Truncation of NH2-terminal amino acid residues increases agonistic potency of leukotactin-1 on CC chemokine receptors 1 and 3

Leukotactin-1 (Lkn-1) is a human CC chemokine that binds to both CC chemokine receptor 1 (CCR1) and CCR3. Structurally, Lkn-1 is distinct from other human CC chemokines in that it has long amino acid residues preceding the first cysteine at the NH(2) terminus, and contains two extra cysteines. NH(2)-terminal amino acids of Lkn-1 were deleted serially, and the effects of each deletion were investigated. In CCR1-expressing cells, serial deletion up to 20 amino acids (Delta20) did not change the calcium flux-inducing activity significantly. Deletion of 24 amino acids (Delta24), however, increased the agonistic potency approximately 100-fold. Deletion of 27 or 28 amino acids also increased the agonistic potency to the same level shown by Delta24. Deletion of 29 amino acids, however, abolished the agonistic activity almost completely showing that at least 3 amino acid residues preceding the first cysteine at the NH(2) terminus are essential for the biological activity of Lkn-1. Loss of agonistic activity was due to impaired binding to CCR1. In CCR3-expressing cells, Delta24 was the only form of Lkn-1 mutants that revealed increased agonistic potency. Our results indicate that posttranslational modification is a potential mechanism for the regulation of biological activity of Lkn-1.     

Interleukin-8 of Cynoglossus semilaevis is a chemoattractant with immunoregulatory property

Interleukin-8 (IL-8), or CXCL8, is a member of the CXC chemokine family that in mammals is known to mediate inflammatory response. In this study, we identified and analyzed an IL-8 orthologue, CsIL8, from half-smooth tongue sole (Cynoglossus semilaevis). The deduced amino acid sequence of CsIL8 contains 100 residues and is closely related to the lineage 1 IL-8 of a number of fish species. In silico analysis identified in CsIL8 a CXC chemokine domain that contains four conserved cysteine residues, two of which form the CXC signature motif of CXC chemokines. Expression of CsIL8 as determined by quantitative real time RT-PCR was detected in a wide range of tissues under normal physiological conditions and was upregulated by bacterial challenge and by vaccination with a subunit vaccine. Purified recombinant CsIL8 (rCsIL8) induced migration of peripheral blood leukocytes and head kidney (HK) lymphocytes and stimulated the proliferation of these cells in a dose-dependent manner. When rCsIL8 was added to the cell culture of HK lymphocytes, it upregulated the expression of interleukin-1β and CsIL8 in a time-dependent fashion. These results indicate that CsIL8 is a biologically active CXC chemokine with immunoregulatory activity and that CsIL8 is involved in pathogen-induced inflammatory response.     

Molecular Cloning and Characterization of a Novel Human CC Chemokine, SCYA26

By searching the Expressed Sequence Tag database, a full-length cDNA for a novel human CC chemokine was cloned. This cDNA encoded a 94-amino-acid protein with a putative signal peptide of 26 amino acids. The deduced mature protein had the four conserved cysteine residues characteristic of CC chemokines and showed 44% identity with MIP-1beta and 40% identity with MIP-1alpha, RANTES, and MCP-4. mRNA for this chemokine was expressed constitutively in human heart and liver and with lesser but detectable levels in skeletal muscle, kidney, and small intestine. To investigate its biological activity, the protein was expressed in mammalian cells and purified by affinity chromatography. The recombinant protein demonstrated chemotactic activity in vitro for T cells and monocytes but not for neutrophils. The gene was mapped to chromosome 7q11.2 by fluorescence in situ hybridization. Based on its structural identity with other CC chemokines and the chemotactic activity and chromosomal location of this chemokine, we designate this chemokine small inducible cytokine subfamily A, member 26 (SCYA26). This gene symbol has been approved by the HUGO Gene Nomenclature Committee.     

Functional analysis of chemically synthesized derivatives of the human CC chemokine CCL15/HCC-2, a high affinity CCR1 ligand.

