Enhanced anti-HIV-1 activity and altered chemotactic potency of NH2-terminally processed macrophage-derived chemokine (MDC) imply an additional MDC receptor

Posttranslational processing of chemokines increases (IL-8) or decreases (monocyte chemotactic protein-1) their chemotactic potency. Macrophage-derived chemokine (MDC) attracts monocytes, dendritic cells, activated lymphocytes, and NK cells and has reportedly anti-HIV-1 activity. Here we report that truncation of MDC by deletion of two NH2-terminal residues resulted in impaired binding to CC chemokine receptor (CCR)4, the only identified MDC receptor so far. Truncated MDC(3-69) failed to desensitize calcium mobilization by MDC(1-69) or thymus- and activation-regulated chemokine (TARC), another CCR4 ligand. MDC(3-69) lacked HUT-78 T cell chemotactic activity but retained its capacity to attract monocytes and to desensitize chemotaxis. Compared with MDC(1-69), MDC(3-69) had weak but enhanced antiviral activity against M- and T-tropic HIV-1 strains. Furthermore, both MDC forms failed to signal through the orphan receptors Bonzo/STRL33 and BOB/GPR15 and to desensitize RANTES and stromal cell-derived factor (SDF)-1 responses in CCR5-transfected and CXC chemokine receptor (CXCR)4-transfected cells, respectively. These findings suggest that MDC recognizes another, yet unidentified, receptor. We conclude that minimal NH2-terminal truncation of MDC differentially affects its various immunologic functions.     

Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic strains of HIV-1. To understand the molecular basis of the binding of chemokines and HIV-1 to CCR5, we developed a number of mAbs that inhibit the various interactions of CCR5, and mapped the binding sites of these mAbs using a panel of CCR5/CCR2b chimeras. One mAb termed 2D7 completely blocked the binding and chemotaxis of the three natural chemokine ligands of CCR5, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta, to CCR5 transfectants. This mAb was a genuine antagonist of CCR5, since it failed to stimulate an increase in intracellular calcium concentration in the CCR5 transfectants, but blocked calcium responses elicited by RANTES, MIP-1alpha, or MIP-1beta. This mAb inhibited most of the RANTES and MIP-1alpha chemotactic responses of activated T cells, but not of monocytes, suggesting differential usage of chemokine receptors by these two cell types. The 2D7 binding site mapped to the second extracellular loop of CCR5, whereas a group of mAbs that failed to block chemokine binding all mapped to the NH2-terminal region of CCR5. Efficient inhibition of an M-tropic HIV-1-derived envelope glycoprotein gp120 binding to CCR5 could be achieved with mAbs recognizing either the second extracellular loop or the NH2-terminal region, although the former showed superior inhibition. Additionally, 2D7 efficiently blocked the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro. These results suggest a complicated pattern of HIV-1 gp120 binding to different regions of CCR5, but a relatively simple pattern for chemokine binding. We conclude that the second extracellular loop of CCR5 is an ideal target site for the development of inhibitors of either chemokine or HIV-1 binding to CCR5.     

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 (Kd = 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-1 alpha, macrophage inflammatory protein-1 beta). 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.     

Bacterial superantigens induce down-modulation of CC chemokine responsiveness in human monocytes via an alternative chemokine ligand-independent mechanism

