Ian Clark-Lewis

Cloning of the human eosinophil chemoattractant, eotaxin. Expression, receptor binding, and functional properties suggest a mechanism for the selective recruitment of eosinophils.

The CC chemokine eotaxin, identified in guinea pigs and also recently in mice, may be a key element for the selective recruitment of eosinophils to certain inflamed tissues. Using a partial mouse eotaxin CDNA probe, the human eotaxin gene was cloned and found to be 61.8 and 63.2% identical at the amino acid level to guinea pig and mouse eotaxin. Human eotaxin protein was a strong and specific eosinophil chemoattractant in vitro and was an effective eosinophil chemoattractant when injected into the skin of a rhesus monkey. Radiolabeled eotaxin was used to identify a high affinity receptor on eosinophils (0.52 nM Kd), expressed at 4.8 x 10(4) sites per cell. This receptor also bound RANTES and monocyte chemotactic protein-3 with lower affinity, but not macrophage inflammatory protein-1 alpha. Eotaxin could desensitize calcium responses of eosinophils to RANTES and monocyte chemotactic protein-3, although RANTES was able to only partially desensitize eosinophil calcium responses to eotaxin. Immunohistochemistry on human nasal polyp with antieotaxin mAbs showed that certain leukocytes as well as respiratory epithelium were intensely immunoreactive, and eosinophil infiltration occurred at sites of eotaxin upregulation. Thus eotaxin in humans is a potent and selective eosinophil chemoattractant that is expressed by a variety cell types in certain inflammatory conditions.

Interleukin-8, a chemotactic and inflammatory cytokine

Interleukin‐8 (IL‐8) belongs to a family of small, structurally related cytokines similar to platelet factor 4. It is produced by phagocytes and mesenchymal cells exposed to inflammatory stimuli (e.g., interleukin‐1 or tumor necrosis factor) and activates neutrophils inducing, chemotaxis, exocytosis and the respiratory burst. In vivo, IL‐8 elicits a massive neutrophil accumulation at the site of injection. Five neutrophil‐activating cytokines similar to IL‐8 in structure and function have been identified recently. IL‐8 and the related cytokines are produced in several tissues upon infection, inflammation, ischemia, trauma etc., and are thought to be the main cause of local noutrophil accumulation.

Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines

During organogenesis, immunosurveillance, and inflammation, chemokines selectively recruit leukocytes by activating seven-transmembrane-spanning receptors. It has been suggested that an important component of this process is the formation of a haptotactic gradient by immobilization of chemokines on cell surface glycosaminoglycans (GAGs). However, this hypothesis has not been experimentally demonstrated in vivo. In the present study we investigated the effect of mutations in the GAG binding sites of three chemokines, monocyte chemoattractant protein-1/CC chemokine ligand (CCL)2, macrophage-inflammatory protein-1β/CCL4, and RANTES/CCL5, on their ability to recruit cells in vivo. These mutant chemokines retain chemotactic activity in vitro, but they are unable to recruit cells when administered intraperitoneally. Additionally, monomeric variants, although fully active in vitro, are devoid of activity in vivo. These data demonstrate that both GAG binding and the ability to form higher-order oligomers are essential for the activity of particular chemokines in vivo, although they are not required for receptor activation in vitro. Thus, quaternary structure of chemokines and their interaction with GAGs may significantly contribute to the localization of leukocytes beyond migration patterns defined by chemokine receptor interactions.

Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1

The three‐dimensional structure of stromal cell‐derived factor‐1 (SDF‐1) was determined by NMR spectroscopy. SDF‐1 is a monomer with a disordered N‐terminal region (residues 1–8), and differs from other chemokines in the packing of the hydrophobic core and surface charge distribution. Results with analogs showed that the N‐terminal eight residues formed an important receptor binding site; however, only Lys‐1 and Pro‐2 were directly involved in receptor activation. Modification to Lys‐1 and/or Pro‐2 resulted in loss of activity, but generated potent SDF‐1 antagonists. Residues 12–17 of the loop region, which we term the RFFESH motif, unlike the N‐terminal region, were well defined in the SDF‐1 structure. The RFFESH formed a receptor binding site, which we propose to be an important initial docking site of SDF‐1 with its receptor. The ability of the SDF‐1 analogs to block HIV‐1 entry via CXCR4, which is a HIV‐1 coreceptor for the virus in addition to being the receptor for SDF‐1, correlated with their affinity for CXCR4. Activation of the receptor is not required for HIV‐1 inhibition.

