CC chemokine ligand 2 and its receptor regulate mucosal production of IL-12 and TGF-beta in high dose oral tolerance

Oral tolerance is the result of a complex immunoregulatory strategy used by the gut and its associated lymphoid tissues to render the peripheral immune system unresponsive to nonpathogenic proteins, such as food or commensal bacteria. The mechanism of oral tolerance induction and maintenance is not well understood. We have previously shown that the chemokine, CC chemokine ligand 2 (CCL2), is important for the induction and maintenance of oral tolerance. To address the role CCL2 plays in oral tolerance, we used both CCL2(-/-) and CCR2(-/-) mice. Cells from the spleen, mesenteric lymph nodes, and peripheral lymph nodes of CCL2(-/-) and CCR2(-/-) mice fed high doses of OVA showed robust proliferative responses compared with cells from Ag-fed wild-type mice. CCL2(-/-) and CCR2(-/-) mice also produced high amounts of Th1 cytokines such as IL-2 and IFN-gamma and very low amounts of IL-4 and IL-10. The ability of APCs from the gut of CCL2(-/-) and CCR2(-/-) OVA-fed mice to stimulate an indicator T cell line was evaluated. APCs from the Peyer's patch of OVA-fed knockout animals could induce a T cell response measured by an increase in proliferation and generation of IL-12 and IFN-gamma with a concomitant reduction of TGF-beta compared with wild-type controls that did not induce a Th1 response. These data indicate that CCL2 and signaling through its receptor CCR2 is critical for the induction of oral tolerance by regulating Ag presentation leading to a disruption in the balance of inflammatory and regulatory cytokines.     

CCR1 and CC chemokine ligand 5 interactions exacerbate innate immune responses during sepsis

CCR1 has previously been shown to play important roles in leukocyte trafficking, pathogen clearance, and the type 1/type 2 cytokine balance, although very little is known about its role in the host response during sepsis. In a cecal ligation and puncture model of septic peritonitis, CCR1-deficient (CCR1(-/-)) mice were significantly protected from the lethal effects of sepsis when compared with wild-type (WT) controls. The peritoneal and systemic cytokine profile in CCR1(-/-) mice was characterized by a robust, but short-lived and regulated antibacterial response. CCR1 expression was not required for leukocyte recruitment, suggesting critical differences extant in the activation of WT and CCR1(-/-) resident or recruited peritoneal cells during sepsis. Peritoneal macrophages isolated from naive CCR1(-/-) mice clearly demonstrated enhanced cytokine/chemokine generation and antibacterial responses compared with similarly treated WT macrophages. CCR1 and CCL5 interactions markedly altered the inflammatory response in vivo and in vitro. Administration of CCL5 increased sepsis-induced lethality in WT mice, whereas neutralization of CCL5 improved survival. CCL5 acted in a CCR1-dependent manner to augment production of IFN-gamma and MIP-2 to damaging levels. These data illustrate that the interaction between CCR1 and CCL5 modulates the innate immune response during sepsis, and both represent potential targets for therapeutic intervention.     

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.     

The chemokine decoy receptor M3 blocks CC chemokine ligand 2 and CXC chemokine ligand 13 function in vivo

Chemokines and their receptors play a key role in immune homeostasis regulating leukocyte migration, differentiation, and function. Viruses have acquired and optimized molecules that interact with the chemokine system. These virus-encoded molecules promote cell entry, facilitate dissemination of infected cells, and enable the virus to evade the immune response. One such molecule in the murine gammaherpesvirus 68 genome is the M3 gene, which encodes a secreted 44-kDa protein that binds with high affinity to certain murine and human chemokines and blocks chemokine signaling in vitro. To test the hypothesis that M3 directly interferes with diverse chemokines in vivo, we examined the interaction of M3 with CCL2 and CXCL13 expressed in the pancreas of transgenic mice. CCL2 expression in the pancreas promoted recruitment of monocytes and dendritic cells; CXCL13 promoted recruitment of B and T lymphocytes. Coexpression of M3 in the pancreas blocked cellular recruitment induced by both CCL2 and CXCL13. These results define M3 as multichemokine blocker and demonstrate its use as a powerful tool to analyze chemokine biology.     

