Polarization of chemokine receptors to the leading edge during lymphocyte chemotaxis.

Leukocyte migration in response to cell attractant gradients or chemotaxis is a key phenomenon both in cell movement and in the inflammatory response. Chemokines are quite likely to be the key molecules directing migration of leukocytes that involve cell polarization with generation of specialized cell compartments. The precise mechanism of leukocyte chemoattraction is not known, however. In this study, we demonstrate that the CC chemokine receptors CCR2 and CCR5, but not cytokine receptors such as interleukin (IL)-2Ralpha, IL-2Rbeta, tumor necrosis factor receptor 1, or transforming growth factor betaR, are redistributed to a pole in T cells that are migrating in response to chemokines. Immunofluorescence and confocal microscopy studies show that the chemokine receptors concentrate at the leading edge of the cell on the flattened cell-substratum contact area, induced specifically by the signals that trigger cell polarization. The redistribution of chemokine receptors is blocked by pertussis toxin and is dependent on cell adhesion through integrin receptors, which mediate cell migration. Chemokine receptor expression on the leading edge of migrating polarized lymphocytes appears to act as a sensor mechanism for the directed migration of leukocytes through a chemoattractant gradient.

Similarities and differences in RANTES- and (AOP)-RANTES-triggered signals: implications for chemotaxis.

Chemokines are a family of proinflammatory cytokines that attract and activate specific types of leukocytes. Chemokines mediate their effects via interaction with seven transmembrane G protein-coupled receptors (GPCR). Using CCR5-transfected HEK-293 cells, we show that both the CCR5 ligand, RANTES, as well as its derivative, aminooxypentane (AOP)- RANTES, trigger immediate responses such as Ca2+ influx, receptor dimerization, tyrosine phosphorylation, and Galphai as well as JAK/STAT association to the receptor. In contrast to RANTES, (AOP)-RANTES is unable to trigger late responses, as measured by the association of focal adhesion kinase (FAK) to the chemokine receptor complex, impaired cell polarization required for migration, or chemotaxis. The results are discussed in the context of the dissociation of the late signals, provoked by the chemokines required for cell migration, from early signals.

Role of the PI3K regulatory subunit in the control of actin organization and cell migration.

Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K. Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen. These observations show the essential role of the PI3K regulatory subunit p85alpha in controlling PDGF receptor-induced cytoskeletal changes and cell migration, illustrating a novel signaling pathway that links receptor stimulation at the cell membrane with actin dynamics.

Activation of Th lymphocytes alters pattern expression and cellular location of VIP receptors in healthy donors and early arthritis patients.

Vasoactive Intestinal Peptide (VIP) is an important immunomodulator of CD4+ cells in normal and pathological conditions, which exerts its anti-inflammatory and immunomodulatory actions through VPAC receptors, VPAC1 and VPAC2. Only a decrease in the expression of VPAC1 mRNA on Th cells upon activation has been reported. Thus, the deepening in the knowledge of the behavior of these receptors may contribute to the design of new therapies based on their activation and/or blockade. In this study, we describe the expression pattern, cellular location and functional role of VIP receptors during the activation of human Th cells in healthy conditions and in early arthritis (EA). The protein expression pattern of VPAC1 did not change with the activation of Th lymphocytes, whereas VPAC2 was up-regulated. In resting cells, VPAC1 was located on the plasma membrane and nucleus, whereas it only appeared in the nucleus in activated cells. VPAC2 was always found in plasma membrane location. VIP receptors signaled through a PKA-dependent pathway in both conditions, and also by a PKA-independent pathway in activated cells. Both receptors exhibit a potent immunomodulatory capacity by controlling the pathogenic profile and the activation markers of Th cells. These results highlight a novel translational view in inflammatory/autoimmune diseases.

Microglia-derived nerve growth factor causes cell death in the developing retina.

While nerve growth factor (NGF) is best known for its trophic functions, recent experiments indicate that it can also cause cell death during development by activating the neurotrophin receptor p75. We now identify microglial cells as the source of NGF as a killing agent in the developing eye. When the retina is separated from the surrounding tissue before colonization by microglial cells, no NGF can be detected, and cell death is dramatically reduced. It is restored by the addition of microglial cells, an effect that is blocked by NGF antibodies. NGF adsorbed at the surface of beads, but not soluble NGF, mimics the killing action of microglial cells. These results indicate an active role for macrophages in neuronal death.