HIV-induced metalloproteinase processing of the chemokine stromal cell derived factor-1 causes neurodegeneration

The mechanisms of neurodegeneration that result in human immunodeficiency virus (HIV) type 1 dementia have not yet been identified. Here, we report that HIV-infected macrophages secrete the zymogen matrix metalloproteinase-2 (MMP-2), which is activated by exposure to MT1-MMP on neurons. Stromal cell–derived factor 1α (SDF-1), a chemokine overexpressed by astrocytes during HIV infection, was converted to a highly neurotoxic protein after precise proteolytic processing by active MMP-2, which removed the N-terminal tetrapeptide. Implantation of cleaved SDF-1(5–67) into the basal ganglia of mice resulted in neuronal death and inflammation with ensuing neurobehavioral deficits that were abrogated by neutralizing antibodies to SDF-1 and an MMP inhibitor drug. Hence, this study identifies a new in vivo neurotoxic pathway in which cleavage of a chemokine by an induced metalloproteinase results in neuronal apoptosis that leads to neurodegeneration.

The chemokine SLC is expressed in T cell areas of lymph nodes and mucosal lymphoid tissues and attracts activated T cells via CCR7

Secondary lymphoid‐tissue chemokine, SLC, also known as exodus‐2 and 6Ckine, is a novel CC chemokine with selectivity for T lymphocytes and preferential expression in lymphoid tissues. We have studied its production, receptor usage and biological activities. High levels of SLC mRNA were detected in lymph nodes, the gastrointestinal tract and several gland tissues, but no expression was found by Northern blot analysis in freshly isolated or stimulated blood monocytes and lymphocytes, or neutrophils and eosinophils. In situ hybridization revealed constitutive expression of SLC in the T cell areas and the marginal zone of follicles in lymph nodes and the mucosa‐associated lymphoid tissue, but not in B cell areas or sinuses. Comparison with immunocytochemical staining showed similarity between the in situ expression of SLC and the distribution of interdigitating dendritic cells but not with sinus‐lining dendritic cells, macrophages or T lymphocytes. SLC induced chemotaxis of T lymphocytes and its activity increased considerably when the cells were conditioned with IL‐2 or phytohemagglutinin (PHA). Under optimal conditions SLC had unusually high efficacy and induced the migration of up to 50 % of input T lymphocytes. SLC also induced Ca2+ mobilization in these cells. Similar responses were obtained with EBI1 ligand chemokine (ELC), and sequential stimulation with both chemokines led to cross‐desensitization, suggesting that SLC acts via the ELC receptor, CCR7. This was confirmed using murine pre‐B cells stably transfected with CCR7 which bound SLC with high affinity and showed chemotaxis and Ca2+ mobilization in response to both SLC and ELC. In T lymphocytes PHA and IL‐2, which enhanced chemotactic responsiveness, also markedly enhanced CCR7 expression. In contrast to all known chemokine receptors, up‐regulation of CCR7 by IL‐2 was transient. A maximum was reached in 2 – 3 days and expression returned to initial levels within 8 – 10 days. The present study shows that SLC is constitutively produced within the T cell areas of secondary lymphoid organs and attracts T lymphocytes via CCR7.

The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP10 and Mig

Integrin‐mediated adhesion to the vascular endothelium is an essential step in leukocyte diapedesis. We show that the chemokines 10‐kDa inflammatory protein (IP10) and monokine induced by IFN (Mig) induce rapid and transient adhesion of human IL‐2‐stimulated T lymphocytes (IL‐2 T cells) to immobilized integrin ligands through their receptor CXCR3, which is selectively expressed on activated T cells. Induction of adhesion by IP10 and Mig was already observed at subnanomolar concentrations and was maximal at 5 – 10 nM, resulting in three‐ to sixfold increase in adhesion of IL‐2 T cells over background. No effect was seen with resting naive/memory T cells which lack CXCR3 and migration responses to IP10 and Mig. Both chemokines are produced in human umbilical vein endothelial cells (HUVEC) upon stimulation with IFN‐γ and TNF‐α. These chemokines induce IL‐2 T cell adhesion also when captured on the surface of endothelial cells. Under conditions of flow, IL‐2 T cells roll and rapidly adhere to IP10/Mig‐expressing HUVEC, and anti‐CXCR3 mAb treatment reduces arrest and firm adhesion. This is the first study that shows chemokine‐induced adhesion in activated memory/effector T cells which represent the fraction of T cells that are selectively mobilized in inflammation. The critical role of IFN‐γ as inducer of IP10/Mig production in HUVEC indicates that these chemokines are essential mediators of effector T cell recruitment to IFN‐γ‐dependent pathologies.

