A Small Molecule Antagonist of Chemokine Receptors CCR1 and CCR3

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We investigated the actions of a panel of nonsteroidal anti-inflammatory drugs on eosinophils, basophils, neutrophils, and monocytes. Indomethacin alone was a potent and selective inducer of eosinophil and basophil shape change. In eosinophils, indomethacin induced chemotaxis, CD11b up-regulation, respiratory burst, and L-selectin shedding but did not cause up-regulation of CD63 expression. Pretreatment of eosinophils with indomethacin also enhanced subsequent eosinophil shape change induced by eotaxin, although treatment with higher concentrations of indomethacin resulted in a decrease in the expression of the major eosinophil chemokine receptor, CCR3. Indomethacin activities and cell selectivity closely resembled those of prostaglandin D 2 (PGD 2 ). Eosinophil shape change in response to eotaxin was inhibited by pertussis toxin, but indomethacin-and PGD 2 -induced shape change responses were not. Treatment of eosinophils with specific inhibitors of phospholipase C (U-73122), phosphatidylinositol 3-kinase (LY-294002), and p38 mitogen-activated protein kinase (SB-202190) revealed roles for these pathways in indomethacin signaling. Indomethacin and its analogues may therefore provide a structural basis from which selective PGD 2 receptor small molecule antago-

supporting

confidence: 68%

Section: Methodsmentioning

confidence: 99%

Indomethacin Causes Prostaglandin D2-like and Eotaxin-like Selective Responses in Eosinophils and Basophils

Stubbs

Schratl

Hartnell

et al. 2002

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“…Similarly, no internalization was induced by natural chemokines acting as antagonists including I-TAC, IP10, and Mig, which bind to CCR3 (35), eotaxin, which binds to CCR2 (44), and MCP-3, which binds to CCR5 (45). Like the chemokine-based antagonists, small-molecule antagonists were unable to down-regulate chemokine receptors, as shown for TAK-779, which binds to CCR5 (66) and for UCB35625, which binds to CCR1 and CCR3 (67). These data indicate that, as a rule, the blockade of chemokine receptors by antagonists mainly depends on receptor occupancy.…”

Section: Inhibition Of Chemotaxis and [Ca 2ϩ ] I Changes By I-tac (4 -mentioning

confidence: 93%

Structure-Function Relationship between the Human Chemokine Receptor CXCR3 and Its Ligands

Clark‐Lewis¹,

Mattioli²,

Gong³

et al. 2003

171

102

I-TAC, IP10, and Mig are interferon-␥ inducible CXC chemokines that share the same G-protein-coupled receptor CXCR3, which is preferentially expressed on Th1 lymphocytes. We have explored the structure-function relationship of the CXCR3 ligands, in particular of I-TAC, which has highest affinity for CXCR3 and is the most potent agonist. A potent antagonist for CXCR3 was obtained by NH 2 -terminal truncation of I-TAC. I-TAC (4 -73), which lacks the first three residues, has no agonistic activity but competes for the binding of I-TAC to CXCR3-bearing cells and inhibits migration and Ca 2؉ changes in such cells in response to stimulation with I-TAC, IP10, and Mig. It does also not induce internalization of CXCR3, which is in support of the lack of agonistic effects. Hybrid chemokines between I-TAC and IP10 were used to identify regions responsible for the higher activity of I-TAC. I-TAC-like IP10 analogs are obtained by substituting the NH 2 terminus (residues 1-8) or N-loop region (residues 12-17) of IP10 with those of I-TAC, suggesting that the differences in function of the CXCR3 ligands can be assigned to distinct regions and that these regions are interchangeable. Structureactivity studies with Mig showed that the extended basic COOH-terminal region, which is not present in I-TAC and IP10, is important for binding and activity.

“…The cells were washed in RPMI ϩ 0.5% BSA ϩ 25 mM HEPES (pH 7.4) (binding buffer), counted, and resuspended in binding buffer at a density of 2 ϫ 10 7 cells/ml. The assay was carried out as previously described (34). Briefly, cells were incubated with 0.1 nM 125 I-huMDC and increasing concentrations of unlabeled huMDC or muMDC for 60 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

The Identification, Characterization, and Distribution of Guinea Pig CCR4 and Epitope Mapping of a Blocking Antibody

Jopling¹,

Sabroe²,

Andrew³

et al. 2002

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Th2 lymphocytes play a central role in the control and maintenance of allergic inflammation. The chemokine receptor CCR4 is preferentially expressed on the surface of Th2 lymphocytes polarised in vitro. However, CCR4 is found on the surface of a significant proportion of circulating memory T lymphocytes, some of which are capable of producing the Th1-associated cytokine interferon ␥. To investigate the function of CCR4 on guinea pig (gp) T lymphocytes, we identified the open-reading frame of gpCCR4, which encodes a 361-amino acid protein with 88 and 81% amino acid identity to human and murine CCR4 sequences, respectively. Cells transfected with gpCCR4 migrated toward the human and murine orthologues of the CCR4 ligands, macrophage-derived chemokine and thymus and activation-regulated chemokine. Surface expression of CCR4, using an anti-human CCR4 monoclonal antibody, 10E4, was detected on ϳ12% of guinea pig peripheral blood T helper cells, and CCR4 ؉ guinea pig thymocytes were detected in low numbers. However, CCR4؉ T helper cells constituted ϳ9% of the T lymphocyte population within the normal guinea pig lung and 52% of the guinea pig bronchoalveolar lavage fluid, which is consistent with a role for CCR4 in T lymphocyte development and trafficking through normal tissues. Subsequent analysis of chimeric chemokine receptors indicated that 10E4, a functional inhibitor of gpCCR4 responses, recognized the amino terminus of CCR4.Allergic inflammation of the lung in diseases such as asthma is largely controlled and maintained by the recruitment of antigen-specific T lymphocytes to the lung tissue (1). The Th2-type lymphocytes that predominate at sites of allergic inflammation secrete cytokines such as IL-4, IL-5 and IL-13, 1 which influence the actions of other leukocyte populations involved in the inflammatory response (2, 3). For example, IL-4 and IL-13 can induce B lymphocyte production of IgE (4, 5) and airway epithelial cell generation of eotaxin, which is central to eosinophil recruitment (6, 7), whereas IL-5 is a potent eosinophil maturation and survival factor (8, 9). Overexpression of IL-13 in the lung causes many of the histopathological and physiological features of asthma (10). Increasing evidence suggests that chemokines are important regulators of the selective trafficking of leukocytes in homeostasis, hematopoiesis, and inflammation (11). Chemokines can be divided into four subfamilies dependent upon the position of their amino-terminal cysteine residues; the two main chemokine groups are the CXCL and the CCL subfamilies (12), which signal via CXCR and CCR receptors belonging to the G-protein coupled receptor superfamily (13,14). The differential expression of chemokine receptors on the surface of leukocytes contributes to the selective recruitment of cells to specific sites, and, consequently, these receptors may provide useful targets for therapeutic intervention in the modulation of inflammatory disorders. Studies of T lymphocytes polarized in vitro have shown that Th1 cells preferentially express CC...