Cell cycle reentry triggers hyperploidization and synaptic dysfunction followed by delayed cell death in differentiated cortical neurons.

Cell cycle reentry followed by neuronal hyperploidy and synaptic failure are two early hallmarks of Alzheimer's disease (AD), however their functional connection remains unexplored. To address this question, we induced cell cycle reentry in cultured cortical neurons by expressing SV40 large T antigen. Cell cycle reentry was followed by hyperploidy in ~70% of cortical neurons, and led to progressive axon initial segment loss and reduced density of dendritic PSD-95 puncta, which correlated with diminished spike generation and reduced spontaneous synaptic activity. This manipulation also resulted in delayed cell death, as previously observed in AD-affected hyperploid neurons. Membrane depolarization by high extracellular potassium maintained PSD-95 puncta density and partially rescued both spontaneous synaptic activity and cell death, while spike generation remained blocked. This suggests that AD-associated hyperploid neurons can be sustained in vivo if integrated in active neuronal circuits whilst promoting synaptic dysfunction. Thus, cell cycle reentry might contribute to cognitive impairment in early stages of AD and neuronal death susceptibility at late stages.

A potential immune escape mechanism by melanoma cells through the activation of chemokine-induced T cell death.

The immune system attempts to prevent or limit tumor growth, yet efforts to induce responses to tumors yield minimal results, rendering tumors virtually invisible to the immune system [1]. Several mechanisms may account for this subversion, including the triggering of tolerance to tumor antigens [2, 3], TGF-alpha or IL-10 production, downregulation of MHC molecules, or upregulation of FasL expression [4, 5]. Melanoma cells may in some instances use FasL expression to protect themselves against tumor-infiltrating lymphocytes (TIL) [4, 5]. Here, we show another, chemokine-dependent mechanism by which melanoma tumor cells shield themselves from immune reactions. Melanoma-inducible CCL5 (RANTES) production by infiltrating CD8 cells activates an apoptotic pathway in TIL involving cytochrome c release into the cytosol and activation of caspase-9 and -3. This process, triggered by CCL5 binding to CCR5, is not mediated by TNFalpha, Fas, or caspase-8. The effect is not unique to CCL5, as other CCR5 ligands such as CCL3 (MIP-1alpha) and CCL4 (MIP-1beta) also trigger TIL cell death, nor is it limited to melanoma cells, as it also operates in activated primary T lymphocytes. The model assigns a role to the CXC chemokine CXCL12 (SDF-1alpha) in this process, as this melanoma cell-produced chemokine upregulates CCL5 production by TIL, initiating TIL cell death.

The amino-terminal domain of the CCR2 chemokine receptor acts as coreceptor for HIV-1 infection.

The chemokines are a homologous serum protein family characterized by their ability to induce activation of integrin adhesion molecules and leukocyte migration. Chemokines interact with their receptors, which are composed of a single-chain, seven-helix, membrane-spanning protein coupled to G proteins. Two CC chemokine receptors, CCR3 and CCR5, as well as the CXCR4 chemokine receptor, have been shown necessary for infection by several HIV-1 virus isolates. We studied the effect of the chemokine monocyte chemoattractant protein 1 (MCP-1) and of a panel of MCP-1 receptor (CCR2)-specific monoclonal antibodies (mAb) on the suppression of HIV-1 replication in peripheral blood mononuclear cells. We have compelling evidence that MCP-1 has potent HIV-1 suppressive activity when HIV-1-infected peripheral blood lymphocytes are used as target cells. Furthermore, mAb specific for the MCP-1R CCR2 which recognize the third extracellular CCR2 domain inhibit all MCP-1 activity and also block MCP-1 suppressive activity. Finally, a set of mAb specific for the CCR2 amino-terminal domain, one of which mimics MCP-1 activity, has a potent suppressive effect on HIV-1 replication in M- and T-tropic HIV-1 viral isolates. We conjecture a role for CCR2 as a coreceptor for HIV-1 infection and map the HIV-1 binding site to the amino-terminal part of this receptor. This concurs with results showing that the CCR5 amino terminus is relevant in HIV-1 infection, although chimeric fusion of various extracellular domains shows that other domains are also implicated. We discuss the importance of CCR2 structure relative to its coreceptor role and the role of anti-CCR2 receptor antibodies in the prevention of HIV-1 infection.