The CCL15 is a human CC chemokine that activates the receptors, CCR1 and CCR3. Unlike other chemokines, it contains an unusually long N-terminal domain of 31 amino acids preceding the first cysteine residue and a third disulfide bond. To elucidate the functional role of distinct structural determinants, a series of sequential amino-terminal truncated and point-mutated CCL15 derivatives as well as mutants lacking the third disulfide bond and the carboxy-terminal alpha-helix were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. We demonstrate that a truncation of 24 amino acid residues (delta24-CCL15) converts the slightly active 92-residue delta0-CCL15 into a potent agonist of CC chemokine receptor 1 (CCR1) and a weak agonist of CCR3 in cell-based assays. The biological activity decreases from delta24-CCL15 to delta29-CCL15, and re-increases from delta29-CCL15 to delta30-CCL15. Thus, an exocyclic N-terminal region of only one amino acid residue is sufficient for efficient CCR1 activation. As none of the peptides investigated except for delta24-CCL15 activates CCR3, we suggest that CCR1 is the major receptor for CCL15 in vivo. Further we demonstrate that the third disulfide bond of CCL15 and an exchange of tyrosine in position 70 by a leucine residue, which is conserved in CXC chemokines, do not alter the interaction with CCR1. In contrast, a CCL15 derivative lacking the carboxy-terminal alpha-helix exhibits a complete loss of tertiary structure and hence loss of CCR1 agonistic and binding activity. This study demonstrates that specific protein residues in chemokines, which contribute to receptor-ligand interaction, vary significantly between chemokines and cannot be extrapolated using data from functionally related chemokines.     

Structural Insights into the Interaction between a Potent Anti-inflammatory Protein,Viral CC Chemokine Inhibitor (vCCI), and the Human CC Chemokine,Eotaxin-1

Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation. Previously, our structure of the vCCI·MIP-1β (macrophage inflammatory protein-1β) complex indicated that vCCI uses negatively charged residues in β-sheet II to interact with positively charged residues in the MIP-1β N terminus, 20s region and 40s loop. However, the interactions between vCCI and other CC chemokines have not yet been fully explored. Here, we used NMR and fluorescence anisotropy to study the interaction between vCCI and eotaxin-1 (CCL11), a CC chemokine that is an important factor in the asthma response. NMR results reveal that the binding pattern is very similar to the vCCI·MIP-1β complex and suggest that electrostatic interactions provide a major contribution to binding. Fluorescence anisotropy results on variants of eotaxin-1 further confirm the critical roles of the charged residues in eotaxin-1. In addition, the binding affinity between vCCI and other wild type CC chemokines, MCP-1 (monocyte chemoattractant protein-1), MIP-1β, and RANTES (regulated on activation normal T cell expressed and secreted), were determined as 1.1, 1.2, and 0.22 nm, respectively. To our knowledge, this is the first work quantitatively measuring the binding affinity between vCCI and multiple CC chemokines.     

NKLP27: A Teleost NK-Lysin Peptide that Modulates Immune Response,Induces Degradation of Bacterial DNA,and Inhibits Bacterial and Viral Infection

NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis) NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides.     

Novel chicken CXC and CC chemokines

Upon stimulation with lipopolysaccharide (LPS) the chicken macrophage cell line HD-11 secretes factors with cytokine activity. To characterize these molecules, representational difference analysis with RNA of LPS-induced and uninduced HD-11 cells was performed. Two cDNA clones were isolated that code for polypeptides with structural features of chemokines. cDNA K60 codes for a novel CXC chemokine of 104 residues including a putative signal peptide of 20 amino acids at the N-terminus. It is 67% identical to the previously cloned chicken chemokine 9E3/CEF4. K60 exhibits a similar degree of sequence identity to human interleukin 8 and other related CXC chemokines (about 50%), rendering straight-forward predictions of its biological properties difficult. cDNA K203 codes for a novel CC chemokine of 89 amino acids including a putative N-terminal signal peptide of 21 residues. It is 43% identical to a previously characterized chicken protein with homology to mammalian macrophage inflammatory protein 1beta (MIP-1beta). K203 exhibits about 50% sequence identity to human MIP-1beta and other related CC chemokines.     

