 69 being 0.44(+/- 0.10) A for the dimer and 0.34(+/- 0.07) A for the individual monomers. The N- and C-terminal residues (1 to 7 and 70 to 73, respectively) are disordered. The overall structure of the MGSA dimer is similar to that reported previously for the NMR and X-ray structures of interleukin-8 (IL-8), and consists of a six-stranded antiparallel beta-sheet packed against two C-terminal antiparallel alpha-helices. A best fit superposition of the NMR structure of MGSA on the X-ray and NMR structures of IL-8 yields backbone atomic r.m.s. differences of 0.99 and 1.28 A, respectively for individual monomers, and 1.08 and 1.82 A, respectively for the dimers (using MGSA residues 8 to 14 and 19 to 69). In general, the MGSA structure resembles the IL-8 X-ray structure more than it does the IL-8 NMR structure. At the tertiary (monomer) level the two main differences between the MGSA solution structure and IL-8 NMR structure involve the loops between residues 14 to 19 and between residues 30 to 38. At the quaternary (dimer) level the difference results from differing angles between the beta-strands which form the dimer interface, and is manifest as a different interhelical separation (distance of closest approach between the two helices is 15.3 A in the IL-8 NMR structure and 11.7 (+/- 0.4) A in the MGSA structure).

The chemokines are a family of immune mediators involved in a wide range of inflammatory processes, most importantly as chemoattractants of monocytes, neutrophils, lymphocytes, and fibroblasts to sites of inflammation. Nuclear magnetic resonance and x-ray crystallographic studies have shown that IL-8 and macrophage-inflammatory protein-1 beta (MIP-1 beta) form noncovalent dimers and that platelet factor-4 (PF-4) forms noncovalent dimers and tetramers, leading to the assumption that, as a family, the chemokines would form multimeric structures. In this study, we analyze the association states of the chemokines IL-8, monocyte chemoattractant protein-1 (MCP-1), and I-309, by using a combination of size exclusion HPLC, sedimentation equilibrium ultracentrifugation, and chemical cross-linking. We find that the association states of MCP-1 and IL-8 are characterized by an equilibrium between monomers and dimers: although dimers predominate at concentrations above 100 microM, these chemokines are almost exclusively monomeric at the nanomolar concentrations at which they display maximal chemotactic activity. I-309, by contrast, remains a monomer at all concentrations tested. I-309 contains two additional cysteine residues (C26 and C68) that are not found in any other members of the chemokine family. We used cyanogen bromide and trypsin digestion strategies to demonstrate that these two residues are linked in a unique intramolecular disulfide bond. Furthermore, by using site-directed mutagenesis, we show that the integrity of this bond is crucial for protein secretion.

To define the structural features important for IL-8 binding to its two known receptors, mutants of IL-8 and melanoma growth-stimulating activity (MGSA) and chimerae consisting of segments of these two chemokines were constructed and purified from the pGEX 2T Escherichia coli expression vector. IL-8 alpha and beta receptors were expressed stably and individually in 293 kidney epithelial cells and HL60 human leukemia cells. The Kd for IL-8 itself and copy numbers for both receptors in transfected cells were comparable. Competition binding with 125I-labeled IL-8, however, showed large differences for several of the IL-8 mutants between alpha and beta receptors. The amino-terminal ELR sequence was important for IL-8 binding to the alpha receptor, but not sufficient for high affinity binding. Both rabbit IL-8 and MGSA share the ELR sequence with human IL-8, but compete poorly with it. The carboxyl terminus distal to amino acid 50 does not seem to mediate high affinity binding to the alpha receptor. A rabbit IL-8/human IL-8 chimera that differs in only eight amino acids from the human IL-8 sequence, was 150-fold lower in its affinity for the alpha receptor than human IL-8. In contrast, both the amino and carboxyl termini appear to be important for binding to the beta receptor. If the ELR sequence of IL-8 was substituted with alanines or if the carboxyl terminus distal to C50 was replaced with the MGSA sequence, a reduction occurred in binding competition. If both changes were introduced simultaneously, binding was abolished. Binding of MGSA was completely prevented by replacement of the ELR sequence with alanines. Ca2+ mobilization in HL60 cells transfected with the alpha or beta receptor was used to assess cell stimulation. The various mutant forms of IL-8 induced receptor activity with a pattern of sensitivity parallel to the competition binding affinities, indicating that both receptors are active.