Lengthening of G2/mitosis in cortical precursors from mice lacking beta-amyloid precursor protein.

The beta-amyloid precursor protein (APP) is expressed within the nervous system, even at the earliest stages of embryonic development when cell growth and proliferation is particularly important. In order to study the function of APP at these early developmental stages, we have studied the development of the cerebral cortex in both wild type and App-/- mutant mice. Here, we demonstrate that APP mRNA is expressed in cortical precursor cells and that APP protein is concentrated within their apical domains during interphase. However, during mitosis, APP re-localizes to the peripheral space surrounding the metaphase plate. In APP-deficient cortical precursors, the duration of mitosis is increased and a higher proportion of cortical precursor cells contained nuclei in late G2. We conclude that during cortical development APP plays a role in controlling cell cycle progression, particularly affecting G2 and mitosis. These observations may have important implications for our understanding of how APP influences the progression of Alzheimer's disease, since degenerating cortical neurons have been shown to up-regulate cell cycle markers and re-enter the mitotic cycle before dying.

DeltahGHR, a novel biosafe cell surface-labeling molecule for analysis and selection of genetically transduced human cells.

We describe a new selectable marker for retroviral transduction and selection of human and murine cells. The molecule expressed on the cell surface of the transduced population is a truncated version of human growth hormone receptor (deltahGHR), capable of ligand (hGH) binding, but devoid of the domains involved in signal triggering. We demonstrate that the engineered molecule is stably expressed in the target cells as an inert protein unable to trigger proliferation or to rescue the cells from apoptosis after ligand binding. This new marker will probably have a wide application spectrum, since hGHR in the human adult is highly expressed only in liver cells, and lower levels have been reported in certain lymphocyte cell populations. The deltahGHR label has high biosafety potential, as it belongs to a well-characterized hormonal system that is nonessential in adults, and there is extensive clinical experience with hGH administration in humans. This record allows us to hypothesize the lack of relevant clinical consequences resulting from massive transgene expression caused by successful replacement of a large tissue with genetically transduced cells. We take advantage of the differential binding properties of several monoclonal antibodies (MAbs) in describing a cell rescue method in which the antibody used to select deltahGHR-transduced cells is eluted by competition with hGH or, alternatively biotinylated hGH is used to capture tagged cells. In the latter system, the final purified population would be recovered free of attached antibodies in hGH (a substance approved for human use)-containing medium, providing additional biosafety relative to currently existing methods that rely on the use of murine MAb to rescue genetically labeled cells.

Characterization of monoclonal antibodies specific for the human growth hormone 22K and 20K isoforms.

We have derived and characterized a set of monoclonal antibodies (mAb) specific for the different human GH (hGH) isoforms. The binding characteristics of each antibody to the hGH isoforms (22K and 20K) were analyzed in direct and competitive immunoassays as well as by Western blot. We studied the effects of these mAb on the biological activity of hGH and showed that they specifically block their respective activities. Using these mAb, we developed several immunoassays that have been applied for the quantitation of the different hGH isoforms in body fluids. Therefore, these mAb may help to unravel the biological function of these variants.

Expression analysis of the thyrotropin-releasing hormone receptor (TRHR) in the immune system using agonist anti-TRHR monoclonal antibodies.