Staphylococcal superantigens (SAgs) are very potent T cell mitogens, but they can also activate monocytes by binding directly to MHC class II molecules in a manner independent of TCR coengagement. Induction of proinflammatory cytokines and chemokine expression in monocytes by superantigens has recently been reported. Here we report that superantigen stimulation of human peripheral blood monocytes results in a rapid, dose-dependent, and specific down-regulation of chemokine (macrophage inflammatory protein-1alpha (MIP-1alpha), monocyte chemotactic protein-1 and MIP-1beta) binding sites (e.g., CCR1, CCR2, and CCR5), which correlates with a concomitant hyporesponsiveness of human monocytes to these CC chemokine ligands. This down-regulation occurs 15-30 min following superantigen stimulation and is specific to chemokine receptors, in that binding and responsiveness of monocytes to the chemoattractant formyl-tripeptide FMLP are not affected. We further demonstrate that SAg-induced down-modulation of chemokine binding and monocyte hyporesponsiveness to the chemokines MIP-1alpha, monocyte chemotactic protein-1, and MIP-1beta is mediated through cellular protein tyrosine kinases, and the down-modulation can be mimicked by an MHC class II-specific mAb. Additionally, our observations indicate that SAg-induced loss of chemokine binding and monocyte responsiveness is probably mediated by secreted serine proteinases. Bacterial SAg-induced down-modulation of chemokine responsiveness represents a previously unrecognized strategy by some bacteria to subvert immune responses by affecting the intricate balance between chemokine and chemokine receptor expression and function.     

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.     

Identification of CCR6, the specific receptor for a novel lymphocyte-directed CC chemokine LARC

Liver and activation-regulated chemokine (LARC) is a recently identified CC chemokine that is expressed mainly in the liver. LARC functions as a selective chemoattractant for lymphocytes that express a class of receptors specifically binding to LARC with high affinity. To identifiy the receptor for LARC, we examined LARC-induced calcium mobilization in cells stably expressing five CC chemokine receptors (CCR1-CCR5) and five orphan seven-transmembrane receptors. LARC specifically induced calcium flux in K562 cells as well as 293/EBNA-1 cells stably expressing an orphan receptor GPR-CY4. LARC induced migration in 293/EBNA-1 cells stably expressing GPR-CY4 with a bi-modal dose-response curve. LARC fused with secreted alkaline phosphatase (LARC-SEAP) bound specifically to Raji cells stably expressing GPR-CY4 with a Kd of 0.9 nM. Only LARC but not five other CC chemokines (MCP-1, RANTES, MIP-1alpha, MIP-1beta, and TARC) competed with LARC-SEAP for binding to GPR-CY4. By Northern blot analysis, GPR-CY4 mRNA was expressed mainly in spleen, lymph nodes, Appendix, and fetal liver among various human tissues. Among various leukocyte subsets, GPR-CY4 mRNA was detected in lymphocytes (CD4(+) and CD8(+) T cells and B cells) but not in natural killer cells, monocytes, or granulocytes. Expression of GPR-CY4 mRNA in CD4(+) and CD8(+) T cells was strongly up-regulated by IL-2. Taken together, GPR-CY4 is the specific receptor for LARC expressed selectively on lymphocytes, and LARC is a unique functional ligand for GPR-CY4. We propose GPR-CY4 to be designated as CCR6.     

CK beta-11/macrophage inflammatory protein-3 beta/EBI1-ligand chemokine is an efficacious chemoattractant for T and B cells

We examined the functional properties of CK beta-11/MIP-3 beta/ELC, a recently reported CC chemokine that specifically binds to a chemokine receptor, EBI1/BLR2/CCR7. CK beta-11/MIP-3 beta/ELC is distantly related to other CC and CXC chemokines in primary amino acid sequence structure. Recombinant human CK beta-11/MIP-3 beta/ELC expressed from a mammalian cell system showed potent chemotactic activity for T cells and B cells but not for granulocytes and monocytes. An optimal concentration of CK beta-11/MIP-3 beta/ELC attracted most input T cells within 3 h, a chemotactic activity comparable with that of stromal cell derived factor 1 (SDF-1), a highly efficacious CXC chemokine. CK beta-11/MIP-3 beta/ELC equally attracted naive CD45RA+ and memory type CD45RO+ T cells. CK beta-11/MIP-3 beta/ELC also strongly attracted both CD4+ and CD8+ T cells, but the attraction for CD4+ T cells was greater. CK beta-11/MIP-3 beta/ELC was also a more efficacious chemoattractant for B cells than MIP-1 alpha, a known B cell chemoattractant. CK beta-11/MIP-3 beta/ELC induced actin polymerization in lymphocytes, and chemotaxis was completely blocked by pertussis toxin showing its receptor, most likely EBI1/BLR2/CCR7, is coupled to a G(alpha i) protein. CK beta-11/MIP-3 beta/ELC induced calcium mobilization in lymphocytes, which could be desensitized by SDF-1, suggesting possible cross-regulation in their signaling. Human CK beta-11/MIP-3 beta/ELC attracted murine splenocytes suggesting functional conservation of CK beta-11/MIP-3 beta/ELC between human and mouse. The efficacy of chemoattraction by CK beta-11/MIP-3 beta/ELC and tissue expression of its mRNA suggest that CK beta-11/MIP-3 beta/ELC may be important in trafficking of T cells in thymus, and T cell and B cell migration to secondary lymphoid organs.     

