Chemokine receptor expression and function in CD4+ T lymphocytes with regulatory activity

We have investigated the chemokine receptor expression and migratory behavior of a new subset of nickel-specific skin-homing regulatory CD4(+) T cells (Th(IL-10)) releasing high levels of IL-10, low IFN-gamma, and undetectable IL-4. These cells inhibit in a IL-10-dependent manner the capacity of dendritic cells to activate nickel-specific Tc1 and Th1 lymphocytes. RNase protection assay and FACS analysis revealed the expression of a vast repertoire of chemokine receptors on resting Th(IL-10), including the Th1-associated CXCR3 and CCR5, and the Th2-associated CCR3, CCR4, and CCR8, the latter at higher levels compared with Th2 cells. The most active chemokines for resting Th(IL-10), in terms of calcium mobilization and in vitro migration, were in order of potency: CCL2 (monocyte chemoattractant protein-1, CCR2 ligand), CCL4 (macrophage-inflammatory protein-1beta, CCR5 ligand), CCL3 (macrophage-inflammatory protein-1alpha, CCR1/5 ligand), CCL17 (thymus and activation-regulated chemokine, CCR4 ligand), CCL1 (I-309, CCR8 ligand), CXCL12 (stromal-derived factor-1, CXCR4), and CCL11 (eotaxin, CCR3 ligand). Consistent with receptor expression down-regulation, activated Th(IL-10) exhibited a reduced or absent response to most chemokines, but retained a significant migratory capacity to I-309, monocyte chemoattractant protein-1, and thymus and activation-regulated chemokine. I-309, which was ineffective on Th1 lymphocytes, attracted more efficiently Th(IL-10) than Th2 cells. I-309 and CCR8 mRNAs were not detected in unaffected skin and were up-regulated at the skin site of nickel-allergic reaction, with an earlier expression kinetics compared with IL-10 and IL-4. Results indicate that skin-homing regulatory Th(IL-10) lymphocytes coexpress functional Th1- and Th2-associated chemokine receptors, and that CCR8/I-309-driven recruitment of both resting and activated Th(IL-10) cells may be critically involved in the regulation of Th1-mediated skin allergic disorders.     

CCL1-CCR8 interactions: an axis mediating the recruitment of T cells and Langerhans-type dendritic cells to sites of atopic skin inflammation

Atopic dermatitis represents a chronically relapsing skin disease with a steadily increasing prevalence of 10-20% in children. Skin-infiltrating T cells, dendritic cells (DC), and mast cells are thought to play a crucial role in its pathogenesis. We report that the expression of the CC chemokine CCL1 (I-309) is significantly and selectively up-regulated in atopic dermatitis in comparison to psoriasis, cutaneous lupus erythematosus, or normal skin. CCL1 serum levels of atopic dermatitis patients are significantly higher than levels in healthy individuals. DC, mast cells, and dermal endothelial cells are abundant sources of CCL1 during atopic skin inflammation and allergen challenge, and Staphylococcus aureus-derived products induce its production. In vitro, binding and cross-linking of IgE on mast cells resulted in a significant up-regulation of this inflammatory chemokine. Its specific receptor, CCR8, is expressed on a small subset of circulating T cells and is abundantly expressed on interstitial DC, Langerhans cells generated in vitro, and their monocytic precursors. Although DC maintain their CCR8+ status during maturation, brief activation of circulating T cells recruits CCR8 from intracytoplamic stores to the cell surface. Moreover, the inflammatory and atopy-associated chemokine CCL1 synergizes with the homeostatic chemokine CXCL12 (SDF-1alpha) resulting in the recruitment of T cell and Langerhans cell-like DC. Taken together, these findings suggest that the axis CCL1-CCR8 links adaptive and innate immune functions that play a role in the initiation and amplification of atopic skin inflammation.     

