Carol J. Raport

Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-1beta, and MIP-1alpha

Chemokines affect leukocyte chemotactic and activation activities through specific G protein-coupled receptors. In an effort to map the closely linked CC chemokine receptor genes, we identified a novel chemokine receptor encoded 18 kilobase pairs downstream of the monocyte chemoattractant protein-1 (MCP-1) receptor (CCR2) gene on human chromosome 3p21. The deduced amino acid sequence of this novel receptor, designated CCR5, is most similar to CCR2B, sharing 71% identical residues. Transfected cells expressing the receptor bind RANTES (regulated on activation normal T cell expressed), MIP-1β, and MIP-1α with high affinity and generate inositol phosphates in response to these chemokines. This same combination of chemokines has recently been shown to potently inhibit human immunodeficiency virus replication in human peripheral blood leukocytes (Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K., Gallo, R. C., and Lusso, P. (1995) Science 270, 1811-1815). CCR5 is expressed in lymphoid organs such as thymus and spleen, as well as in peripheral blood leukocytes, including macrophages and T cells, and is the first example of a human chemokine receptor that signals in response to MIP-1β.

Macrophage-derived Chemokine Is a Functional Ligand for the CC Chemokine Receptor 4

Macrophage-derived chemokine (MDC) is a recently identified member of the CC chemokine family. MDC is not closely related to other chemokines, sharing most similarity with thymus- and activation-regulated chemokine (TARC), which contains 37% identical amino acids. Both chemokines are highly expressed in the thymus, with little expression seen in other tissues. In addition, the genes for MDC and TARC are encoded by human chromosome 16. To explore this relationship in greater detail, we have more precisely localized the MDC gene to chromosome 16q13, the same position reported for the TARC gene. We have also examined the interaction of MDC with CC chemokine receptor 4 (CCR4), recently shown to be a receptor for TARC. Using a fusion protein of MDC with secreted alkaline phosphatase, we observed high affinity binding of MDC-secreted alkaline phosphatase to CCR4-transfected L1.2 cells (K d = 0.18 nm). MDC and TARC competed for binding to CCR4, while no binding competition was observed for six other chemokines (MCP-1, MCP-3, MCP-4, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein-1α, macrophage inflammatory protein-1β). MDC was tested for calcium mobilization in L1.2 cells tranfected with seven different CC chemokine receptors. MDC induced a calcium flux in CCR4-transfected cells, but other receptors did not respond to MDC. TARC, which also induced calcium mobilization in CCR4 transfectants, was unable to desensitize the response to MDC. In contrast, MDC fully desensitized a subsequent response to TARC. Both MDC and TARC functioned as chemoattractants for CCR4 transfectants, confirming that MDC is also a functional ligand for CCR4. Since MDC and TARC are both expressed in the thymus, one role for these chemokines may be to attract CCR4-bearing thymocytes in the process of T cell education and differentiation.

New members of the chemokine receptor gene family

Chemokines are relatively small peptides with potent chemoattractant and activation activities for leukocytes. Several chemokine receptors have been cloned and characterized and all are members of the G protein‐coupled receptor superfamily. Using degenerate oligonucleotides and polymerase chain reaction, we have identified seven novel receptors with significant homology to chemokine receptors. Two of these sequences are presented here for the first time. We have shown, with gene mapping studies, that receptors with the highest sequence similarity are closely linked on human chromosomes. This close genetic association suggests a functional relationship as well.

The orphan G-protein-coupled receptor-encoding gene V28 is closely related to genes for chemokine receptors and is expressed in lymphoid and neural tissues

A polymerase chain reaction (PCR) strategy with degenerate primers was used to identify novel G-protein-coupled receptor-encoding genes from human genomic DNA. One of the isolated clones, termed V28, showed high sequence similarity to the genes encoding human chemokine receptors for monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1 alpha (MIP-1 alpha)/RANTES, and to the rat orphan receptor-encoding gene RBS11. When RNA was analyzed by Northern blot, V28 was found to be most highly expressed in neural and lymphoid tissues. Myeloid cell lines, particularly THP.1 cells, showed especially high expression of V28. We have mapped V28 to human chromosome 3p21-3pter, near the MIP-1 alpha/RANTES receptor-encoding gene.

Macrophage-Derived Chemokine and EBI1-Ligand Chemokine Attract Human Thymocytes in Different Stage of Development and Are Produced by Distinct Subsets of Medullary Epithelial Cells: Possible Implications for Negative Selection

The chemoattractant activity of macrophage-derived chemokine (MDC), EBI1-ligand chemokine (ELC), and secondary lymphoid tissue chemokine (SLC) on human thymocytes was analyzed. Both ELC and SLC caused the accumulation of CD4+CD8− or CD4−CD8+ CD45RA+ thymocytes showing high CD3 expression. By contrast, a remarkable proportion of MDC-responsive thymocytes were CD4+CD8+ cells exhibiting reduced levels of CD8 or CD4+CD8− cells showing CD3 and CD45R0, but not CD45RA. MDC-responsive thymocyte suspensions were enriched in cells expressing the MDC receptor, CCR4, selectively localized to the medulla, and in CD30+ cells, whereas ELC-responsive thymocytes never expressed CD30. Reactivity to both MDC and ELC was localized to cells of the medullary areas, but never in the cortex. Double immunostaining showed no reactivity for either MDC or ELC by T cells, macrophages, or mature dendritic cells, whereas many medullary epithelial cells were reactive to MDC or ELC. However, MDC reactivity was consistently localized to the outer wall of Hassal’s corpuscles, whereas ELC reactivity was often found in cells surrounding medullary vessels, but not in Hassal’s corpuscles. Moreover, while most MDC-producing cells also stained positive for CD30L, this molecule was never found on ELC-producing cells. We suggest therefore that CD30L-expressing MDC-producing medullary epithelial cells attract CCR4-expressing thymocytes, thus favoring the CD30/CD30L interaction, and therefore the apoptosis, of cells that are induced to express CD30 by autoantigen activation. By contrast, ELC production by CD30L-lacking medullary epithelial cells may induce the migration into periphery of mature thymocytes that have survived the process of negative selection.