Evidence for the presence of a kininogen-like species in a case of total deficiency of low and high molecular weight kininogens

EBI1-ligand chemokine (ELC) is a CC chemokine constitutively expressed in various lymphoid tissues and a high-affinity functional ligand for EBI1/CCR7, a seven transmembrane G-protein-coupled receptor originally identified as an Epstein-Barr virus (EBV)-inducible gene. Here we examined chemotactic activity of ELC on peripheral blood leukocytes. ELC attracted both CD4+ and CD8+ T cells, particularly efficiently after activation with IL-2 or with phytohemagglutinin (PHA) plus IL-2, as well as CD19+ B cells, but not CD16+ NK cells, CD14+ monocytes or neutrophils. Among CD3+ T cells, ELC attracted both CD45RO- naive and CD45RO+ memory subsets. ELC also induced vigorous calcium mobilization in T cells stimulated with IL-2 with an ED50 of 3 nM. ELC fused with the secreted form of alkaline phosphatase (ELC-SEAP) specifically bound to lymphocytes and this binding was blocked only by ELC among 10 CC chemokines so far tested. Notably, lymphocytes stimulated with IL-2 or T cells expanded by PHA plus IL-2 showed much higher levels of binding than fresh lymphocytes. Consistently, CCR7 mRNA was detected in CD4+ and CD8+ T cells as well as B cells, but not in NK cells, monocytes or neutrophils, and was dramatically increased in T cells upon treatment with IL-2 or with PHA plus IL-2. Like ELC mRNA, CCR7 mRNA was expressed in various lymphoid tissues. By in situ hybridization, ELC and CCR7 mRNA were detected in the parafollicular and inner cortical regions of a lymph node, and in the parafollicular regions of an appendix. Collectively, ELC and CCR7 may be involved in the trafficking of a broad spectrum of lymphocytes, especially activated T cells, into and within various lymphoid tissues.     

Cloning and characterization of the guinea pig eosinophil eotaxin receptor, C-C chemokine receptor-3: blockade using a monoclonal antibody in vivo

Certain C-C chemokines, signaling via the eotaxin receptor C-C chemokine receptor-3 (CCR3), are thought to be central mediators of eosinophil accumulation in allergic inflammation. To investigate the role of CCR3 in vivo, we cloned the guinea pig eotaxin receptor (guinea pig CCR3) from a genomic DNA library. We isolated a single-exon open reading frame coding for a 358-amino acid chemokine receptor protein with 67 and 69% homology to human and murine CCR3, respectively. When expressed in stable transfectants, this receptor bound 125I-labeled guinea pig eotaxin, 125I-labeled human monocyte chemotactic protein-3, and 125I-labeled human RANTES. In chemotaxis assays, guinea pig CCR3 transfectants responded only to guinea pig eotaxin, with a maximal effect at 100 nM. mAbs were raised that bound selectively to both guinea pig CCR3 transfectants and guinea pig eosinophils. One of these mAbs, 2A8, blocked both ligand binding to transfectants and their chemotaxis in response to eotaxin. The Ab also inhibited chemotaxis and the elevation of cytosolic calcium in guinea pig eosinophils in response to eotaxin. F(ab')2 fragments of 2A8 were prepared that retained the ability to inhibit eosinophil calcium responses to eotaxin. Pretreatment of (111)In-labeled eosinophils in vitro with F(ab')2 2A8 selectively inhibited their accumulation in response to eotaxin in vivo. These data demonstrate that functional blockade of eosinophil chemokine receptors can be achieved in vivo and provide further support for the development of novel anti-inflammatory drugs targeting eosinophil recruitment through chemokine receptor antagonism.     

