Characterization of the CCR2 chemokine receptor: functional CCR2 receptor expression in B cells

We have derived anti-human CCR2-specific mAbs by immunization with synthetic peptides corresponding to CCR2 sequences presumably involved in the interaction with its ligand(s). The characterization of these mAbs includes the ability to recognize the CCR2 receptor specifically, as well as the function based on their ability to promote Ca2+ influx or to block MCP-1-induced Ca2+ influx and chemotaxis. One mAb (MCP-1 R02) that is directed to the NH2 terminal domain of the CCR2 receptor has MCP-1 agonist activity, and two that recognize the third extracellular domain (MCP-1R04 and MCP-1 R05) have MCP-1 antagonist activity. We analyzed the presence of CCR2 in several PBL and tonsil-derived leukocyte populations and found expression of this receptor in monocytes, activated T cells, and, surprisingly, in B cells. CCR2 receptor expression in B cells was further corroborated in Southern blot using CCR2-specific probes. Moreover, both MCP-1 and the agonist mAb trigger specific B cell migration via a PTX-sensitive mechanism, indicating the presence of a functional CCR2 receptor in these cells.     

The N-terminal extracellular segments of the chemokine receptors CCR1 and CCR3 are determinants for MIP-1alpha and eotaxin binding, respectively, but a second domain is essential for efficient receptor activation

CCR1 and CCR3 are seven-transmembrane domain G protein-coupled receptors specific for members of the CC chemokine subgroup of leukocyte chemoattractants. Both have been implicated in the inflammatory response, and CCR3, through its expression on eosinophils, basophils, and Th2 lymphocytes, may be especially important in allergic inflammation. CCR1 and CCR3 are 54% identical in amino acid sequence and share some ligands but not others. In particular, macrophage inflammatory protein 1alpha (MIP-1alpha) is a ligand for CCR1 but not CCR3, and eotaxin is a ligand for CCR3 but not CCR1. To map ligand selectivity determinants and to guide rational antagonist design, we analyzed CCR1:CCR3 chimeric receptors. When expressed in mouse pre-B cells, chimeras in which the N-terminal extracellular segments were switched were both able to bind both MIP-1alpha and eotaxin, but in each case, binding occurred via separate sites. Nevertheless, neither MIP-1alpha nor eotaxin were effective agonists at either chimeric receptor in either calcium flux or chemotaxis assays. These data are consistent with a multi-site model for chemokine-chemokine receptor interaction in which one or more subsites determine chemokine selectivity, but others are needed for receptor activation. Agents that bind to the N-terminal segments of CCR1 and CCR3 may be useful in blocking receptor function.     

Blockade of CC chemokine receptor 5 (CCR5)-tropic human immunodeficiency virus-1 replication in human lymphoid tissue by CC chemokines

The CC chemokines MIP-1alpha, MIP-1beta, and RANTES suppress replication of certain HIV-1 strains in cultured PBMC and T cell lines by blocking interaction of gp120 with CC chemokine receptor 5 (CCR5). However, the same chemokines can enhance HIV-1 replication in cultured macrophages. The net effect of chemokines on HIV-1 infection in intact lymphoid tissue, the major reservoir of HIV-1 in vivo, is unknown and unpredictable since the tissue contains both T lymphocytes and macrophages. Here we show that exogenous MIP-1alpha, MIP-1beta, and RANTES markedly suppressed replication of CCR5-tropic HIV-1 strains in blocks of human lymphoid tissue infected ex vivo. Moreover, endogenous MIP-1alpha, MIP-1beta, and RANTES were upregulated in tissues infected ex vivo with CXC chemokine receptor 4-tropic but not CCR5-tropic HIV-1. Such an upregulation may contribute to the virus phenotype shift in the course of HIV disease in vivo.     

Interferon-gamma increases expression of chemokine receptors CCR1, CCR3, and CCR5, but not CXCR4 in monocytoid U937 cells

Chemokine receptors (CR), which can mediate migration of immune cells to the site of inflammation, also function as coreceptors for human immunodeficiency virus (HIV) entry into CD4+ T lymphocytes and antigen-presenting cells. We demonstrate here that interferon-gamma (IFN-gamma) increases the expression of chemokine receptors CCR1, CCR3, and CCR5 in monocytoid U937 cells as detected by cell surface molecule labeling and mRNA expression, as well as by intracellular calcium mobilization and cell migration in response to specific ligands. The increased expression of these chemokine receptors also results in an enhanced HIV-1 entry into cells. Our data provide evidence for a relationship of cellular pathways that are induced by IFN-gamma with those that regulate chemokine receptor expression.     