GRO alpha, a protein structurally related to interleukin-8 (IL-8) and originally described as a melanoma growth stimulatory factor, possesses potent neutrophil-stimulating activity. Recently, two closely related genes, gro beta and gro gamma, were identified. In the present work, the three GRO proteins were chemically synthesized, and their biological activities on human neutrophils and other leukocytes were compared. GRO alpha, GRO beta, and GRO gamma, like IL-8, induced chemotaxis, shape change, a rise in intracellular free calcium levels, exocytosis, and the respiratory burst in neutrophils. The GRO proteins were also active toward basophils as shown by chemotaxis and intracellular calcium concentration changes. The order of potency in neutrophils and basophils was IL-8 > GRO alpha > or = GRO gamma > GRO beta. Of the two IL-8 receptors expressed on human neutrophils, one binds GRO alpha with high and the other with low affinity. Competition binding experiments using radiolabeled IL-8 and GRO alpha revealed the same characteristics for GRO beta and GRO gamma. Similarly, cross-desensitization, as assessed by the stimulus-dependent changes in intracellular calcium concentration, indicated that all three GRO proteins interact with common receptors. From these results, it can be concluded that GRO alpha, GRO beta, and GRO gamma have the same pattern of activity toward human granulocytes and that the differences in amino acid sequence among these proteins have only minor effects on biological activity.

Potent chemotactic activity for neutrophils was detected in rat inflammatory exudate induced by a subcutaneous injection of lipopolysaccharide in a carboxymethyl-cellulose suspension. We purified and characterized chemoattractants from the exudate by the following procedures: carboxymethyl-Sephadex C-25 ion-exchange chromatography; G3000SW gel-filtration chromatography; preparative reverse-phase high-pressure liquid chromatography; rechromatography on reverse-phase HPLC. Two chemotactic factors were purified and their N-terminal amino acid sequences were determined. One factor was a protein in which the first 20 N-terminal amino acids were identical to those of rat cytokine-induced neutrophil chemoattractant (CINC), a counterpart of human gro/melanoma growth-stimulating activity (MGSA). The other factor was highly similar to mouse macrophage inflammatory protein 2 (MIP-2). Mouse MIP-2, a chemotactic factor for neutrophils, is a member of the interleukin-8 family; however the protein we purified had higher similarity to human gro/MGSA than to human interleukin-8. These results indicate that, in rats, chemotactic factors for neutrophils induced by lipopolysaccharide stimulation are not counterparts of interleukin-8, but are gro/CINC-related peptides.

Alveolar macrophages (AM) mediate lung inflammation by producing lipid and peptide molecules that attract neutrophils (PMN) to the lung. Recently we described two porcine proteins called alveolar macrophage-derived chemotactic factors, AMCF-I and -II, that are potent, efficacious, and specific PMN chemoattractants both in vitro and in vivo. We report here the cloning of the full-length cDNAs which code for each protein. Porcine AM were stimulated for 4 h in vitro with Escherichia coli endotoxin (LPS), and a cDNA library was created from poly(A)(+)-selected mRNA. Specific oligonucleotide probes for AMCF-I and AMCF-II were amplified from the porcine AM cDNA library by the polymerase chain reaction using degenerate oligonucleotide primer pairs derived from the N-terminal amino acid sequences of the proteins. These probes were used to isolate 2 full-length cDNAs of 1466 (AMCF-I) and 1515 (AMCF-II) base pairs. Both cDNAs code for proteins with four cysteine residues containing the C-X-C sequence characteristic of the intercrine-alpha family of neutrophil chemoattractants. AMCF-I shares 74% identity with human IL-8 and 84% identity with rabbit IL-8, and likely represents the porcine homologue of IL-8. By contrast, AMCF-II has no obvious human homologue. AMCF-II shares 53% identity with human neutrophil activating peptide 2. Its shared identity with the GRO-related proteins is as high as 61% (rat CINC/GRO), and its shared identity with the 78 amino acid epithelial cell-derived neutrophil activator (ENA-78) is 67%. AMCF-II may represent a new member of the intercrine-alpha family of neutrophil chemoattractants.(ABSTRACT TRUNCATED AT 250 WORDS)