Differential effects of CC chemokines on CC chemokine receptor 5 (CCR5) phosphorylation and identification of phosphorylation sites on the CCR5 carboxyl terminus

The binding of CC chemokines to CC chemokine receptor 5 (CCR5) triggers cellular responses that, generally, are only transient in nature. To explore the potential role of G protein-coupled receptor kinases (GRKs) in the regulation of CCR5, we performed phosphorylation experiments in a rat basophilic leukemia cell line stably expressing CCR5. The ability of various CCR5 ligands to stimulate calcium mobilization in these cells correlated with their ability to induce receptor phosphorylation, desensitization, internalization, and GRK association with the receptor. Aminooxypentane-RANTES, a potent inhibitor of human immunodeficiency virus infection, has been proposed to act through enhanced CCR5 internalization and inhibition of receptor recycling. Aminooxypentane-RANTES profoundly induced CCR5 phosphorylation, but had no effect on CCR1. In permeabilized rat basophilic leukemia CCR5 cells, monoclonal antibodies with specificity for GRK2/3 inhibited RANTES-induced receptor phosphorylation. Consistent with a role for these kinases in CCR5 regulation, 1-2 x 10(5) copies of GRK2 or GRK3 were found to be expressed in peripheral blood leukocytes. Phosphoamino acid analysis revealed that RANTES-induced CCR5 phosphorylation selectively occurs on serine residues. Our findings with receptor mutants indicate that serine residues at positions 336, 337, 342, and 349 represent GRK phosphorylation sites on CCR5. This study demonstrates that chemokines differ in their ability to induce CCR5 phosphorylation and desensitization and provides a molecular mechanism for the agonist-induced attenuation of CCR5 signaling.     

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.     

Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity

CCR2 and its major ligand, chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1, have been found to influence T1/T2 immune response polarization. Our objective was to directly compare the roles of CCR2 and CCL2 in T1/T2 immune response polarization using a model of pulmonary Cryptococcus neoformans infection. Either deletion of CCR2 or treatment of wild-type mice with CCL2 neutralizing Ab produced significant and comparable reductions in macrophage and T cell recruitment into the lungs following infection. Both CCL2 neutralization and CCR2 deficiency resulted in significantly diminished IFN-gamma production, and increased IL-4 and IL-5 production by lung leukocytes (T1 to T2 switch), but only CCR2 deficiency promoted pulmonary eotaxin production and eosinophilia. In the lung-associated lymph nodes (LALN), CCL2-neutralized mice developed Ag-specific IFN-gamma-producing cells, while CCR2 knockout mice did not. LALN from CCR2 knockout mice also had fewer MHCII(+)CD11c(+) and MHCII(+)CD11b(+) cells, and produced significantly less IL-12p70 and TNF-alpha when stimulated with heat-killed yeast than LALN from wild-type or CCL2-neutralized mice, consistent with a defect in APC trafficking in CCR2 knockout mice. Neutralization of CCL2 in CCR2 knockout mice did not alter immune response development, demonstrating that the high levels of CCL2 in these mice did not play a role in T2 polarization. Therefore, CCR2 (but not CCL2) is required for afferent T1 development in the lymph nodes. In the absence of CCL2, T1 cells polarize in the LALN, but do not traffic from the lymph nodes to the lungs, resulting in a pulmonary T2 response.     

Expression of a functional eotaxin (CC chemokine ligand 11) receptor CCR3 by human dendritic cells

Critical to the function of Ag-presenting dendritic cells (DCs) is their capacity to migrate to lymphoid organs and to sites of inflammation. A final stage of development, termed maturation, yields DCs that are strong stimulators of T cell-mediated immunity and is associated with a remodeling of the cell surface that includes a change in the levels of expression of many molecules, including chemokine receptors. We show in this study that CCR3, a chemokine receptor initially discovered on eosinophils, is also expressed by human DCs that differentiate from blood monocytes, DCs that emigrate from skin (epidermal and dermal DCs), and DCs derived from CD34+ hemopoietic precursors in bone marrow, umbilical cord blood, and cytokine-elicited peripheral blood leukapheresis. Unlike other chemokine receptors, such as CCR5 and CCR7, the expression of CCR3 is not dependent on the state of maturation. All DC subsets contain a large intracellular pool of CCR3. The surface expression of CCR3 is not modulated following uptake of particulate substances such as zymosan or latex beads. CCR3 mediates in vitro chemotactic responses to the known ligands, eotaxin and eotaxin-2, because the DC response to these chemokines is inhibited by CCR3-specific mAbs. We postulate that expression of CCR3 may underlie situations where both DCs and eosinophils accumulate in vivo, such as the lesions of patients with Langerhans cell granulomatosis.     

Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice

The chemokine receptors CCR2 and CCR5 play important roles in the recruitment of monocytes/macrophages and T cells. To better understand the role of both receptors in murine models of inflammatory diseases and to recognize potential problems when correlating these data to humans, we have generated mAbs against murine CCR2 and CCR5. In mice CCR2 is homogeneously expressed on monocytes and on 2--15% of T cells, closely resembling the expression pattern in humans. In contrast to humans, murine NK cells are highly CCR5 positive. In addition, CCR5 is expressed on 3--10% of CD4 and 10--40% of CD8-positive T cells and is weakly detectable on monocytes. Using a model of immune complex nephritis, we examined the effects of inflammation on chemokine receptor expression and found a 10-fold enrichment of CCR5(+) and CCR2(+) T cells in the inflamed kidneys. The activity of various chemokines and the antagonistic properties of the mAbs were measured by ligand-induced internalization of CCR2 and CCR5 on primary leukocytes. The Ab MC-21 (anti-CCR2) reduced the activity of murine monocyte chemotactic protein 1 by 95%, whereas the Ab MC-68 (anti-CCR5) blocked over 99% of the macrophage-inflammatory protein 1alpha and RANTES activity. MC-21 and MC-68 efficiently blocked the ligand binding to CCR2 and CCR5 with an IC(50) of 0.09 and 0.6--1.0 microg/ml, respectively. In good correlation to these in vitro data, MC-21 almost completely prevented the influx of monocytes in thioglycollate-induced peritonitis. Therefore, both Abs appear as useful reagents to further study the role of CCR2 and CCR5 in murine disease models.     

The role of the Th2 CC chemokine ligand CCL17 in pulmonary fibrosis

Increasing evidence suggests that the development of pulmonary fibrosis is a Th2-mediated process. We hypothesized that the CC chemokines that are associated with a Th2 profile (CCL17 and CCL22) have an important role in the development of pulmonary fibrosis. We measured CCL17 and CCL22 during the pathogenesis of bleomycin-induced pulmonary fibrosis. We found that both CCL17 and CCL22 were significantly elevated through day 20 as compared with control mice. Peak expression of CCL22 preceded the peak levels of CCL17, as measured by real-time quantitative PCR. CCR4 is the receptor for CCL17 and CCL22 therefore, to further characterize the role of CCL17 and CCL22, we measured CCR4 mRNA in lung tissue of bleomycin-treated mice by real-time quantitative PCR. CCR4 was significantly elevated in bleomycin-treated mice as compared with control mice. Immunolocalization demonstrated that CCR4 was expressed predominantly on macrophages. Neutralization of CCL17, but not CCL22, led to a reduction in pulmonary fibrosis. Immunolocalization of bleomycin-treated lung tissue and human idiopathic pulmonary fibrosis tissue specimens showed that epithelial cells expressed CCL17. These findings demonstrate a central role for Th2 chemokines and the macrophage in the pathogenesis of pulmonary fibrosis and are further support for the role of a Th2 phenotype in the pathogenesis of pulmonary fibrosis.     