Regulation of cytokine expression and leukotriene formation in human basophils by growth factors, chemokines and chemotactic agonists

Basophils stimulated with IL-3 plus C5a selectively express IL-4 and IL-13 and continuously produce leukotrienes (LT) for hours. C5a combined with IL-5 or granulocyte-macrophage colony-stimulated factor was, however, much less effective in promoting cytokine expression and a late continuous phase of LTC4 production, possibly due to lower expression levels of their receptor alpha chains. Basophils also express several chemoattractant receptors, including high levels of C5a receptors, macrophage chemotactic protein (MCP) receptors (CCR2) and eotaxin receptors (CCR3), intermediate levels of CXCR1, CXCR2 and platelet-activating factor receptors, and lower levels of N-formyl-Met-Leu-Phe (fMLP) receptors. However, among the corresponding agonists, only C5a, fMLP and much more weakly MCP1, were found to induce cytokine expression and continuous LTC4 release, and only when combined with IL-3. CCR3, which is highly expressed on basophils and has been shown to mediate strong migratory but weak release responses, does not regulate cytokine expression. The weakly expressed fMLP receptor is an efficient activator of several cell functions including LTC4 formation, while CXCR2 hardly affects basophil function despite considerable expression. Thus, chemoattractant-receptors mediate different cellular responses unrelated to their expression levels.     

Selective up-regulation of chemokine receptors CCR4 and CCR8 upon activation of polarized human type 2 Th cells

Polarized Th1 and Th2 cells differentially express adhesion molecules and chemokine receptors, endowing these cells with distinct tissue homing capabilities. Here we report that, in contrast to other chemokine receptors, the expression of CCR4 and CCR8 on Th2 cells is transiently increased following TCR and CD28 engagement. IL-4 is not required for this activation-induced up-regulation of CCR4 and CCR8. In accordance with receptor expression, the response of Th2 cells to I-309 (CCR8 ligand) and thymus- and activation-regulated chemokine (CCR4 and CCR8 ligand) is enhanced upon activation. Moreover, activated Th1 cells up-regulate CCR4 expression and functional responsiveness to thymus- and activation-regulated chemokine. Analysis of polarized subsets of CD8+ T cells reveals a similar pattern of chemokine receptor expression and modulation of responsiveness. Taken together, these findings suggest that an up-regulation of CCR4 and CCR8 following Ag encounter may contribute to the proper positioning of activated T cells within sites of antigenic challenge and/or specialized areas of lymphoid tissues.     

Up-regulation of CCR1 and CCR3 and induction of chemotaxis to CC chemokines by IFN-gamma in human neutrophils

Human neutrophils (polymorphonuclear leukocytes; PMN) respond to some CXC chemokines but do not migrate to CC chemokines. Recent work has shown that chemokine receptors can be modulated by inflammatory cytokines. In this study, the effect of IFN-gamma, a prototypic Th1 cytokine, on chemokine receptor expression in PMN was investigated. IFN-gamma caused a rapid (approximately 1 h) and concentration-dependent increase of CCR1 and CCR3 mRNA. The expression of CCR2, CCR5, and CXCR1-4 was not augmented. IFN-gamma-treated PMN, but not control cells, expressed specific binding sites for labeled monocyte-chemotactic protein (MCP)-3 and migrated to macrophage-inflammatory protein (MIP)-1alpha, RANTES, MCP-3, MIP-5/HCC2, and eotaxin. 7B11, a mAb for CCR3, inhibited the chemotactic response of IFN-gamma-treated PMN to eotaxin, and aminoxypentane-RANTES blocked PMN migration to RANTES. These results suggest that the selectivity of certain chemokines for their target cells may be altered by cytokines produced within an inflammatory context. Since PMN may play a role in orienting immunity toward Th1 responses, it is possible to speculate that IFN-gamma not only promotes Th1 differentiation directly, but also reorients the functional significance of Th2 effector cytokines by broadening the spectrum of their action to include PMN.     

Molecular cloning, functional characterization and mRNA expression analysis of the murine chemokine receptor CCR6 and its specific ligand MIP-3alpha

We have cloned the murine CCR6 receptor and its ligand, the beta-chemokine mMIP-3alpha. Calcium mobilization assays performed with mCCR6 transfectants showed significant responses upon addition of mMIP-3alpha. Murine MIP-3alpha RNA is expressed in thymus, small intestine and colon, whereas mCCR6 RNA is expressed in spleen and lymph nodes. RT-PCR analysis of FACS-sorted lymphoid and antigen presenting cell subsets showed mCCR6 expression mainly in B cells, CD8- splenic dendritic cells and CD4+ T cells. The cloning and functional characterization of the mCCR6 and mMIP-3alpha will allow the study of the role of these proteins in mouse models of inflammation and immunity.     

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.     

