CC chemokine receptor (CCR)4, a high affinity receptor for the CC chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), is expressed in the thymus and spleen, and also by peripheral blood T cells, macrophages, platelets, and basophils. Recent studies have shown that CCR4 is the major chemokine receptor expressed by T helper type 2 (Th2) polarized cells. To study the in vivo role of CCR4, we have generated CCR4-deficient (CCR4−/−) mice by gene targeting. CCR4−/− mice developed normally. Splenocytes and thymocytes isolated from the CCR4−/− mice failed to respond to the CCR4 ligands TARC and MDC, as expected, but also surprisingly did not undergo chemotaxis in vitro in response to macrophage inflammatory protein (MIP)-1α. The CCR4 deletion had no effect on Th2 differentiation in vitro or in a Th2-dependent model of allergic airway inflammation. However, CCR4−/− mice exhibited significantly decreased mortality on administration of high or low dose bacterial lipopolysaccharide (LPS) compared with CCR4+/+ mice. After high dose LPS treatment, serum levels of tumor necrosis factor α, interleukin 1β, and MIP-1α were reduced in CCR4−/− mice, and decreased expression of MDC and MIP-2 mRNA was detected in peritoneal exudate cells. Analysis of peritoneal lavage cells from CCR4−/− mice by flow cytometry also revealed a significant decrease in the F4/80+ cell population. This may reflect a defect in the ability of the CCR4−/− macrophages to be retained in the peritoneal cavity. Taken together, our data reveal an unexpected role for CCR4 in the inflammatory response leading to LPS-induced lethality.

Dendritic cells are potent antigen-presenting cells involved in the initiation of immune responses. The trafficking of these cells to tissues and lymph nodes is mediated by members of the chemokine family. Recently, a novel CC chemokine known as MIP-3α or liver and activation-regulated chemokine has been identified from the EMBL/GenBank/DDBJ expressed sequence tag database. In the present study, we have shown that the messenger RNA for MIP-3α is expressed predominantly in inflamed and mucosal tissues. MIP-3α produced either synthetically or by human embryonic kidney 293 cells is chemotactic for CD34+-derived dendritic cells and T cells, but is inactive on monocytes and neutrophils. MIP-3α was unable to displace the binding of specific CC or CXC chemokines to stable cell lines expressing their respective high affinity receptors, namely CCR1–5 and CXCR1 and CXCR2, suggesting that MIP-3α acts through a novel CC chemokine receptor. Therefore, we used degenerate oligonucleotide-based reverse transcriptase PCR to identify candidate MIP-3α receptors in lung dendritic cells. Our results show that the orphan receptor known as GCY-4, CKRL-3, or STRL-22 is a specific receptor for MIP-3α, and that its activation leads to pertussis toxin–sensitive and phospholipase C–dependent intracellular Ca2+ mobilization when it is expressed in HEK 293 cells.

We report the cloning and characterization of a novel basophil CC chemokin receptor, K5-5, from the human immature basophilic cell line KU-812. The predicted protein sequence of K5-5 shows only 49% identity to the macrophage inflammatory protein-1 alpha/RANTES receptor (CC CKR-1) and 47% identity to monocyte chemotactic protein-1 receptor (b form), suggesting that this cDNA encodes a novel member of the CC chemokine receptor family. Analysis of K5-5 mRNA expression indicates that it is restricted to leukocyte-rich tissues. In addition, we have shown significant levels of K5-5 mRNA in human basophils, which were up-regulated by treatment with interleukin-5. The CC chemokines, Macrophage inflammatory protein-1 alpha, RANTES, and monocyte chemotactic protein-1 were able to stimulate a Ca(2+)-activated chloride channel in Xenopus laevis oocytes injected with K5-5 cRNA, whereas no signal was detected in response to monocyte chemotactic protein-2, macrophage inflammatory protein-1 beta, or the CXC chemokine, interleukin-8. Taken together, these results indicate for the first time the presence of a CC chemokine receptor on basophils, which functions as a "shared" CC chemokine receptor and may therefore be implicated in the pathogenesis of basophil-mediated allergic diseases.