Human NK cells express CC chemokine receptors 4 and 8 and respond to thymus and activation-regulated chemokine, macrophage-derived chemokine, and I-309

NK cells respond to various chemokines, suggesting that they express receptors for these chemokines. In this paper, we show that IL-2-activated NK (IANK) cells express CC chemokine receptor 4 (CCR4) and CCR8, as determined by flow cytometric, immunoblot, and RNase protection assays. Macrophage-derived chemokine (MDC), the ligand for CCR4, induces the phosphorylation of CCR4 within 0.5 min of activating IANK cells with this ligand. This is corroborated with the recruitment of G protein-coupled receptor kinases 2 and 3 and their association with CCR4 in IANK cell membranes. Also, CCR4 is internalized between 5 and 45 min but reappears in the membranes after 60 min of stimulation with MDC. MDC, thymus and activation-regulated chemokine (TARC), and I-309 induce the chemotaxis of IANK cells, an activity that is inhibited upon pretreatment of these cells with pertussis toxin, suggesting that receptors for these chemokines are coupled to pertussis toxin-sensitive G proteins. In the calcium release assay, cross-desensitization experiments showed that TARC completely desensitizes the calcium flux response induced by MDC or I-309, whereas both MDC and I-309 partially desensitize the calcium flux response induced by TARC. These results suggest that TARC utilizes CCR4 and CCR8. Our results are the first to show that IL-2-activated NK cells express CCR4 and CCR8, suggesting that these receptors are not exclusive for Th2 cells.     

Thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of Th2-attracting chemokines and disease severity

Thymic stromal lymphopoietin (TSLP) is said to increase expression of chemokines attracting Th2 T cells. We hypothesized that asthma is characterized by elevated bronchial mucosal expression of TSLP and Th2-attracting, but not Th1-attracting, chemokines as compared with controls, with selective accumulation of cells bearing receptors for these chemokines. We used in situ hybridization and immunohistochemistry to examine the expression and cellular provenance of TSLP, Th2-attracting (thymus and activation-regulated chemokine (TARC)/CCL17, macrophage-derived chemokine (MDC)/CCL22, I-309/CCL1) and Th1-attracting (IFN-gamma-inducible protein 10 (IP-10)/CXCL10, IFN-inducible T cell alpha-chemoattractant (I-TAC)/CXCL11) chemokines and expression of their receptors CCR4, CCR8, and CXCR3 in bronchial biopsies from 20 asthmatics and 15 normal controls. The numbers of cells within the bronchial epithelium and submucosa expressing mRNA for TSLP, TARC/CCL17, MDC/CCL22, and IP-10/CXCL10, but not I-TAC/CXCL11 and I-309/CCL1, were significantly increased in asthmatics as compared with controls (p 相似文献   

Human CC chemokine I-309, structural consequences of the additional disulfide bond

I-309 is a member of the CC subclass of chemokines and is one of only three human chemokines known to contain an additional, third disulfide bond. The three-dimensional solution structure of I-309 was determined by (1)H nuclear magnetic resonance spectroscopy and dynamic simulated annealing. The structure of I-309, which remains monomeric at high concentrations, was determined on the basis of 978 experimental restraints. The N-terminal region of I-309 was disordered, as has been previously observed for the CC chemokine eotaxin but not others such as MCP-1 and RANTES. This was followed in I-309 by a well-ordered region between residues 13 and 69 that consisted of a 3(10)-helix, a triple-stranded antiparallel beta-sheet, and finally a C-terminal alpha-helix. Root-mean-square deviations of 0.61 and 1.16 were observed for the backbone and heavy atoms, respectively. A comparison of I-309 to eotaxin and HCC-2 revealed a significant structural change in the C-terminal region of the protein. The alpha-helix normally present in chemokines was terminated early and was followed by a short section of extended strand. These changes were a direct result of the additional disulfide bond present in this protein. An examination of the I-309 structure will aid in an understanding of the specificity of this protein with its receptor, CCR8.     

CCR8 on human thymocytes functions as a human immunodeficiency virus type 1 coreceptor

To determine whether human immunodeficiency virus type 1 (HIV-1) coreceptors besides CXCR4 and CCR5 are involved in HIV-1 infection of the thymus, we focused on CCR8, a receptor for the chemokine I-309, because of its high expression in the thymus. Similar levels of CCR8 mRNA were detected in immature and mature primary human thymocytes. Consistent with this, [(125)I]I-309 was shown to bind specifically and with similar affinity to the surface of immature and mature human thymocytes. Fusion of human thymocytes with cells expressing HIV-1 X4 or X4R5 envelope glycoprotein was inhibited by I-309 in a dose-dependent manner. In addition, I-309 partially inhibited productive infection of human thymocytes by X4, R5, and X4R5 HIV-1 strains. Our data provide the first evidence that CCR8 functions as an HIV-1 coreceptor on primary human cells and suggest that CCR8 may contribute to HIV-1-induced thymic pathogenesis.     