Monocyte chemotactic protein-4: tissue-specific expression and signaling through CC chemokine receptor-2.

Chemokines constitute a family of low‐molecular‐weight proteins that attract or activate a variety of cell types, including leukocytes, endothelial cells, and fibroblasts. An electronic search of the GenBank Expressed Sequence Tags database uncovered a partial cDNA sequence with homology to the chemokine monocyte chemotactic protein‐1 (MCP‐1). Isolation of the full‐length clone revealed that it encodes the chemokine MCP‐4, an eosinophil chemoattractant recently described by Uguccioni et al. [J. Exp. Med. 183, 2379–2384]. Recombinant MCP‐4 was expressed in mammalian cells and purified by heparin‐Sepharose chromatography. Sequencing the amino terminus of this protein corroborated the reported sequence of recombinant MCP‐4 produced in insect cells. As shown by calcium flux assays, MCP‐4 activated the cloned G protein‐coupled receptor CCR‐2, which also recognizes MCP‐1 and MCP‐3. Northern hybridization indicated that MCP‐4 is constitutively expressed at high levels in the small intestine, colon, and lung. This expression profile is consistent with its role as a chemoattractant for eosinophils, which can be rapidly mobilized to the lung or intestine in response to invading pathogens. In marked contrast to MCP‐1, MCP‐4 was not induced in cell lines treated with pro‐inflammatory stimuli such as lipopolysaccharide or tumor necrosis factor α. J. Leukoc. Biol. 61: 353–360; 1997.

Profile of human macrophage transcripts: insights into macrophage biology and identification of novel chemokines.

High throughput partial sequencing of randomly selected cDNA clones has proven to be a powerful tool for examining the relative abundance of mRNAs and for the identification of novel gene products. Because of the important role played by macrophages in immune and inflammatory responses, we sequenced over 3000 randomly selected cDNA clones from a human macrophage library. These sequences represent a molecular inventory of mRNAs from macrophages and provide a catalog of highly expressed transcripts. Two of the most abundant clones encode recently identified CC chemokines. Macrophage‐derived chemokine (MDC) plays a complex role in immunoregulation and is a potent chemoattractant for dendritic cells, T cells, and natural killer cells. The chemokine receptor CCR4 binds MDC with high affinity and also responds by calcium flux and chemotaxis. CCR4 has been shown to be expressed by Th2 type T cells. Recent studies also implicate MDC as a major component of the host defense against human immunodeficiency virus. J. Leukoc. Biol. 64: 49–54; 1998.

Chemokines and Chemokine Receptors: Structure and Function

Publisher Summary This chapter provides a brief introduction to chemokine structure and activities. It explores the many functions of immune modulators, the family of chemokines. Chemokines are encoded by a large gene family with at least 45 members. The receptors for chemokines also belong to a gene family with at least 18 members, and all are G-protein-coupled receptors. The sequence similarities found in the chemokine gene family are reflected in their similar three-dimensional structures; however, chemokines display a diverse range of activities. Originally identified as potent leukocyte attractants in inflammatory diseases, chemokines have now been found to play critical roles in the natural development and regulation of the immune system. In addition, chemokines and their receptors have been utilized by pathogens to subvert the host immune system. Furthermore, this chapter provides a tabular description of the comprehensive chemokine nomenclature along with the receptors and cell types with which they interact. It also discusses the classification of chemokines. Chemokines can be divided into two general classes based on whether they are induced by pro-inflammatory cytokines or are constitutively expressed. The induced chemokines are upregulated very quickly at sites of infection or trauma and control the recruitment of leukocytes to the affected area. The constitutive chemokines are generally more involved in controlling migration of leukocytes through various tissues. Finally, it deals with chemokine receptors.

Chapter 24 – Chemokines and Chemokine Receptors: Structure and Function

Publisher Summary This chapter provides a brief introduction to chemokine structure and activities. It explores the many functions of immune modulators, the family of chemokines. Chemokines are encoded by a large gene family with at least 45 members. The receptors for chemokines also belong to a gene family with at least 18 members, and all are G-protein-coupled receptors. The sequence similarities found in the chemokine gene family are reflected in their similar three-dimensional structures; however, chemokines display a diverse range of activities. Originally identified as potent leukocyte attractants in inflammatory diseases, chemokines have now been found to play critical roles in the natural development and regulation of the immune system. In addition, chemokines and their receptors have been utilized by pathogens to subvert the host immune system. Furthermore, this chapter provides a tabular description of the comprehensive chemokine nomenclature along with the receptors and cell types with which they interact. It also discusses the classification of chemokines. Chemokines can be divided into two general classes based on whether they are induced by pro-inflammatory cytokines or are constitutively expressed. The induced chemokines are upregulated very quickly at sites of infection or trauma and control the recruitment of leukocytes to the affected area. The constitutive chemokines are generally more involved in controlling migration of leukocytes through various tissues. Finally, it deals with chemokine receptors.