Human immunodeficiency virus type 1 (HIV-1) entry is governed by the interaction of the viral envelope glycoprotein (Env) with its receptor. The HIV-1 receptor is composed of two molecules, the CD4 binding receptor and a coreceptor. The seven-membrane-spanning chemokine receptor CCR-5 is one of the coreceptors used by primary isolates of HIV-1. We demonstrate that the mouse homolog of CCR-5 (mCCR-5) does not function as an HIV-1 coreceptor. A set of chimeras of human CCR-5 and mCCR-5 was studied for Env-induced cell fusion and HIV-1 infection. Using the HIV-1 ADA envelope glycoprotein in a syncytium formation assay, we show that replacement of any fragment containing extracellular domains of mCCR-5 by its human counterparts is sufficient to allow Env-induced fusion. Conversely, replacement of any fragment containing human extracellular domains by its murine counterpart did not lead to coreceptor function loss. These results show that several domains of CCR-5 participate in coreceptor function. In addition, using a panel of primary nonsyncytium-inducing and syncytium-inducing isolates that use CCR-5 or both CXCR-4 and CCR-5 as coreceptors, we show that the latter dual-tropic isolates are less tolerant to changes in CCR-5 than strains with a more restricted coreceptor use. Thus, different strains are likely to have different ways of interacting with the CCR-5 coreceptor.

We have cloned a novel CC chemokine receptor cDNA from mouse thymus. The deduced amino acid sequence shows 74% identity to the human monocyte chemotactic protein (MCP)-1 receptor (CC CKR-2b) and 54% to a recently cloned murine macrophage inflammatory protein (MIP)-1alpha receptor (Gao, J. L., and Murphy, P. M.(1995) J. Biol. Chem. 270, 17494-17501). Northern blot analysis of mouse tissues showed that the mRNA was also expressed in heart, spleen and liver, and to a lesser extent in lung and brain. The rank order of CC chemokine competition for 125I-labeled human RANTES (regulated on activation, normal T-cell expressed and secreted) binding to human embryonic kidney (HEK) 293 cells stably transfected with the receptor cDNA was murine MIP-1alpha >> human MIP-1beta > human RANTES > murine RANTES > murine MIP-1beta > human MCP-2 > murine MCP-1 (JE) > human MIP-1alpha > human MCP-3 > human MCP-1. Of the chemokines tested, only murine MIP-1alpha, human and murine MIP-1beta and RANTES, human MCP-2, and JE were able to induce mobilization of intracellular Ca2+ from fura-2-loaded HEK 293 cells expressing the receptor. These results suggest that this receptor functions as a high affinity murine MIP-1alpha receptor; however, it is likely to be an important target for the biological activities of several CC chemokines in mouse.

The 3′-terminal stem-loop (3′SL) of the RNA genome of the flavivirus West Nile (WNV) harbors, in its stem, one of the sequence elements that are required for genome cyclization. As cyclization is a prerequisite for the initiation of viral replication, the 3′SL was proposed to act as a replication silencer. The lower part of the 3′SL is metastable and confers a structural flexibility that may regulate the switch from the linear to the circular conformation of the viral RNA. In the human system, we previously demonstrated that a cellular RNA-binding protein, AUF1 p45, destabilizes the 3′SL, exposes the cyclization sequence, and thus promotes flaviviral genome cyclization and RNA replication. By investigating mutant RNAs with increased 3′SL stabilities, we showed the specific conformation of the metastable element to be a critical determinant of the helix-destabilizing RNA chaperone activity of AUF1 p45 and of the precision and efficiency of the AUF1 p45-supported initiation of RNA replication. Studies of stability-increasing mutant WNV replicons in human and mosquito cells revealed that the cultivation temperature considerably affected the replication efficiencies of the viral RNA variants and demonstrated the silencing effect of the 3′SL to be temperature dependent. Furthermore, we identified and characterized mosquito proteins displaying similar activities as AUF1 p45. However, as the RNA remodeling activities of the mosquito proteins were found to be considerably lower than those of the human protein, a potential cell protein-mediated destabilization of the 3′SL was suggested to be less efficient in mosquito cells. In summary, our data support a model in which the 3′SL acts as an RNA thermometer that modulates flavivirus replication during host switching.

Introduction: Communities situated in protected areas generate conflicts among park administrators, residents and scientists. Should they stay or should they go? This article presents a positive example of a community existing in a state park. The study describes the community's governance process as well as how the park administration and the community solve the conflicts that arise and achieve a method of co-management in a multi-level governance process. Methods: The analysis is based on the Management and Transition Framework (MTF). We used a case study approach and collected data via document study, participatory observation and qualitative interviews.

Synthetic biology (SB) is a new techno-scientific field surrounded by an aura of hope, hype and fear. Currently it is difficult to predict which way the public debate – and thus the social shaping of technology – is heading. With limited hard evidence at hand, we resort to a strategy that takes into account speculative design and diegetic prototyping, accessing the Bio:Fiction science film festival, and its 52 short films from international independent filmmakers. Our first hypothesis was that these films could be used as an indicator of a public debate to come. The second hypothesis was that SB would most likely not follow the debate around genetic engineering (framing technology as conflict) as assumed by many observers. Instead, we found good evidence for two alternative comparators, namely nanotechnology (technology as progress) and information technology (technology as gadget) as stronger attractors for an upcoming public debate on SB.