Coordinated involvement of mast cells and T cells in allergic mucosal inflammation: critical role of the CC chemokine ligand 1:CCR8 axis

CCL1 is the predominant chemokine secreted from IgE-activated human and mouse mast cells in vitro, colocalizes to mast cells in lung biopsies, and is elevated in asthmatic airways. CCR8, the receptor for CCL1, is expressed by approximately 70% of CD4(+) T lymphocytes recruited to the asthmatic airways, and the number of CCR8-expressing cells is increased 3-fold in the airways of asthmatic subjects compared with normal volunteers. In vivo, CCL1 expression in the lung is reduced in mast cell-deficient mice after aeroallergen provocation. Neutralization of CCL1 or CCR8 deficiency results in reduced mucosal lung inflammation, airway hyperresponsiveness, and mucus hypersecretion to a similar degree as detected in mast cell-deficient mice. Adenoviral delivery of CCL1 to the lungs of mast cell-deficient mice restores airway hyperresponsiveness, lung inflammation, and mucus hypersecretion to the degree observed in wild-type mice. The consequences of CCR8 deficiency, including a marked reduction in Th2 cytokine levels, are comparable with those observed by depletion of CD4(+) T lymphocytes. Thus, mast cell-derived CCL1- and CCR8-expressing CD4(+) effector T lymphocytes play an essential role in orchestrating lung mucosal inflammatory responses.     

Structure/function relationships of CCR8 agonists and antagonists. Amino-terminal extension of CCL1 by a single amino acid generates a partial agonist

We describe here the interactions of CCR8 with its ligands using both CCR8 transfectants and a T-cell line expressing the receptor endogenously. Of the CCR8 agonists reported previously, only CCL1 and vMIP-I exhibited potency in assays of intracellular calcium flux, chemotaxis, and receptor internalization, this latter mechanism being dependent upon the expression of beta-arrestins 1 and 2 but independent of Galpha(i) signaling. NH(2)-terminal extension of the mature CCL1 sequence by a serine residue (Ser-CCL1) resulted in a partial agonist with a reduced affinity for CCR8, suggesting that the NH(2) terminus of the ligand plays a role in ligand binding to an intrahelical site. Attempts to identify key residues within this site revealed that the conserved glutamic acid residue in transmembrane helix 7, Glu-286, is crucial for trafficking of the receptor to the cell surface, while Asp-97 of transmembrane helix 2 is dispensable. CCL7 was found to inhibit both Ser-CCL1 and vMIP-I responses but not those of CCL1 itself. Similarly, vMIP-I responses were more than 2 orders of magnitude more sensitive to the specific CCR8 antagonist MC148 than those induced by CCL1, which is difficult to reconcile with the reported affinities for the receptor. Collectively, these data suggest that the CCR8 ligands are allotropic, binding to distinct sites within CCR8 and that the human immune system may have evolved to use CCL7 as a selective antagonist of viral chemokine activity at CCR8 but not those of the host ligand.     

Expression and cellular provenance of thymic stromal lymphopoietin and chemokines in patients with severe asthma and chronic obstructive pulmonary disease

Asthma and chronic obstructive pulmonary disease (COPD) are associated with Th2 and Th1 differentiated T cells. The cytokine thymic stromal lymphopoietin (TSLP) promotes differentiation of Th2 T cells and secretion of chemokines which preferentially attract them. We hypothesized that there is distinct airways expression of TSLP and chemokines which preferentially attract Th1- and Th2-type T cells, and influx of T cells bearing their receptors in asthma and COPD. In situ hybridization, immunohistochemistry, and ELISA were used to examine the expression and cellular provenance of TSLP, Th2-attracting (TARC/CCL17, MDC/CCL22, I-309/CCL1), and Th1-attracting (IP-10/CXCL10, I-TAC/CXCL11) chemokines in the bronchial mucosa and bronchoalveolar lavage fluid of subjects with moderate/severe asthma, COPD, and controls. Cells expressing mRNA encoding TSLP, TARC/CCL17, MDC/CCL22, and IP-10/CXCL10, but not I-TAC/CXCL11 and I-309/CCL1, were significantly increased in severe asthma and COPD as compared with non-smoker controls (p < 0.02). This pattern was reflected in bronchoalveolar lavage fluid protein concentrations. Expression of the same chemokines was also increased in ex- and current smokers. The cellular sources of TSLP and chemokines were strikingly similar in severe asthma and COPD. The numbers of total bronchial mucosal T cells expressing the chemokine receptors CCR4, CCR8, and CXCR3 did not significantly differ in asthma, COPD, and controls. Both asthma and COPD are associated with elevated bronchial mucosal expression of TSLP and the same Th1- and Th2-attracting chemokines. Increased expression of these chemokines is not, however, associated with selective accumulation of T cells bearing their receptors.     