A lymphocyte-specific CC chemokine, secondary lymphoid tissue chemokine (SLC), is a highly efficient chemoattractant for B cells and activated T cells

Secondary lymphoid tissue chemokine (SLC) is a CC chemokine expressed mainly in lymph nodes, appendix and spleen, and specifically chemotactic for lymphocytes (Nagira et al., J. Biol. Chem. 1997. 272: 19518-19524). Here, we carried out transendothelial migration assays to determine the classes and subsets of lymphocytes migrating toward SLC. SLC attracted freshly isolated B cells with high efficiency and T cells modestly. Thus, SLC is the first CC chemokine with a strong chemotactic activity on fresh B cells. Among T cell types and subsets, SLC broadly attracted CD4+ and CD8+ cells, CD45RO- (naive) and CD45RO+ (memory) cells, and CD26high (activated) and CD26low- (resting) cells. SLC also attracted both L-selectin+ and L-selectin- subpopulations of various T cell subsets and B cells. Furthermore, mitogenic stimulation strongly enhanced migratory responses of T cells and B cells toward SLC. By in situ hybridization, SLC mRNA was detected in the cortical parafollicular regions (the T cell areas) of a lymph node and an appendix. Collectively, SLC may be a basic chemokine supporting homeostatic migration of a broad spectrum of lymphocytes into the secondary lymphoid tissues. SLC may also be involved in immune responses by inducing highly efficient migration of T and B cells following antigenic stimulation.     

A RANTES-antibody fusion protein retains antigen specificity and chemokine function

The successful eradication of cancer cells in the setting of minimal residual disease may require targeting of metastatic tumor deposits that evade the immune system. We combined the targeting flexibility and specificity of mAbs with the immune effector function of the chemokine RANTES to target established tumor deposits. We describe the construction of an Ab fusion molecule with variable domains directed against the tumor-associated Ag HER2/neu, linked to sequences encoding the chemokine RANTES (RANTES.her2.IgG3). RANTES is a potent chemoattractant of T cells, NK cells, monocytes, and dendritic cells, and expression of RANTES has been shown to enhance immune responses against tumors in murine models. RANTES.her2.IgG3 fusion protein bound specifically to HER2/neu Ag expressed on EL4 cells and on SKBR3 breast cancer cells as assayed by flow cytometry. RANTES.her2.IgG3 could elicit actin polymerization of THP-1 cells and transendothelial migration of primary T lymphocytes. RANTES.her2.IgG3 prebound to SKBR3 cells also facilitated migration of T cells. RANTES.her2.IgG3 bound specifically to the CCR5 chemokine receptor, as demonstrated by flow cytometry, and inhibited HIV-1 infection via the CCR5 coreceptor. RANTES.her2.IgG3, alone or in combination with other chemokine or cytokine fusion Abs, may be a suitable reagent for recruitment and activation of an expanded repertoire of effector cells to tumor deposits.     