Intracellular signalling pathways induced by chemokines in natural killer cells.

Chemokines are small peptides involved in the recruitment of various cell types into inflammatory sites. They are divided into four sub-families depending on the presence of amino acids separating the cysteine residues in their N-terminal region. These are the alpha (CXC), beta (CC), gamma (C) and delta (CX)C) chemokines. In addition, five CXC chemokine (CXCR1-5), nine CC chemokine (CCR1-9), one C chemokine (XCR1) and one C-X3C chemokine (CX3CR1) receptors have been identified. These receptors belong to the seven transmembrane spanning domain family, and are coupled to the heterotrimeric guanine nucleotide binding (G) proteins. Chemokines activate various immune cells, and in particular the anti-viral/anti-tumour effectors, the natural killer (NK) cells by activating members of the heterotrimeric G proteins. The importance of the family of chemokines is highlighted by the ability of its members to inhibit the replication of HIV-1 strains in CD4+ cells, where chemokine receptors act as HIV-1 co-receptors. This review discusses the intracellular signalling pathways induced by chemokines in NK and other cell types, and the relationships to HIV-1 signalling in these cells.     

Molecular identification and expression analysis of the CC chemokine gene in rock bream (Oplegnathus fasciatus) and the biological activity of the recombinant protein

We identified the CC chemokine cDNA designated as RbCC1 (CC chemokine 1 in rock bream, Oplegnathus fasciatus), which was isolated using expressed sequence tag (EST) analysis of a lipopolysaccharide (LPS)-stimulated rock bream liver cDNA library. The full-length RbCC1 cDNA (850 bp) contained an open reading frame (ORF) of 366 bp encoding 122 amino acids. Results from our phylogenetic analysis demonstrated that the RbCC1 was closest relationship to the orange-spotted grouper and Mi-iyu croaker CC chemokines located within the fish CC chemokine group. RbCC1 was significantly expressed in the intestine, spleen, liver, and PBLs (peripheral blood leukocytes). Rock bream PBLs were stimulated with several mitogens, LPS and Con A/PMA which significantly induced the expression of RbCC1 mRNA in the PBLs. The RbCC1 mRNA expression in several tissues under conditions of bacterial and viral challenge was examined. The experimental challenge revealed that the kidney and spleen of fish infected with Streptococcus iniae showed the most significant increases in RbCC1 expression compared to the control. In the case of RSIV infection, the RbCC1 mRNA expression was markedly up-regulated in the liver. In this study, recombinant RbCC1 (approximately 53 kDa) was produced using an Escherichia coli expression system followed by purification. Subsequently, the addition of purified rRbCC1 was examined to investigate the impact on the proliferative and chemotactic activity on kidney leukocytes from rock bream. The results demonstrated that the rRbCC1 induces significant biological activity on kidney leukocyte proliferation and attraction at concentrations in the range of 10–300 μg/mL and suggests that rRbCC1 could be utilized as an immune-stimulant and/or molecular adjuvant to enhance the immune effects of vaccines.     

Structural determinants of chemokine binding by an Ectromelia virus-encoded decoy receptor