Role of the CTLA-4 receptor in T cell activation and immunity. Physiologic function of the CTLA-4 receptor

Near-death experience (NDE) was studied in a series of 48 consecutive patients who were admitted to hospital in a deep coma (level III on the III-3 coma scale) due to cardiac arrest, pulmonary failure, cerebrovascular accident, and/or other life-threatening disease. When the patients recovered from coma without complications such as aphasia, dementia or mental disturbance, they were interviewed by the same physician following the same protocol consisting of 25 questions about their experience during the period of deep coma. Of 48 patients interviewed, 14 (37%) had a vivid and undeniably personal experience during their unconscious state. Factors attributable to the NDE were assessed by the following three methods. First, the frequency and odds ratio were examined in terms of gender, age, underlying disease, occupation, religion, education, site of accident, duration of comatose state, drugs and treatment for resuscitation, and drugs being taken at the time of interview. There were no specific factors significantly related to the NDE. Next, background factors were compared between the NDE positive and negative groups to detect a particular factor related to the NDE. However, there were no factors that showed significant frequency in the NDE-positive group. Finally, discriminatory analysis was performed to detect discriminatory factors in the occurrence of NDE by selecting NDE as an objective variable and background factors as explanatory variables. However, the discriminatory equation gained was not significant. Thus, there were no background factors that could explain the occurrence of NDE. Among the NDE reported, there were such elements as flying in a dark void space with dim light ahead, encountering dead relatives or friends, standing at the boundary of brook, river or pond, and returning to the world in response to a voice calling from behind. These elements are common to those reported by investigators abroad, except for the lack of a tunnel experience. As for the influence of the NDE on life subsequent to the experience, the majority of patients who had had a NDE stated that they became more sincere to towards every aspect of life and held spiritual values in high esteem than before. This was quite a contrast to the attitudes in the non-NDE patients who looked upon the comatose episode as arising from an underlying disease and considered it a health problem only. Most of the NDE patients considered that death was neither fearful nor difficult, but calm and peaceful if it occurs in a manner similar to that in their NDE. From this study, a picture can be down of the dying process, based on empirical information, it can also be seen that a NDE causes the individual to develop a sincere introspective depth. It is possible that these findings may be applicable to elderly patients in terminal care.     

Peripheral blood-derived CD34+ progenitor cells: CXC chemokine receptor 4 and CC chemokine receptor 5 expression and infection by HIV

The present study demonstrates cell surface expression of both CXC chemokine receptor 4 (CXCR4) and CC chemokine receptor 5 (CCR5), major coreceptors for T cell-tropic and macrophage-tropic strains of HIV, respectively, on CD34+ progenitor cells derived from the peripheral blood. CD34+ progenitor cells were susceptible to infection by diverse strains of HIV, and infection could be sustained for prolonged periods in vitro. HIV entry into CD34+ progenitor cells could be modulated by soluble CD4, HIV gp120 third variable loop neutralizing mAb and the cognate ligands for the CXCR4 and CCR5 HIV coreceptors. This study suggests that a significant proportion of the circulating progenitor cell pool may serve as a reservoir for HIV that is capable of trafficking the virus to diverse anatomic compartments. Furthermore, the infection and ultimate destruction of these progenitor cells may explain in part the defective lymphopoiesis in certain HIV-infected individuals despite effective control of virus replication during highly active antiretroviral therapy.     

Protein tyrosine kinases in activation signal of human basophils through the immunoglobulin E receptor type l

Human basophils activated through high-affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) are involved in the late phase of the allergic reaction. To investigate the possible involvement of protein-tyrosine kinases in this activation we used human acute basophilic leukemia (ABL) cells in culture as well as a pure population of normal basophils in vitro-derived from human bone marrow precursor cells (HBMB). ABL cells were 50-80% basophils at various stages of maturation as assessed by staining, morphology, ultrastructure, and flow cytometry analysis, and only basophils in ABL cells expressed Fc epsilon RI. Aggregation of Fc epsilon RI by IgE and anti-IgE, IgE and antigen, or anti-Fc epsilon RI monoclonal antibodies on ABL cells or on HBMB, led to increased tyrosine phosphorylation of 120-, 100-, 80-, 72-, 50- to 65-, and 38-kDa substrates. Tyrosine phosphorylations in ABL cells were in basophils because 1) they were detected after a 5-s stimulation, 2) they were observed under conditions where mediator release is minimal, i.e., in the absence of extracellular calcium, 3) hapten addition during antigen stimulation resulted in almost total disappearance of tyrosine phosphorylations within 30 s. There was correlation between histamine release and tyrosine phosphorylation in anti-IgE dose-responses and in dose-responses of the tyrosine kinase inhibitor genistein. The tyrosine kinase p72syk was detected in the cells. Stimulation of ABL cells for 1 min resulted in extracellular calcium-independent tyrosine phosphorylation and activation of p72syk. Therefore, tyrosine kinases are involved in the early steps of human Fc epsilon RI signaling in basophils. Tyrosine kinases and their substrates could represent new potential therapeutic targets to prevent the development of the allergic reaction.     