Expression of chemokine receptors CXCR4 and CCR5 in HIV-1-infected and uninfected individuals

The chemokine receptors CXCR4 and CCR5 have been identified as major coreceptors for HIV-1 entry into CD4+ T cells. The majority of primary HIV-1 isolates in early disease use CCR5 as a coreceptor, whereas during disease progression with the emergence of syncytium-inducing viruses, CXCR4 is also used. We performed a cross-sectional study in which we evaluated the expression of two HIV-1 coreceptors, CCR5 and CXCR4, in whole blood samples taken from HIV-1-infected and uninfected individuals. We demonstrate that CXCR4 on CD4+ and CD8+ T cells, and CD14+ monocytes is significantly down-regulated, and CCR5 expression on CD4+ T cells is up-regulated in HIV-infected individuals compared with uninfected controls. Coreceptor expression correlated with the level of cellular activation in vivo in both HIV-infected and uninfected individuals, with CXCR4 being expressed predominantly on quiescent (HLA-DR-) T cells and CCR5 being expressed predominantly on activated (HLA-DR+) T cells. Lower expression of CXCR4 and higher expression of CCR5 on CD4+ T cells correlated with advancing disease. In addition, a tendency for greater activation of CXCR4+CD4+ T cells in patients with advanced disease was observed. Patients who harbored syncytium-inducing viruses, however, could not be distinguished from those who harbored nonsyncytium-inducing viruses based on the level of CD4+ T cell activation or chemokine receptor expression.     

EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation

Dendritic cells (DC) that are stimulated with inflammatory mediators can maturate and migrate from nonlymphoid tissues to lymphoid organs to initiate T cell-mediated immune responses. This migratory step is closely related to the maturation of the DC. In an attempt to identify chemokine receptors that might influence migration and are selectively expressed in mature DC, we have discovered that the chemokine receptor, EBI1/CCR7, is strikingly up-regulated upon maturation in three distinct culture systems: 1) mouse bone marrow-derived DC, 2) mouse epidermal Langerhans cells, and 3) human monocyte-derived DC. The EBI1/CCR7 expressed in mature DC is functional because ELC/MIP-3beta, recently identified as a ligand of EBI1/CCR7, induces a rise in intracellular free calcium concentrations and directional migration of human monocyte-derived mature DC (HLA-DRhigh, CD1a(low), CD14-, CD25+, CD83+, and CD86high) in a dose-dependent manner, but not of immature DC (HLA-DRlow, CD1a(high), CD14-, CD25-, CD83-, and CD86-). In contrast, macrophage inflammatory protein-1alpha (MIP-1alpha), monocyte chemotactic protein-3 (MCP-3), and RANTES are active on immature DC but not on mature DC. Thus, it seems likely that MIP-1alpha, MCP-3, and RANTES can mediate the migration of immature DC located in peripheral sites, whereas ELC/MIP-3beta can direct the migration of Ag-carrying DC from peripheral inflammatory sites, where DC are stimulated to up-regulate the expression of EBI1/CCR7, to lymphoid organs. It is postulated that different chemokines and chemokine receptors are involved in DC migration in vivo, depending on the maturation state of DC.     

The T cell-directed CC chemokine TARC is a highly specific biological ligand for CC chemokine receptor 4

Thymus and activation-regulated chemokine (TARC) is a recently identified CC chemokine that is expressed constitutively in thymus and transiently in stimulated peripheral blood mononuclear cells. TARC functions as a selective chemoattractant for T cells that express a class of receptors binding TARC with high affinity and specificity. To identify the receptor for TARC, we produced TARC as a fusion protein with secreted alkaline phosphatase (SEAP) and used it for specific binding. By stably transfecting five orphan receptors and five known CC chemokine receptors (CCR1 to -5) into K562 cells, we found that TARC-SEAP bound selectively to cells expressing CCR4. TARC-SEAP also bound to K562 cells stably expressing CCR4 with a high affinity (Kd = 0.5 nM). Only TARC and not five other CC chemokines (MCP-1 (monocyte chemoattractant protein-1), RANTES (regulated upon activation, normal T cells expressed and secreted), MIP-1alpha (macrophage inflammatory protein-1alpha), MIP-1beta, and LARC (liver and activation-regulated chemokine)) competed with TARC-SEAP for binding to CCR4. TARC but not RANTES or MIP-1alpha induced migration and calcium mobilization in 293/EBNA-1 cells stably expressing CCR4. K562 cells stably expressing CCR4 also responded to TARC in a calcium mobilization assay. Northern blot analysis revealed that CCR4 mRNA was expressed strongly in human T cell lines and peripheral blood T cells but not in B cells, natural killer cells, monocytes, or granulocytes. Taken together, TARC is a specific functional ligand for CCR4, and CCR4 is the specific receptor for TARC selectively expressed on T cells.     

