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.

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.

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.

Single- and multi-analyte determination of gonadotropic hormones in urine by Surface Plasmon Resonance immunoassay.

Single- and multi-analyte detection of two gonadotropic hormones (follicle stimulating hormone (hFSH) and luteinizing hormone (hLH)) was achieved by a Surface Plasmon Resonance (SPR) immunoassay on untreated human urine samples. Multi-analyte detection was accomplished using two alternative formats which are based in the individual or simultaneous immobilization of the hormones on the sensor surface. The lowest detection limit for both hormones in urine was found to be 1 ng mL(-1), which in international units (IU) in terms of the World Health Organization (WHO) standards represents 8 mIU mL(-1) of hLH and 14 mIU mL(-1) of hFSH, respectively. The reliability of the assay was demonstrated by intra- and inter-assay variabilities < 6%, chip-to-chip variabilities < 5%, recoveries in the range of 80-120% and stability of the sensor response through more than 100 measurements. The sensitivity of this biosensing methodology renders it in a useful technique for the diagnosis of reproductive disorders, as well as for fertility monitoring.

Determination of human growth hormone in human serum samples by surface plasmon resonance immunoassay.

A surface plasmon resonance immunoassay has been developed to determine human growth hormone (hGH) directly and without pre-treatment in human serum samples. A binding inhibition immunoassay was employed. Antibody concentration, assay buffer and regeneration solution have been optimized in order to reach the best performance and the lower non-specific binding of the matrix components to the sensor surface. The lowest detection limit was 6 ng/mL, with a working range covering the physiological range. Reproducibility of the assay was excellent with both intra-assay and inter-assay relative standard deviations <5%, while a variation of 2.19% was obtained employing different sensor chips. Reutilization of the sensor surface allows its continuous use over 50 measurements with a signal drop <20%. The SPR immunoassay results were validated using enzyme-linked immunosorbent assay (ELISA) showing an excellent correlation (R(2)=0.985). A portable and fully automated system (Sensia SL) was employed in this work. This is the first SPR biosensor assay capable of detecting relevant concentrations of a clinical analyte in serum. This study shows the potentials of this device as a diagnostic tool for the detection of multiple clinical analytes.

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.

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.

Chemokine receptor dimerization: two are better than one.

The chemokines participate in an exceptional range of physiological and pathological processes, including the control of lymphocyte trafficking, tumor growth, wound healing, allograft rejection, regulation of T-cell differentiation, asthma, infection with HIV and atherosclerosis. This vast array of activities is triggered by the interaction of nearly 50 different chemokines with a relatively modest number of 20 G-protein-coupled receptors. The asymmetry between the number of receptors and ligands suggests an underlying, shared control mechanism activated at a very early stage of the response. One of the first events triggered by the binding of chemokines is the homo- and hetero-dimerization of their receptors; here, we outline these events and their consequences in chemokine signaling.

Functional expression of chemokine receptor 2 by normal human eosinophils.

BACKGROUND: Within the granulocytes, the CC chemokines preferentially activate basophils and eosinophils on binding to chemokine receptors (CCRs). In vivo administration of neutralizing anti-monocyte chemoattractant protein 1 (MCP-1) antibodies can block accumulation of eosinophils in the lungs of antigen-challenged animals. OBJECTIVE: We studied a panel of chemokines for chemotactic activity in normal human eosinophils from healthy donors with a special focus on MCP-1, identified the respective receptor required for the biological response of eosinophils, and investigated mediators used for signal transduction. METHODS: Cells were enriched by magnetic cell sorting. Receptor expression in eosinophils was shown by RT-PCR and fluorescence-activated cell sorting. The biological response was tested in chemotaxis and calcium mobilization assays. RESULTS: Eosinophils have detectable mRNA for CCR2, and the receptor protein is expressed on cell surfaces. MCP-1 induces chemotaxis and calcium mobilization in eosinophils. The chemotactic activity of MCP-1 revealed a double-peaked dose-response curve; one of the peaks is abolished by addition of a blocking antibody to CCR2, but it is insensitive to blocking of CCR1 or CCR3. Specific enzyme inhibitors ruled out signaling characteristics of CCR2 in eosinophils. CONCLUSION: Normal human eosinophils express functional CCR2 on cell surfaces.

Expression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cells.