Proinflammatory proteases liberate a discrete high-affinity functional FPRL1 (CCR12) ligand from CCL23

Most chemokines have been found to bind to and signal through single or highly related chemokine receptors. However, a single chemokine protein, a processed form of the alternatively spliced CCL23 (CKbeta8/MPIF-1) gene product, potently engages both the "classical" chemokine receptor CCR1, as well as FPRL1, a type of pattern recognition receptor on innate immune cells. However, the mechanism by which the alternative form of CCL23 is processed is unknown. In this study, we show that proteases associated with inflammation cleave CCL23 immediately N-terminal to the 18-residue domain encoded by the alternatively spliced nucleotides, resulting in potent CCR1 and FPRL1 activity. The proteases also cleave CCL23 immediately C-terminal to the inserted domain, producing a typical CC chemokine "body" containing even further-increased CCR1 potency and a released approximately 18-aa peptide with full FPRL1 activity but no activity for CCR1. This peptide, which we term SHAAGtide, is by itself an attractant of monocytes and neutrophils in vitro, recruits leukocytes in vivo, and is 50- to 100-fold more potent than all other natural agents posited to act on FPRL1. The appearance of SHAAGtide appears to be transient, however, as the proinflammatory proteases subsequently cleave within the peptide, abolishing its activity for FPRL1. The sequential activation of a transient FPRL1 ligand and a longer-lived CCR1 ligand within a single chemokine may have important consequences for the development of inflammation or the link between innate and adaptive immunity.     

In vitro selection of RNA aptamers that block CCL1 chemokine function

CCL1, the CCR8 ligand, is a CC chemokine secreted by activated monocytes and lymphocytes and is a potent chemoattractant for these cell types. The in vivo role of the CCL1/CCR8 axis in Th2-mediated inflammation is far from clear. Ligand neutralisation studies reported discrepancies in the effect of CCL1/CCR8 and CCR8 knockout studies showed very different insights into the functional role of the CCR8. To further study the biological function of CCL1, we focused on the generation and characterisation of RNA aptamers. We report here the in vitro isolation of the first nuclease resistant and selective RNA aptamer (T48) with high-binding affinity for human and mouse CCL1. The T48 aptamer but not a random control aptamer antagonises CCL1 function in a dose-dependent fashion in both heparin binding and chemotaxis assays. To our knowledge, the T48 aptamer constitutes one of the most potent CCL1 antagonists reported to date and is an excellent tool to dissect CCL1-specific function in vivo. The T48 aptamer may also have potential as new generation of therapeutic tools.     

Eotaxin/CCL11 suppresses IL-8/CXCL8 secretion from human dermal microvascular endothelial cells

The CC chemokine eotaxin/CCL11 is known to bind to the receptor CCR3 on eosinophils and Th2-type lymphocytes. In this study, we demonstrate that CCR3 is expressed on a subpopulation of primary human dermal microvascular endothelial cells and is up-regulated by TNF-alpha. We found that incubation of human dermal microvascular endothelial cells with recombinant eotaxin/CCL11 suppresses TNF-alpha-induced production of the neutrophil-specific chemokine IL-8/CXCL8. The eotaxin/CCL11-suppressive effect on endothelial cells was not seen on IL-1beta-induced IL-8/CXCL8 release. Eotaxin/CCL11 showed no effect on TNF-alpha-induced up-regulation of growth-related oncogene-alpha or IFN-gamma-inducible protein-10, two other CXC chemokines tested, and did not affect production of the CC chemokines monocyte chemoattractant protein-1/CCL2 and RANTES/CCL5, or the adhesion molecules ICAM-1 and E-selectin. These results suggest that eotaxin/CXCL11 is not effecting a general suppression of TNF-alphaR levels or signal transduction. Suppression of IL-8/CXCL8 was abrogated in the presence of anti-CCR3 mAb, pertussis toxin, and wortmannin, indicating it was mediated by the CCR3 receptor, G(i) proteins, and phosphatidylinositol 3-kinase signaling. Eotaxin/CCL11 decreased steady state levels of IL-8/CXCL8 mRNA in TNF-alpha-stimulated cells, an effect mediated in part by an acceleration of IL-8 mRNA decay. Eotaxin/CCL11 may down-regulate production of the neutrophil chemoattractant IL-8/CXCL8 by endothelial cells in vivo, acting as a negative regulator of neutrophil recruitment. This may play an important biological role in the prevention of overzealous inflammatory responses, aiding in the resolution of acute inflammation or transition from neutrophilic to mononuclear/eosinophilic inflammation.     