The EVM1 protein encoded by Ectromelia virus is a member of a highly conserved family of poxvirus chemokine binding proteins that interfere with host immune surveillance processes. EVM1 is abundantly expressed early during mousepox infection and is able to selectively bind CC chemokines and inhibit their interactions with host receptors. Here, we characterize the interaction between EVM1 and the human and murine chemokines CCL3 (MIP-1alpha), CCL2 (MCP-1), and CCL5 (RANTES). Each of these CC chemokines binds EVM1 with 1:1 stoichiometry and equilibrium dissociation constants ranging from 29 pM to 20 nM. The interactions are characterized by rapid-association kinetics between acidic EVM1 and generally basic chemokines with half-lives enduring up to 30 min. The 2.6-A crystal structure of EVM1 reveals a globular beta sandwich with a large, sequence-conserved surface patch encircled by acidic residues on one face of the protein. To determine whether this conserved cluster of residues is involved in chemokine engagement, a structure-based mutational analysis of EVM1 was employed. Mapping of the mutational results onto the surface of EVM1 reveals that a cluster of five residues (I173, S171, S134, N136, and Y69) emanating from one beta sheet is critical for CCL2 and CCL3 sequestration. Additionally, we find that the extended beta2-beta4 loop flanking this conserved cluster is also essential for high-affinity, lasting interactions with chemokines. This analysis provides insight into the mechanism of CC-chemokine inhibition employed by the poxvirus family of chemokine decoy receptors.     

Coevolution of the mammalian chemokines and their receptors

 A phylogeny of mammalian chemokines revealed two major clusters, corresponding to the CC and CXC chemokines; the C chemokines appeared to be more closely related to the former. In a phylogeny of chemokine receptors, there were also two major clusters: one containing CC chemokine receptors plus other receptors of unknown function and another containing CXC receptors and other receptors of unknown function. However, within the CC receptors, there was not a close correspondence between the phylogenies of chemokines and their receptors. The CC chemokines contained two major subfamilies: (1) the MIP subfamily (including MIP-1α, MIP-1β, and RANTES); and (2) the MCP subfamily (including MCP-1,-2,-3, and -4 and eotaxin). Receptors having preferred ligands in the MCP subfamily did not constitute a monophyletic group but rather evolved twice independently. Reconstruction of ancestral amino acid sequences suggested that these two groups of MCP receptors did not convergently evolve any amino acid residues; rather, they convergently lost sequence features found in the third and fourth extracellular domains of known receptors for MIP-subfamily chemokines. Received: 1 May 1998 / Revised: 3 July 1998     

The C-reactive protein of tongue sole Cynoglossus semilaevis is an acute phase protein that interacts with bacterial pathogens and stimulates the antibacterial activity of peripheral blood leukocytes

Pentraxins are a family of evolutionarily conserved proteins that play an important part in innate immunity. C-reactive protein (CRP) is a member of the pentraxin family and in humans is known to be the major acute phase protein. In this work, we report the identification and analysis of a CRP, CsCRP, from half-smooth tongue sole (Cynoglossus semilaevis). CsCRP is composed of 228 amino acid residues and possesses a Pentraxin/CRP domain. Expression of CsCRP occurred in a wide range of tissues and was upregulated by pathogen infection in kidney, spleen, blood, and, in particular, liver. Following bacterial infection, CsCRP level in blood rose rapidly within 12 h and was approximately 3.8 fold of that of the basal level. Purified recombinant CsCRP (rCsCRP) was able to interact with Gram-negative and Gram-positive bacteria including those of pathogenic nature in a dose-dependent manner. When peripheral blood leukocytes (PBL) were infected with bacterial pathogen in the presence of rCsCRP, the respiratory burst and phagocytic capacity of the cells were increased to significant extents. Taken together, these results indicate that CsCRP is an acute phase protein that plays a role in innate immune defense against bacterial infection.     

Modulation of immunity against herpes simplex virus infection via mucosal genetic transfer of plasmid DNA encoding chemokines

In this study, we examined the effects of murine chemokine DNA, as genetic adjuvants given mucosally, on the systemic and distal mucosal immune responses to plasmid DNA encoding gB of herpes simplex virus (HSV) by using the mouse model. The CC chemokines macrophage inflammatory protein 1beta (MIP-1beta) and monocyte chemotactic protein 1 (MCP-1) biased the immunity to the Th2-type pattern as judged by the ratio of immunoglobulin isotypes and interleukin-4 cytokine levels produced by CD4(+) T cells. The CXC chemokine MIP-2 and the CC chemokine MIP-1alpha, however, mounted immune responses of the Th1-type pattern, and such a response rendered recipients more resistant to HSV vaginal infection. In addition, MIP-1alpha appeared to act via the upregulation of antigen-presenting cell (APC) function and the expression of costimulatory molecules (B7-1 and B7-2), whereas MIP-2 enhanced Th1-type CD4(+) T-cell-mediated adaptive immunity by increasing gamma interferon secretion from activated NK cells. Our results emphasize the value of using the mucosal route to administer DNA modulators such as chemokines that function as adjuvants by regulating the activity of innate immunity. Our findings provide new insight into the value of CXC and CC chemokines, which act on different innate cellular components as the linkage signals between innate and adaptive immunity in mucosal DNA vaccination.     