Analysis of ligand-stimulated CC chemokine receptor 5 (CCR5) phosphorylation in intact cells using phosphosite-specific antibodies

Human CC chemokine receptor 5 (CCR5), a member of the superfamily of G protein-coupled receptors, regulates the activation and directed migration of leukocytes and serves as the main coreceptor for the entry of R5 tropic strains of human immunodeficiency viruses. We have previously shown that RANTES/CCL5 binding to CCR5 induces GPCR kinase (GRK)- and protein kinase C (PKC)-mediated phosphorylation of four distinct C-terminal serine residues. To study these phosphorylation events in vivo, we have generated monoclonal antibodies, which specifically react only with either phosphorylated or nonphosphorylated CCR5. These phosphosite-specific antibodies reveal that following ligand stimulation of the receptor serine 337 is exclusively phosphorylated by a PKC-mediated mechanism, while GRKs phosphorylate serine 349. GRK-mediated receptor phosphorylation proceeds in a regular time-dependent manner (t(12) approximately 2 min) with an apparent EC(50) of 5 nm. In contrast, PKC phosphorylates serine 337 at 50-fold lower concentrations and in a very rapid, albeit transient manner. Protein phosphatases that are active at neutral pH and are inhibited by okadaic acid rapidly dephosphorylate phosphoserine 337, but less efficiently phosphoserine 349, in intact cells and in an in vitro assay. Immunofluorescence microscopy demonstrates that phosphorylated receptors accumulate in a perinuclear compartment, which resembles recycling endosomes. This study is the first to analyze in detail the spatial and temporal dynamics of GRK- versus PKC-mediated phosphorylation of a G protein-coupled receptor and its subsequent dephosphorylation on the level of individual phosphorylation sites.     

Allosteric and orthosteric sites in CC chemokine receptor (CCR5), a chimeric receptor approach

Chemokine receptors play a major role in immune system regulation and have consequently been targets for drug development leading to the discovery of several small molecule antagonists. Given the large size and predominantly extracellular receptor interaction of endogenous chemokines, small molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5 chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5 chemokines (CCL3 and CCL5), with CCR2-like high affinities and potencies throughout the CCR5 signaling unit. Concomitantly, high affinity binding of small molecule CCR5 agonists and antagonists was retained in the transmembrane region. Importantly, whereas the agonistic and antagonistic properties were preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units.     

Induction of low dose oral tolerance in monocyte chemoattractant protein-1- and CCR2-deficient mice

The chemokine monocyte chemoattractant protein-1 (MCP-1) and its receptor CCR2 have been shown to play an important role in the migration and trafficking of macrophages and Th1 effector cells in experimental autoimmune encephalomyelitis. Also, MCP-1 has been reported to regulate oral tolerance induction by inhibition of Th1 cell-related cytokines and by the ability of Abs to MCP-1 to inhibit oral tolerance. This study demonstrates that neither MCP-1 nor its receptor CCR2 is required for the induction of oral tolerance. Mice deletional for either MCP-1 or CCR2 had suppressed cell-proliferative and Th1 responses following oral administration and immunization with myelin oligodendrocyte glycoprotein (MOG(35-55)). TGF-beta was up-regulated in fed and immunized deletional mice, while IL-4 was absent from deletional mice, but up-regulated in controls. Decreased experimental autoimmune encephalomyelitis severity was found in MOG(35-55)-fed MCP-1 deletional mice, indicating induction of oral tolerance. These results demonstrate that MCP-1 is not required for induction of oral tolerance and that MCP-1 and CCR2 are essential for up-regulation of IL-4 in tolerized mice.     

Structure modeling of the chemokine receptor CCR5: implications for ligand binding and selectivity

The G-protein coupled receptor CCR5 is the main co-receptor for macrophage-tropic HIV-1 strains. I have built a structural model of the chemokine receptor CCR5 and used it to explain the binding and selectivity of the antagonist TAK779. Models of the extracellular (EC) domains of CCR5 have been constructed and used to rationalize current biological data on the binding of HIV-1 and chemokines. Residues spanning the transmembrane region of CCR5 have been modeled after rhodopsin, and their functional significance examined using the evolutionary trace method. The receptor cavity shares six residues with CC-chemokine receptors CCR1 through CCR4, while seven residues are unique to CCR5. The contribution of these residues to ligand binding and selectivity is tested by molecular docking simulations of TAK779 to CCR1, CCR2, and CCR5. TAK779 binds to CCR5 in the cavity formed by helices 1, 2, 3, and 7 with additional interactions with helices 5 and 6. TAK779 did not dock to either CCR1 or CCR2. The results are consistent with current site-directed mutagenesis data and with the observed selectivity of TAK779 for CCR5 over CCR1 and CCR2. The specific residues responsible for the observed selectivity are identified. The four EC regions of CCR5 have been modeled using constrained simulated annealing simulations. Applied dihedral angle constraints are representative of the secondary structure propensities of these regions. Tertiary interactions, in the form of distance constraints, are generated from available epitope mapping data. Analysis of the 250 simulated structures provides new insights to the design of experiments aimed at determining residue-residue contacts across the EC domains and for mapping CC-chemokines on the surface of the EC domains.     