Inhibition of CD26/dipeptidyl peptidase IV enhances CCL11/eotaxin-mediated recruitment of eosinophils in vivo

Chemokines mediate the recruitment of leukocytes to the sites of inflammation. N-terminal truncation of chemokines by the protease dipeptidyl peptidase IV (DPPIV) potentially restricts their activity during inflammatory processes such as allergic reactions, but direct evidence in vivo is very rare. After demonstrating that N-terminal truncation of the chemokine CCL11/eotaxin by DPPIV results in a loss of CCR3-mediated intracellular calcium mobilization and CCR3 internalization in human eosinophils, we focused on the in vivo role of CCL11 and provide direct evidence for specific kinetic and rate-determining effects by DPPIV-like enzymatic activity on CCL11-mediated responses of eosinophils. Namely, it is demonstrated that i.v. administration of CCL11 in wild-type F344 rats leads to mobilization of eosinophils into the blood, peaking at 30 min. This mobilization is significantly increased in DPPIV-deficient F344 rats. Intradermal administration of CCL11 is followed by a dose-dependent recruitment of eosinophils into the skin and is significantly more effective in DPPIV-deficient F344 mutants as well as after pharmacological inhibition of DPPIV. Interestingly, CCL11 application leads to an up-regulation of DPPIV, which is not associated with negative feedback inhibition via DPPIV-cleaved CCL11((3-74)). These findings demonstrate regulatory effects of DPPIV for the recruitment of eosinophils. Furthermore, they illustrate that inhibitors of DPPIV have the potential to interfere with chemokine-mediated effects in vivo including but not limited to allergy.     

Inhibition of airway inflammation by amino-terminally modified RANTES/CC chemokine ligand 5 analogues is not mediated through CCR3

Chemokines play a key role in the recruitment of activated CD4(+) T cells and eosinophils into the lungs in animal models of airway inflammation. Inhibition of inflammation by N-terminally modified chemokines is well-documented in several models but is often reported with limited dose regimens. We have evaluated the effects of doses ranging from 10 ng to 100 micro g of two CC chemokine receptor antagonists, Met-RANTES/CC chemokine ligand 5 (CCL5) and aminooxypentane-RANTES/CCL5, in preventing inflammation in the OVA-sensitized murine model of human asthma. In the human system, aminooxypentane-RANTES/CCL5 is a full agonist of CCR5, but in the murine system neither variant is able to induce cellular recruitment. Both antagonists showed an inverse bell-shaped inhibition of cellular infiltration into the airways and mucus production in the lungs following allergen provocation. The loss of inhibition at higher doses did not appear to be due to partial agonist activity because neither variant showed activity in recruiting cells into the peritoneal cavity at these doses. Surprisingly, neither was able to bind to the major CCR expressed on eosinophils, CCR3. However, significant inhibition of eosinophil recruitment was observed. Both analogues retained high affinity binding for murine CCR1 and murine CCR5. Their ability to antagonize CCR1 and CCR5 but not CCR3 was confirmed by their ability to prevent RANTES/CCL5 and macrophage inflammatory protein-1beta/CCL4 recruitment in vitro and in vivo, while they had no effect on that induced by eotaxin/CCL11. These results suggest that CCR1 and/or CCR5 may be potential targets for asthma therapy.     