The chemokine monocyte chemotactic protein 1 triggers Janus kinase 2 activation and tyrosine phosphorylation of the CCR2B receptor

Gelsolin is an actin filament-capping protein that has been shown to play a key role in cell migration. Here we have studied the involvement of phosphoinositide 3-kinase (PI 3-kinase) and GTP-binding proteins (G-proteins) in the regulation of gelsolin-actin interactions in neutrophils. Inhibition of PI 3-kinase activity in vivo by wortmannin did not affect the dissociation of actin-gelsolin (1:1) complexes induced by neutrophil stimulation with N-formyl-Met-Leu-Phe. Guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) indirectly promoted the dissociation of actin-gelsolin complexes in a cell-free system using neutrophil cytosol, and this effect was blocked by the GDP dissociation inhibitor for Rho (Rho-GDI). The GTPgammaS-loaded ialpha2 and the beta1gamma2 subunits of heterotrimeric G-proteins (Gialpha2 and Gbeta1gamma2) also triggered actin-gelsolin dissociation in a Rho-GDI-sensitive manner. GTP-loaded activated Rac, but not activated Rho, induced the dissociation of cytosolic actin-gelsolin complexes. The guanine nucleotide exchange on Rac was increased by addition of GTPgammaS-loaded Gialpha2 or Gbeta1gamma2 to neutrophil cytosol. These findings suggest that activation of Rac by G-protein-coupled receptors in neutrophils triggers uncapping of actin filaments, independently of PI 3-kinase.     

Morphological alteration of cultured tracheobronchial epithelial cells is accompanied by the expression of chemokines, MCP-1 and CINC/gro, in rats

Research on regressive group processes such as Janis' (1982) "groupthink" phenomenon has rarely focused on work groups in authentic settings. In this study, teams from six different organisations (n = 308) were studied by using a groupthink questionnaire constructed in accordance with the symptoms of groupthink described by Janis. It was hypothesised that groupthink could be described as a bipolar construct identifying either an omnipotent or a depressive variant of a group's delusions about its own and other groups' features. The questionnaire showed reasonably good reliability as a whole and a factor analysis identified three factors in line with the proposed theoretical model in which the two different types of groupthink can be distinguished. We propose that any group might have a tendency or predisposition to react in either of the two directions during provocative circumstances. The six different organisations exhibited different types of groupthink to a varying degree. A religious sect was the one most characterised by omnipotent groupthink, while a technological company and a psychiatric team seemed to be the ones with most features of depressive groupthink.     

Detection of MCP-4 in dermal fibroblasts and its activation of the respiratory burst in human eosinophils

CC-chemokines are an important family of proinflammatory mediators that promote the recruitment and activation of human eosinophils in chronic inflammatory diseases. Recently, a novel human CC-chemokine, monocyte chemotactic protein 4 (MCP-4), has been reported that shows amino acid sequence similarities with eotaxin and RANTES, induces chemotaxis of eosinophils, and signals through specific chemokine receptors. In this study, we investigated the effect of MCP-4 on different eosinophil effector functions leading to the activation of the respiratory burst. In human eosinophils, MCP-4 dose dependently induced the production of reactive oxygen species and actin polymerization as a related event. Pretreatment of eosinophils with different enzyme inhibitors interacting with the signal transduction cascade revealed that Gi protein, protein kinase C, tyrosine kinase, and phosphatidylinositol-3-kinase are involved in the signaling following stimulation with MCP-4. In addition, cytokine-stimulated human dermal fibroblasts expressed high levels of MCP-4 mRNA, suggesting that fibroblasts are a physiologic source of MCP-4. Therefore, this study demonstrates that there is an important role of MCP-4 in the activation of eosinophils and that the interaction between dermal fibroblasts and human eosinophils may play an important role within the cytokine network.     

Patterns of CCR5, CXCR4, and CCR3 usage by envelope glycoproteins from human immunodeficiency virus type 1 primary isolates

Coreceptor usage by Envs from diverse primary human immunodeficiency virus type 1 isolates was analyzed by a vaccinia virus-based expression and assay system. Usage of recombinant CCR5 and CXCR4 correlated closely with fusogenicity toward macrophages and T-cell lines expressing endogenous coreceptors. Surprisingly, recombinant CCR3 was utilized by most primary and T-cell-line-adapted Envs. Endogenous CXCR4 in macrophages was functional as a coreceptor.