Posttranslational modifications affect the activity of the human monocyte chemotactic proteins MCP-1 and MCP-2: identification of MCP-2(6-76) as a natural chemokine inhibitor

Chemokines are important mediators in infection and inflammation. The monocyte chemotactic proteins (MCPs) form a subclass of structurally related C-C chemokines. MCPs select specific target cells due to binding to a distinct set of chemokine receptors. Recombinant and synthetic MCP-1 variants have been shown to function as chemokine antagonists. In this study, posttranslationally modified immunoreactive MCP-1 and MCP-2 were isolated from mononuclear cells. Natural forms of MCP-1 and MCP-2 were biochemically identified by Edman degradation and mass spectrometry and functionally characterized in chemotaxis and Ca2+-mobilization assays. Glycosylated MCP-1 (12 and 13.5 kDa) was found to be two- to threefold less chemotactic for monocytes and THP-1 cells than nonglycosylated MCP-1 (10 kDa). Natural, NH2-terminally truncated MCP-1(5-76) and MCP-1(6-76) were practically devoid of bioactivity, whereas COOH-terminally processed MCP-1(1-69) fully retained its chemotactic and Ca2+-inducing capacity. The capability of naturally modified MCP-1 forms to desensitize the Ca2+ response induced by intact MCP-1 in THP-1 cells correlated with their agonistic potency. In contrast, naturally modified MCP-2(6-76) was devoid of activity, but could completely block the chemotactic effect of intact MCP-2 as well as that of MCP-1, MCP-3, and RANTES. Carboxyl-terminally processed MCP-2(1-74) did retain its chemotactic potency. Although comparable as a chemoattractant, natural intact MCP-2 was found to be 10-fold less potent than MCP-1 in inducing an intracellular Ca2+ increase. It can be concluded that under physiologic or pathologic conditions, posttranslational modification affects chemokine potency and that natural MCP-2(6-76) is a functional C-C chemokine inhibitor that might be useful as an inhibitor of inflammation.     

STRL22 is a receptor for the CC chemokine MIP-3alpha

STRL22 is a human seven transmembrane domain orphan receptor related to known chemokine receptors and expressed in peripheral blood lymphocytes, tumor infiltrating lymphocytes and lymphoid tissues. MIP-3alpha/LARC/Exodus is a CC chemokine that is chemotactic for lymphocytes and that is expressed in activated cells, including monocytes, T cells, endothelial cells, and fibroblasts, and in liver, lung, and some lymphoid tissues. We report here that STRL22-transfected human embryonic kidney 293 cells demonstrated specific binding for MIP-3alpha and that MIP-3alpha, but no other chemokines, produced a calcium flux in the STRL22-transfected cells. We show that MIP-3alpha, unlike other chemokines, produced a calcium flux in freshly-isolated peripheral blood lymphocytes and we show that MIP-3alpha also produced a signal in tumor infiltrating lymphocytes that express STRL22. Since STRL22 is the sixth functional CC chemokine receptor identified, it should be re-named CCR6.     

Characterization of the signal transduction pathway activated in human monocytes and dendritic cells by MPIF-1, a specific ligand for CC chemokine receptor 1