Probing the Role of CXC Motif in Chemokine CXCL8 for High Affinity Binding and Activation of CXCR1 and CXCR2 Receptors

All chemokines share a common structural scaffold that mediate a remarkable variety of functions from immune surveillance to organogenesis. Chemokines are classified as CXC or CC on the basis of conserved cysteines, and the two subclasses bind distinct sets of GPCR class of receptors and also have markedly different quaternary structures, suggesting that the CXC/CC motif plays a prominent role in both structure and function. For both classes, receptor activation involves interactions between chemokine N-loop and receptor N-domain residues (Site-I), and between chemokine N-terminal and receptor extracellular/transmembrane residues (Site-II). We engineered a CC variant (labeled as CC-CXCL8) of the chemokine CXCL8 by deleting residue X (CXC → CC), and found its structure is essentially similar to WT. In stark contrast, CC-CXCL8 bound poorly to its cognate receptors CXCR1 and CXCR2 (K_i > 1 μm). Further, CC-CXCL8 failed to mobilize Ca²⁺ in CXCR2-expressing HL-60 cells or recruit neutrophils in a mouse lung model. However, most interestingly, CC-CXCL8 mobilizes Ca²⁺ in neutrophils and in CXCR1-expressing HL-60 cells. Compared with the WT, CC-CXCL8 binds CXCR1 N-domain with only ∼5-fold lower affinity indicating that the weak binding to intact CXCR1 must be due to its weak binding at Site-II. Nevertheless, this level of binding is sufficient for receptor activation indicating that affinity and activity are separable functions. We propose that the CXC motif functions as a conformational switch that couples Site-I and Site-II interactions for both receptors, and that this coupling is critical for high affinity binding but differentially regulates activation.     

A soluble chemokine-binding protein from vaccinia virus reduces virus virulence and the inflammatory response to infection

Many poxviruses express a secreted protein that binds CC chemokines with high affinity and has been called viral CC chemokine inhibitor (vCCI). This protein is unrelated to any known cellular protein, yet can compete with host cellular CC chemokine receptors to modulate host inflammatory and immune responses. Although several strains of vaccinia virus (VV) express a vCCI, the best characterized VV strains Western Reserve and Copenhagen do not. In this study, we have expressed the vCCI from VV strain Lister in a recombinant Western Reserve virus (v Delta B8R-35K) and characterized its binding properties in vitro and its effect on virulence in vivo relative to wild-type virus (v Delta B8R) or a revertant virus (v Delta B8R-R) where Lister 35-kDa had been removed. Cells infected with v Delta B8R-35K secreted a 35-kDa protein that bound the CC chemokine macrophage-inflammatory protein 1 alpha. Expression of vCCI attenuated the virus in a murine intranasal model, characterized by reduced mortality and weight loss, decreased virus replication and spread, and a reduced recruitment of inflammatory cells into the lungs of VV-infected mice. The CC chemokines macrophage-inflammatory protein 1 alpha, eotaxin, and macrophage chemotactic protein 1 were detected in bronchoalveolar lavage fluids from v Delta B8R-infected mice; however, bronchoalveolar lavage fluids from v Delta B8R-35K-infected mice had lower levels of chemokines and a reduced chemotactic activity for murine leukocytes in vitro. These observations suggest that vCCI plays an important role in regulating leukocyte trafficking to the lungs during VV infection by binding to CC chemokines and blocking their chemotactic activities.     