Human cord blood-derived mast cells synthesize and release I-309 in response to IgE

Mast cells are the central mediating cells of allergic reactions. Binding of allergen specific IgE to high affinity IgE receptor (Fcepsilon RI) and subsequent binding of allergen by the IgE causes receptor cross-linking and activation. In a study examining the differential gene expression in human cord blood-derived mast cells (CBMCs) mediated by activation of Fcepsilon RI both with IgE and IgE followed by cross-linking with alpha-IgE, the chemokine I-309 was found to be upregulated. I-309 is the ligand for the CCR8 receptor and is responsible for chemoattraction of TH2 type T-cells. Interestingly, I-309 RNA and protein levels were elevated not only in response to IgE/alpha-IgE activation but also by IgE alone. In addition, the I-309 levels were augmented by growth of the CBMCs in the presence of the proinflammatory cytokine IL-4. GM-CSF and MIP-1alpha secretion was also induced by IgE. These results suggest that IgE, through the production and release of cytokines such as I-309, GM-CSF and MIP-1alpha could promote an inflammatory reaction in the absence of antigen stimulation of mast cells.     

Identification and characterization of U83A viral chemokine, a broad and potent beta-chemokine agonist for human CCRs with unique selectivity and inhibition by spliced isoform

Leukotropic human herpesvirus 6 (HHV-6) establishes a persistent infection associated with inflammatory diseases and encodes chemokines that could chemoattract leukocytes for infection or inflammation. HHV-6 variant A encodes a distant chemokine homolog, U83A, and a polymorphism promoting a secreted form was identified. U83A and three N-terminal modifications were expressed and purified, and activities were compared with a spliced truncated isoform, U83A-Npep. U83A efficiently and potently induced calcium mobilization in cells expressing single human CCR1, CCR4, CCR6, or CCR8, with EC50 values <10 nM. U83A also induced chemotaxis of Th2-like leukemic cells expressing CCR4 and CCR8. High-affinity binding, 0.4 nM, was demonstrated to CCR1 and CCR5 on monocytic/macrophage cells, and pretreatment with U83A or modified forms could block responses for endogenous ligands. U83A-Npep acted only as antagonist, efficiently blocking binding of CCL3 to CCR1 or CCR5 on differentiated monocytic/macrophage leukemic cells. Furthermore, CCL3 induction of calcium signaling via CCR1 and CCL1 induced chemotaxis via CCR8 in primary human leukocytes was inhibited. Thus, this blocking by the early expressed U83A-Npep could mediate immune evasion before finishing the replicative cycle. However, late in infection, when full-length U83A is made, chemoattraction of CCR1-, CCR4-, CCR5-, CCR6-, and CCR8-bearing monocytic/macrophage, dendritic, and T lymphocyte cells can facilitate dissemination via lytic and latent infection of these cells. This has further implications for neuroinflammatory diseases such as multiple sclerosis, where both cells bearing CCR1/CCR5 plus their ligands, as well as HHV-6A, have been linked. Applications also discussed include novel vaccines/immunotherapeutics for cancer and HIV as well as anti-inflammatories.     

Unusual chemokine receptor antagonism involving a mitogen-activated protein kinase pathway

Antagonism of chemokines on chemokine receptors constitutes a new regulatory principle in inflammation. Eotaxin (CCL11), an agonist for CCR3 and an attractant of eosinophils, basophils, and Th2 lymphocytes, was shown to act as an antagonist for CCR2, which is widely expressed on leukocytes and is essential for inflammatory responses. In this report we provide direct evidence for a novel mechanism how chemokine receptor function can be arrested by endogenous ligands. We show that binding of eotaxin to CCR2 stimulates the mitogen-activated protein kinases extracellular signal-regulated kinase 1/2 (ERK1/2). Activation of the mitogen-activated protein kinase kinase 1/2-ERK pathway is indispensable for eotaxin-mediated attenuation of CCR2 function, as inhibition of ERK phosphorylation abolishes the arresting effect. ERK is also activated by CCR2 agonists, e.g., monocyte chemoattractant protein-1 (CCL2). However, the involved pathways are different, although in either case coupling of CCR2 to pertussis toxin-sensitive heterotrimeric G proteins is necessary. The results are in agreement with the view that CCR2 could assume different activation states depending on the ligand it encounters. With respect to actin polymerization and calcium mobilization, the different activation states lead to agonistic and antagonistic responses. It is conceivable that the intracellular signal transduction pathway that is activated by eotaxin could cause an attenuation of proinflammatory responses mediated by CCR2.     