The receptor specificity and signal transduction pathway has been identified and characterized for a truncated form of myeloid progenitor inhibitory factor-1 (MPIF-1(24-99)). MPIF-1 binds specifically to sites, in particular CCR1, shared with macrophage inflammatory protein-1alpha (MIP-1alpha) on the surface of human monocytes and dendritic cells, as inferred by its ability to compete for [125I]MIP-1alpha, but not for [125I]MIP-1beta or [125I]monocyte chemotactic protein-1(MCP-1) binding to intact cells. Based on calcium flux, MPIF-1 is an agonist on CCR1-transfected HEK-293 cells, monocytes, and dendritic cells, but not on CCR5-, CCR8-, or CX3CR1-transfected cells. The inhibitory effect of guanosine 5'-O-(3-thio-triphosphate) (GTP-gammaS) or pertussis toxin pretreatment on MPIF-1 binding and calcium mobilization, respectively, indicates the involvement of G proteins in the interaction of MPIF-1 and its receptor(s). The increase in intracellular free calcium concentration following MPIF-1 treatment is mainly due to the influx of calcium from an extracellular pool. However, a portion of the intracellular free calcium concentration is derived from a phospholipase C inhibitor-sensitive intracellular pool. MPIF-1 induces a rapid dose-dependent release of [3H]arachidonic acid from monocytes that is dependent on extracellular calcium and is blocked by phospholipase A2 (PLA2) inhibitors. Furthermore, PLA2 activation is shown to be necessary for filamentous actin formation in monocytes. Thus, the MPIF-1 signal transduction pathway appears to include binding to CCR1; transduction by G proteins; effector function by phospholipase C, protein kinase C, calcium flux, and PLA2; and cytoskeletal remodeling.     

Characterization of a novel CC chemokine, HCC-4, whose expression is increased by interleukin-10

We have identified and characterized a human beta (CC) chemokine, designated HCC-4, that is most closely related to HCC-1 and which demonstrates chemotactic activity for monocytes. Northern analysis of multiple tissue blots and of activated monocytes mRNA shows expression of a 500-bp mRNA. A 1,500-bp mRNA was highly expressed in monocytes activated 12 hours in the presence of interleukin-10 (IL-10) but was absent in monocytes activated for only 1 hour regardless of the presence or absence of IL-10. The upregulation of expression in the presence of IL-10 is in contrast to the downregulatory effects of IL-10 on expression of most other chemokines. Recombinant HCC-4 demonstrated chemotactic activity for human monocytes and THP-1 monocyte cells but not for resting lymphocytes or neutrophils. HCC-4 also induced a Ca2+ flux in THP-1 cells that was desensitized by prior exposure to RANTES. Taken together, these data indicate that HCC-4 is a novel chemokine whose expression is uniquely upregulated by IL-10.     

Prenatal onset of axonopathy in Dystonia musculorum mice

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic HIV-1 strains to enter the host cells. In this study, we aim to better understand the ligand-binding profiles of CCR5 and the chemokine-receptor usage on leukocyte cells. We found that MCP-2 could bind to CCR5 transfectants with high affinity and cross-compete effectively with RANTES, MIP-1alpha, and MIP-1beta. MCP-2 is a true agonist for CCR5, eliciting a robust chemotactic response in CCR5 transfectants similar to that of the three known CCR5 ligands and exhibiting cross-desensitization with RANTES in the Ca2+ flux response. MCP-4 also bound to CCR5 with high affinity and was efficiently displaced by other CCR5 ligands. However, MCP-4 only partially displaced the binding of radiolabeled MIP-1alpha and caused a chemotactic response only at high concentrations. Furthermore, MCP-2 inhibited the binding of the M-tropic HIV-1 gp120 envelope glycoprotein to CCR5 and HIV-1 infection of peripheral blood mononuclear cells. More importantly, we found that MCP-2 could bind and elicit chemotaxis in CD3-activated and IL-2-maintained T cells, and most of these functions could be specifically inhibited by the anti-CCR5 mAb 2D7, whereas the responses mediated by MIP-1alpha or MCP-4 were only partially inhibited by 2D7. Thus, although MCP-2 can bind to and signal through CCR1, CCR2b, and CCR5, among which both CCR2 and CCR5 are expressed at high levels on activated T cells, it appears to preferably utilize CCR5 on these cells. In contrast, MIP-1alpha and MCP-4 seem to activate multiple receptors on the same cells.