Differential immobilization and hierarchical involvement of chemokines in monocyte arrest and transmigration on inflamed endothelium in shear flow

Monocyte extravasation into areas of inflammation involves sequential interactions of multiple adhesion molecules. However, differential contribution of chemokines produced by cytokine‐stimulated endothelium and their receptors to leukocyte attachment and transmigration under flow conditions remains to be elucidated. The activation of endothelial cells with TNF‐α up‐regulated mRNA and protein expression of the CXC chemokine GRO‐α and the CC chemokine monocyte chemotactic protein (MCP)‐1, which act through the receptors CXCR2 and CCR2 expressed on monocytes, respectively. Whereas GRO‐α was immobilized to endothelial cells via heparan sulfate proteoglycans, MCP‐1 was secreted in a soluble form. Consequently, inhibition experiments with blocking peptide analogues and monoclonal antibodies revealed that GRO‐α and its receptor CXCR2 but not MCP‐1 and its receptors substantially contributed to conversion of rolling into firm, shear‐resistant arrest of monocytes to TNF‐α‐stimulated endothelium in physiological flow. In contrast, MCP‐1 and CCR2 but not GRO‐α and CXCR2 mediated spreading, shape change and subsequent transendothelial migration, which was evident in flow but rarely in stasis and may thus require the establishment of a diffusible MCP‐1 gradient. Differential patterns of presentation may determine a functional specialization and hierarchy of chemokines and their receptors in sequential steps of monocyte emigration on inflamed endothelium and shear flow.

Identification of CCR8, the Receptor for the Human CC Chemokine I-309

The nucleotide sequence for a putative chemokine receptor, termed TER1, ChemR1, or CKR-L1, was recently obtained by a polymerase chain reaction-based cloning technique. It encodes a protein of 355 amino acids that shows 32–45% sequence identity with human chemokine receptors. The gene was localized on human chromosome 3p21–24, the site for the genes for the five known CC chemokine receptors, suggesting that the natural ligand may be a CC chemokine. We have stably expressed this receptor in murine pre-B cells 300-19 and have tested their responsiveness to 20 human chemokines and some other potential agonists. The CC chemokine I-309 was the only agonist that selectively induced intracellular Ca2+ mobilization and chemotaxis in receptor-transfected 300-19 cells. Stromal cell-derived factor 1, which binds to murine CXCR4 expressed in parental as well as transfected 300-19 cells, served as positive control in the functional screening. The interaction of I-309 with TER1 was of high affinity as shown by125I-I-309 binding (K d of 1.2 nm) and transient [Ca2+] i changes at subnanomolar concentrations of agonist. Migration responses in receptor-transfected 300-19 cells was typically bimodal with maximal activity at 10 nm of I-309. These data demonstrate that TER1 (ChemR1 or CKR-L1) is the receptor for I-309, and we propose to call this receptor CCR8 in agreement with the current nomenclature for chemokine receptors. The expression of CCR8 in blood leukocytes and lymphocytes was analyzed by Northern blot. No transcripts were found in RNA from freshly isolated blood neutrophils, monocytes, cultured macrophages, and phytohemagglutinin-stimulated T lymphocytes, and a faint hybridization signal corresponding to the RNA species of 4 kb was obtained only with RNA from interleukin-2-treated T lymphocytes. CCR8 is unusual for its selectivity for a single chemokine, previously shown only for CXCR1 and CXCR4, which bind interleukin-8 and stromal cell-derived factor 1, respectively. Identification of the receptor for I-309 represents a significant progress in determining the function of I-309 in inflammation and disease.

Interferon-γ induces the expression of H-2 and Ia antigens on brain cells *

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