Monoclonal anti-rat thyrotropin-releasing hormone (TRH) receptor (TRHR)-specific antibodies (mAb) were generated by immunization with synthetic peptides of rat TRHR partial amino acid sequences; one (TRHR01) was directed against a sequence (84-98) in the extracellular portion of the rat TRHR reported to be constant among different species, including man, and the second (TRHR02) recognizes the C-terminal region sequence 399-412. In lysates from GH4C1 cells, a clonal rat pituitary cell line, both mAb recognize the TRHR in Western blot analysis, and TRHR02 immunoprecipitates the TRHR. Incubation of GH4C1 cells with the mAb causes a fluorescence shift in fluorescence-activated cell sorting analysis. The cells were stained specifically by both mAb using immunocytochemical techniques. Furthermore, TRHR01 is agonistic in its ability to trigger Ca2+ flux, and desensitizes the TRH receptor. We tested for TRHR in several rat organs and found expression in lymphoid tissues. TRHR01 recognizes the human TRHR, and analysis of human peripheral blood lymphocyte and tonsil-derived leukocyte populations showed receptor expression in non-activated and phytohemagglutinin-activated T and B cells.

Role of neurotrophins in the control of neural development: neurotrophin-3 promotes both neuron differentiation and survival of cultured chick retinal cells.

The effects of neurotrophins brain-derived neurotrophic factor and neurotrophin-3 on cultured dissociated cells from chick retina were studied at several embryonic ages from day 4 to day 13. Precursor cells from days 4-7 retinas proliferated in vitro and, after 20 h in culture, a proportion of them underwent spontaneous differentiation, as judged by both [3H]thymidine uptake and acquisition of neuronal morphology and neuron-specific markers. Brain-derived neurotrophic factor did not affect neuronal differentiation, although this factor supports survival of differentiated retinal ganglion cells [Rodríguez-Tébar et al. (1989) Devl Biol. 136, 296-303]. However, in cultures from young undifferentiated retinas, neurotrophin-3 produced up to a 2.5-fold increase in the number of [3H]thymidine-positive neurons, i.e. those that in vitro replicated their DNA. Moreover, in older retinas, neurotrophin-3, like brain-derived neurotrophic factor, supported the survival of differentiated retinal ganglion cells over a short developmental period. This effect was negligible at embryonic day 5, maximal at day 9, decreased at day 11 and was absent at embryonic day 13. Neurotrophin-3 also supported the survival of a population of amacrine neurons. This effect was modest at embryonic day 9, and increased at days 11 and 13. Our results show that, whereas the action of brain-derived neurotrophic factor is restricted to differentiated neurons, neurotrophin-3 exerts two distinct successive actions on retinal cells in vitro: first, this factor promotes either differentiation of neuroepithelial cells or maturation of recently differentiated neurons, and later in development, this factor supports the survival of differentiated retinal ganglion and amacrine cells but only during a discrete post-differentiation period.

Developmentally regulated vitronectin influences cell differentiation, neuron survival and process outgrowth in the developing chicken retina.

Vitronectin is a multifunctional protein involved in the regulation of the immune system and blood coagulation. Here we report that the expression of vitronectin is developmentally regulated in the embryonic retina of the chicken. Vitronectin immunoreactivity was detected in chicken retinas from embryonic day 5, encompassing the cell bodies of most neuroepithelial cells. At this developmental stage, alpha v integrin subunit expression was distributed across the retina, suggesting a ligand/receptor interaction. Expression of both vitronectin and alpha v increased during development and reached a maximum at embryonic day 9, a time when most differentiated neurons grow processes and initiate synapse formation. At this age, vitronectin immunoreactivity appeared to be located predominantly in the fiber and inner plexiform layers of the differentiated stratified retina. alpha v immunoreactivity and mRNA expression was seen associated with all layers formed by differentiated neurons, being most abundant in the ganglion cell and inner nuclear layers. Later in development, levels of vitronectin decreased and immunoreactivity appeared exclusively associated with the fiber layer. In accordance with this pattern of expression, vitronectin as a substrate sustained both proliferation and differentiation of cultured neuroepithelial cells from embryonic day 5 retinas. At later stages, vitronectin supported survival and neurite outgrowth of most differentiated neurons. Our data suggest that vitronectin is a ubiquitous component of the retinal extracellular matrix, serving as a substrate for developmental processes such as proliferation, differentiation of neuron progenitors, cell survival, and axonal and dendritic growth of differentiated neurons.

Control of the cell cycle by neurotrophins: lessons from the p75 neurotrophin receptor.