Effect of posttranslational processing on the in vitro and in vivo activity of chemokines

The CXC and CC chemokine gene clusters provide an abundant number of chemotactic factors selectively binding to shared G protein-coupled receptors (GPCR). Hence, chemokines function in a complex network to mediate migration of the various leukocyte subsets, expressing specific GPCRs during the immune response. Further fine-tuning of the chemokine system is reached through specific posttranslational modifications of the mature proteins. Indeed, enzymatic processing of chemokines during an early phase of inflammation leads to activation of precursor molecules or cleavage into even more active or receptor specific chemokine isoforms. At a further stage, proteolytic processing leads to loss of GPCR signaling, thereby providing natural chemokine receptor antagonists. Finally, further NH₂-terminal cleavage results in complete inactivation to dampen the inflammatory response. During inflammatory responses, the two chemokines which exist in a membrane-bound form may be released by proteases from the cellular surface. In addition to proteolytic processing, citrullination and glycosylation of chemokines is also important for their biological activity. In particular, citrullination of arginine residues seems to reduce the inflammatory activity of chemokines in vivo. This goes along with other positive and negative regulatory mechanisms for leukocyte migration, such as chemokine synergy and scavenging by decoy receptors.     

Identification of a gammaherpesvirus selective chemokine binding protein that inhibits chemokine action

Chemokines are involved in recruitment and activation of hematopoietic cells at sites of infection and inflammation. The M3 gene of gammaHV68, a gamma-2 herpesvirus that infects and establishes a lifelong latent infection and chronic vasculitis in mice, encodes an abundant secreted protein during productive infection. The M3 gene is located in a region of the genome that is transcribed during latency. We report here that the M3 protein is a high-affinity broad-spectrum chemokine scavenger. The M3 protein bound the CC chemokines human regulated upon activation of normal T-cell expressed and secreted (RANTES), murine macrophage inflammatory protein 1alpha (MIP-1alpha), and murine monocyte chemoattractant protein 1 (MCP-1), as well as the human CXC chemokine interleukin-8, the murine C chemokine lymphotactin, and the murine CX(3)C chemokine fractalkine with high affinity (K(d) = 1. 6 to 18.7 nM). M3 protein chemokine binding was selective, since the protein did not bind seven other CXC chemokines (K(d) > 1 microM). Furthermore, the M3 protein abolished calcium signaling in response to murine MIP-1alpha and murine MCP-1 and not to murine KC or human stromal cell-derived factor 1 (SDF-1), consistent with the binding data. The M3 protein was also capable of blocking the function of human CC and CXC chemokines, indicating the potential for therapeutic applications. Since the M3 protein lacks homology to known chemokines, chemokine receptors, or chemokine binding proteins, these studies suggest a novel herpesvirus mechanism of immune evasion.     

A Teleost Bactericidal Permeability-Increasing Protein Kills Gram-Negative Bacteria,Modulates Innate Immune Response,and Enhances Resistance against Bacterial and Viral Infection

Bactericidal/permeability-increasing protein (BPI) is an important factor of innate immunity that in mammals is known to take part in the clearance of invading Gram-negative bacteria. In teleost, the function of BPI is unknown. In the present work, we studied the function of tongue sole (Cynoglossus semilaevis) BPI, CsBPI. We found that CsBPI was produced extracellularly by peripheral blood leukocytes (PBL). Recombinant CsBPI (rCsBPI) was able to bind to a number of Gram-negative bacteria but not Gram-positive bacteria. Binding to bacteria led to bacterial death through membrane permeabilization and structural destruction, and the bound bacteria were more readily taken up by PBL. In vivo, rCsBPI augmented the expression of a wide arrange of genes involved in antibacterial and antiviral immunity. Furthermore, rCsBPI enhanced the resistance of tongue sole against bacterial as well as viral infection. These results indicate for the first time that a teleost BPI possesses immunoregulatory effect and plays a significant role in antibacterial and antiviral defense.