The small cytokine monocyte chemoattractant protein-1 has structural similarity to the neutrophil chemoattractant interleukin-8, but each protein is specific in attracting its own target cell. To investigate the structural basis of this cell type specificity, we have developed an Escherichia coli expression system for the monocyte chemoattractant and mutagenized selected amino acid residues to ones found at the corresponding positions of interleukin-8. We find that a double mutation of tyrosine 28 and arginine 30 to leucine and valine, respectively, causes a drastic decrease in chemotactic activity toward monocytes with the appearance of a novel (interleukin-8-like) neutrophil chemotactic activity. Computer graphic analysis predicts that, with the double substitution, a putative receptor binding groove of the monocyte chemoattractant protein would become topographically similar to that of interleukin-8. We therefore postulate that one or both of these amino acid residues are part of the binding contact of these small cytokines and their receptors.

A model of the three-dimensional structure of the monocyte chemo-attractant and activating protein MCAF/MCP-1 is presented. The model is predicted based on the previously determined solution structure of interleukin-8 (IL-8/NAP-1) [Clore, G.M., Appella, E., Yamada, M., Matsushima, K. and Gronenborn, A.M. (1990) Biochemistry 29, 1689-1696]. Both proteins belong to a superfamily of cytokine proteins involved in cell-specific chemotaxis, host defense and the inflammatory response. The amino acid sequence identity between the two proteins is 24%. It is shown that the regular secondary structure elements of the parent structure can be retained in the modeled structure, such that the backbone hydrogen bonding pattern is very similar in the two structures. The polypeptide backbone is superimposable with an atomic r.m.s. difference of 0.9 A and all side chains can be modeled by transferring the parent side chain conformation to the new structure. Thus, the deduced structure, like the parent one, is a dimer and consists of a six-stranded antiparallel beta-sheet, formed by two three-stranded Greek keys, one from each monomer, upon which lie two symmetry-related antiparallel alpha-helices, approximately 24 A long and separated by approximately 14 A. All amino acid sequence changes can be accommodated within the parent polypeptide framework without major rearrangements. This is borne out by the fact that the IL-8/NAP-1 and modeled MCAF/MCP-1 structures have similar non-bonding energies. These results strongly suggest that both proteins and all other members of the superfamily most likely have the same tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

An inflammatory reaction was induced in vivo by injection of zymosan into the peritoneal cavity of the rabbit. The inflammatory exudate was found to contain oedema-inducing and neutrophil chemoattractant activity when assayed in rabbit skin in vivo, using 125I-albumin and 111In-neutrophils. This activity was additional to that of complement fragment C5a, which was removed by an affinity gel. Two chemoattractants were isolated by cation-exchange, gel-filtration and reversed-phase h.p.l.c. One of these, which ran as a single band of 6-8 kDa on SDS/PAGE, was subjected to N-terminal sequence analysis without reduction and alkylation of cysteine residues. Positive identification of 28 of the first 31 amino acids revealed a rabbit homologue of interleukin-8 (75% sequence identity with human interleukin-8). The demonstration of interleukin-8 as a major neutrophil chemoattractant in an inflammatory reaction in vivo provides the basis for further investigations into the role of this cytokine in the inflammatory process.