Although traditionally little attention has been paid to the interplay between neurotrophins and the cell cycle, a number of recent findings suggest an important role for these growth factors in the regulation of this aspect of the cellular physiology. In this article, we review the evidence from a number of studies that neurotrophins can influence cell cycle progression or mitotic cycle arrest both in the nervous system as well as in other cell types. The contrary response of different cells to neurotrophins in terms of cell cycle regulation derives in part from the fact that these factors use two different receptor types to transmit their signals: members of the Trk family and the p75 neurotrophin receptor (p75NTR). With this in mind, we outline the current state of our knowledge regarding the molecular basis underlying the control of cell cycle progression by neurotrophins. We focus our interest on the receptors that transduce these signals and, in particular, the striking finding that p75NTR interacts with proteins that can promote mitotic cycle arrest. Finally, we discuss the mechanisms of cell death mediated by p75NTR in the context of cell cycle regulation.

Neurotrophins and other growth factors in the generation of retinal neurons.

The generation of neurons in the vertebrate retina, as in other areas of the developing nervous system, largely depends on extracellular signals. Of the known signaling molecules, neurotrophins play decisive, defined, and distinct roles. The three neurotrophins identified in the chick, namely, neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF), are expressed in either the pigment epithelium (NT-3 and BDNF) or in the neural retina (NGF) at the onset of neuron birth. In addition, trkC and trkB, receptors for NT-3 and BDNF, respectively, together with p75, the low-affinity neurotrophin receptor, are expressed in the retina at the same developmental period. The role of these three neurotrophins in the differentiation of neurons in the chick retina has been elucidated by a combination of in vitro and in vivo experiments. Thus, NT-3 promotes the conversion of neuroepithelial cells into neurons, whereas BDNF and NGF control the programmed cell death (apoptosis) that affects early postmitotic neuroblasts. BDNF, acting via its trkB receptor, is a survival factor for these cells, whereas NGF, binding to p75 receptor, acts as a killing factor, thereby controlling the provisional number of newly generated neurons.

Implementation of a SPR immunosensor for the simultaneous detection of the 22K and 20K hGH isoforms in human serum samples.

We have implemented a Surface Plasmon Resonance (SPR) immunosensor based on a sandwich assay for the simultaneous detection of the two main hGH isoforms, of 22 kDa (22K) and 20 kDa (20K). An oriented-antibody sensor surface specific for both hormone isoforms was assembled by using the biotin-streptavidin system. The immunosensor functionality was checked for the direct detection of the 22K hGH isoform in buffer, which gave high specificity and reproducibility (intra and inter-assay mean coefficients of variation of 8.23% and 9% respectively). The selective determination of the 22K and 20K hGH isoforms in human serum samples in a single assay was possible by using two specific anti-hGH monoclonal antibodies. The detection limit for both hormone isoforms was 0.9 ng mL(-1) and the mean coefficient of variation was below 7.2%. The excellent reproducibility and sensitivity obtained indicate the high performance of this immunosensor for implementing an anti-doping test.

Technical advance: Surface plasmon resonance-based analysis of CXCL12 binding using immobilized lentiviral particles.

Use of SPR-based biosensors is an established method for measuring molecular interactions. Their application to the study of GPCRs is nonetheless limited to detergent-solubilized receptors that can then be reconstituted into a lipid environment. Using the chemokine receptor CXCR4 and its specific ligand CXCL12, we outline here a highly reproducible biosensor method based on receptor presentation on the surface of lentiviral particles; the approach is simple and does not require the use of antibodies to achieve correct receptor orientation on the sensorchip surface. We measured the kinetic parameters of CXCR4/CXCL12 binding in a single step and in real time and evaluated the effect of GAG presentation of chemokines on this interaction. The data indicate that at low concentrations, soluble heparin modulates CXCR4/CXCL12 interaction and at high concentrations, abrogates binding. These observations suggest that in addition to their known role in modulating local chemokine availability, GAG affect the receptor/ligand interaction, although their influence on affinity parameters is very limited. The method will also be useful for quantifying these biomarkers in biological fluids and for the development of high-throughput screening for their antagonists.