A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues

Mucosae-associated epithelial chemokine (MEC) is a novel chemokine whose mRNA is most abundant in salivary gland, with strong expression in other mucosal sites, including colon, trachea, and mammary gland. MEC is constitutively expressed by epithelial cells; MEC mRNA is detected in cultured bronchial and mammary gland epithelial cell lines and in epithelia isolated from salivary gland and colon using laser capture microdissection, but not in the endothelial, hemolymphoid, or fibroblastic cell lines tested. Although MEC is poorly expressed in skin, its closest homologue is the keratinocyte-expressed cutaneous T cell-attracting chemokine (CTACK; CCL27), and MEC supports chemotaxis of transfected lymphoid cells expressing CCR10, a known CTACK receptor. In contrast to CTACK, however, MEC also supports migration through CCR3. Consistent with this, MEC attracts eosinophils in addition to memory lymphocyte subsets. These results suggest an important role for MEC in the physiology of extracutaneous epithelial tissues, including diverse mucosal organs.     

Increase of CXC chemokine CXCL10 and CC chemokine CCL2 serum levels in normal ageing

No study has evaluated contemporaneously serum CXC and CC chemokines changes in normal ageing. Serum levels of CXCL10 (sCXCL10) (CXC) and CCL2 (sCCL2) (CC) prototype chemokines have been measured in 164 healthy subjects, from 10 to 79 years of age (82 males/82 females). By simple regression analysis, sCXCL10 and sCCL2 were significantly related with increasing age (r=0.32, p<0.001; r=0.31, p<0.0001, respectively), and with each other (r=0.30, p=0.0004). In a multiple linear regression model, only age and sCCL2 were significantly related to sCXCL10 levels (p<0.001); age and sCXCL10 were significantly related to sCCL2 levels (p<0.001). Subjects with high sCXCL10 levels (>150 pg/ml) were not significantly associated with those with high sCCL2 levels (>559 pg/ml). This study, performed in healthy subjects on an age gradient, demonstrates an increase of sCXCL10 and sCCL2 with advancing age; the differential increase of sCXCL10 or sCCL2 may reflect a general shift towards Th1 or Th2 cytokines pattern, respectively.     

The human CC chemokine MIP-1beta dimer is not competent to bind to the CCR5 receptor

Chemokine dimerization has been the subject of much interest in recent years as evidence has accumulated that different quaternary states of chemokines play different biological roles; the monomer is believed to be the receptor-binding unit, whereas the dimer has been implicated in binding cell surface glycosaminoglycans. However, although several studies have provided evidence for this paradigm by making monomeric chemokine variants or dimer-impaired chemokines, few have provided direct evidence of the receptor function of a chemokine dimer. We have produced a covalent dimer of the CC chemokine macrophage inflammatory protein-1beta (MIP-1beta) by placing a disulfide bond at the center of its dimer interface through a single amino acid substitution (MIP-1beta-A10C). This variant was shown to be a nondissociating dimer by SDS-PAGE and analytical ultracentrifugation. NMR reveals a structure largely the same as the wild type protein. In studies of glycosaminoglycan binding, MIP-1beta-A10C binds to a heparin-Sepharose column as tightly as the wild type protein and more tightly than monomeric variants. However, MIP-1beta-A10C neither binds nor activates the MIP-1beta receptor CCR5. It was found that the ability to activate CCR5 was recovered upon reduction of the intermolecular disulfide cross-link by incubation with 1 mm dithiothreitol. This work provides the first definitive evidence that the CC chemokine MIP-1beta dimer is not able to bind or activate its receptor and implicates the CC chemokine monomer as the sole receptor-interacting unit.