Eotaxin-3/CCL26 is a natural antagonist for CC chemokine receptors 1 and 5. A human chemokine with a regulatory role

Eotaxin-3 (CCL26), like eotaxin (CCL11) and eotaxin-2 (CCL24), has long been considered a specific agonist for CC chemokine receptor 3 (CCR3), attracting and activating eosinophils, basophils, and Th2 type T lymphocytes. Although not characterized extensively yet, its expression profile coincides with a potential role in allergic inflammation. We recently reported that eotaxin-3 is an antagonist for CCR2 (Ogilvie, P., Paoletti, S., Clark-Lewis, I., and Uguccioni, M. (2003) Blood 102, 789-784). In the present report, we provide evidence that eotaxin-3 acts as a natural antagonist on CCR1 and -5 as well. Eotaxin-3 bound to cells transfected with either CCR1 or -5 as well as to monocytes expressing both receptors. Further, it inhibited chemotaxis, the release of free intracellular calcium, and actin polymerization when cells were stimulated with known agonists of CCR1 and -5. An analysis of its three-dimensional structure indicated the presence of two distinct epitopes that may be involved in specific binding to CCR1, -2, -3, and -5. Taken together, our data thus indicate eotaxin-3 to be the first human chemokine that features broadband antagonistic activities, suggesting that it may have a modulatory rather than an inflammatory function. Further, eotaxin-3 may play an unrecognized role in the polarization of cellular recruitment by attracting Th2 lymphocytes as well as eosinophils and basophils via CCR3, while concomitantly blocking the recruitment of Th1 lymphocytes and monocytes via CCR1, -2, and -5.     

Crystal structure of viral macrophage inflammatory protein I encoded by Kaposi's sarcoma-associated herpesvirus at 1.7A

Viral macrophage inflammatory protein I (vMIP-I) is a chemokine encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) that selectively activates the CC chemokine receptor 8 (CCR8), for which the endogenous ligand is CCL1. The crystal structure of vMIP-I was determined at 1.7A for comparison with other chemokines, especially those that bind CCR8, such as vMIP-II from KSHV, a CCR8 antagonist and the closest homolog (40% identical). vMIP-I has a typical chemokine fold consisting of an extended N-terminal loop, followed by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. The four molecules in the asymmetric unit comprise two MIP-1beta-like dimers. Electrostatic surface representations of CCR8-binding chemokines reveal only minor areas of correlating surface potential, which must be reconciled with promiscuity in receptor and glycosaminoglycan (GAG) binding. In addition, the biological relevance of chemokine oligomerization is examined by comparing the oligomeric states of all chemokine structures deposited to date in the RCSB PDB.     

Differential recognition and scavenging of native and truncated macrophage-derived chemokine (macrophage-derived chemokine/CC chemokine ligand 22) by the D6 decoy receptor

The promiscuous D6 receptor binds several inflammatory CC chemokines and has been recently proposed to act as a chemokine-scavenging decoy receptor. The present study was designed to better characterize the spectrum of CC chemokines scavenged by D6, focusing in particular on CCR4 ligands and analyzing the influence of NH(2)-terminal processing on recognition by this promiscuous receptor. Using D6 transfectants, it was found that D6 efficiently bound and scavenged most inflammatory CC chemokines (CCR1 through CCR5 agonists). Homeostatic CC chemokines (CCR6 and CCR7 agonists) were not recognized by D6. The CCR4 agonists CC chemokine ligand 17 (CCL17) and CCL22 bound to D6 with high affinity. CCL17 and CCL22 have no agonistic activity for D6 (chemotaxis and calcium fluxes), but were rapidly scavenged, resulting in reduced chemotactic activity on CCR4 transfectants. CD26 mediates NH(2) terminus processing of CCL22, leading to the production of CCL22 (3-69) and CCL22 (5-69) that do not interact with CCR4. These NH(2)-terminal truncated forms of CCL22 were not recognized by D6. The results presented in this study show that D6 recognizes and scavenges a wide spectrum of inflammatory CC chemokines, including the CCR4 agonists CCL22 and CCL17. However, this promiscuous receptor is not engaged by CD26-processed, inactive, CCL22 variants. By recognizing intact CCL22, but not its truncated variants, D6 expressed on lymphatic endothelial cells may regulate the traffic of CCR4-expressing cells, such as dendritic cells.