Chemokines are secreted into the tumor microenvironment by tumor-infiltrating inflammatory cells as well as by tumor cells. Chemokine receptors mediate agonist-dependent cell responses, including migration and activation of several signaling pathways. In the present study we show that several human melanoma cell lines and melanoma cells on macroscopically infiltrated lymph nodes express the chemokine receptors CXCR3 and CXCR4. Using the highly invasive melanoma cell line BLM, we demonstrate that the chemokine Mig, a ligand for CXCR3, activates the small GTPases RhoA and Rac1, induces a reorganization of the actin cytoskeleton, and triggers cell chemotaxis and modulation of integrin VLA-5- and VLA-4-dependent cell adhesion to fibronectin. Furthermore, the chemokine SDF-1alpha, the ligand of CXCR4, triggered modulation of beta(1) integrin-dependent melanoma cell adhesion to fibronectin. Additionally, Mig and SDF-1alpha activated MAPKs p44/42 and p38 on melanoma cells. Expression of functional CXCR3 and CXCR4 receptors on melanoma cells indicates that they might contribute to cell motility during invasion as well as to regulation of cell proliferation and survival.

Receptor dimerization: a key step in chemokine signaling.

Chemokines exert their effects through their interaction with seven transmembrane domain receptors coupled to G-proteins, GPCRs. Such receptor ligation leads to the regulation of numerous activities where chemokines play a key role, including hematopoiesis, T-cell activation, angiogenesis, inflammatory diseases or HIV-1 infection. Here we discuss the molecular mechanisms that underlie chemokine receptor activation. As occurs with other GPCRs, chemokines initiate the signaling cascades by inducing receptor dimerization. This dimerization enables the activation of the JAK/STAT pathway which allows the subsequent triggering of G-protein dependent signaling events. This mechanism provides a new context to explain some of the activities exerted by chemokines and introduces new targets for the development of drugs to fight those diseases were chemokines are implicated, such as inflammation and AIDS.

Chemokine receptor homo- or heterodimerization activates distinct signaling pathways.

Chemokine receptors of both the CC and CXC families have been demonstrated to undergo a ligand-mediated homodimerization process required for Ca2+ flux and chemotaxis. We show that, in the chemokine response, heterodimerization is also permitted between given receptor pairs, specifically between CCR2 and CCR5. This has functional consequences, as the CCR2 and CCR5 ligands monocyte chemotactic protein-1 (MCP-1) and RANTES (regulated upon activation, normal T cell-expressed and secreted) cooperate to trigger calcium responses at concentrations 10- to 100-fold lower than the threshold for either chemokine alone. Heterodimerization results in recruitment of each receptor-associated signaling complex, but also recruits dissimilar signaling path ways such as G(q/11) association, and delays activation of phosphatidyl inositol 3-kinase. The consequences are a pertussis toxin-resistant Ca2+ flux and trig gering of cell adhesion rather than chemotaxis. These results show the effect of heterodimer formation on increasing the sensitivity and dynamic range of the chemokine response, and may aid in understanding the dynamics of leukocytes at limiting chemokine concentrations in vivo.

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.

CXCR3 chemokine receptor distribution in normal and inflamed tissues: expression on activated lymphocytes, endothelial cells, and dendritic cells.

Using new human CXCR3 chemokine receptor-specific monoclonal antibodies, we studied human CXCR3 tissue distribution in lymphoid and nonlymphoid organs, as well as in inflammatory conditions, including rheumatoid arthritis, Hashimoto's thyroiditis, and dermal vasculitis. CXCR3 was expressed by certain dendritic cell subsets, specifically myeloid-derived CD11c positive cells, not only in those present in normal lymphoid organs, but also in germinal centers generated in inflammatory conditions. CXCR3 expression was also detected in some lymphocyte subsets such as intraepithelial lymphocytes of secondary lymphoid organs and infiltrating lymphocytes in inflammatory conditions. In addition, CXCR3 was constitutively expressed by endothelial cells (EC) of vessels of medium and large caliber but not in small vessels from different organs. Finally, enhanced CXCR3 expression was found in EC and in infiltrating lymphocytes with an activated phenotype in inflammatory diseases. The CXCR3 chemokine receptor may play a role in the regulation of leukocyte migration to inflammatory sites.

Chemokine signaling and functional responses: the role of receptor dimerization and TK pathway activation.

A broad array of biological responses, including cell polarization, movement, immune and inflammatory responses, and prevention of HIV-1 infection, are triggered by the chemokines, a family of structurally related chemoattractant proteins that bind to specific seven-transmembrane receptors linked to G proteins. Here we discuss one of the early signaling pathways activated by chemokines, the JAK/STAT pathway. Through this pathway, and possibly in conjunction with other signaling pathways, the chemokines promote changes in cellular morphology, collectively known as polarization, required for chemotactic responses. The polarized cell expresses the chemokine receptors at the leading cell edge, to which they are conveyed by rafts, a cholesterol-enriched membrane fraction fundamental to the lateral organization of the plasma membrane. Finally, the mechanisms through which the chemokines promote their effect are discussed in the context of the prevention of HIV-1 infection.

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.

Unscheduled re-entry into the cell cycle induced by NGF precedes cell death in nascent retinal neurones.

During their early postmitotic life, a proportion of the nascent retinal ganglion cells (RGCs) are induced to die as a result of the interaction of nerve growth factor (NGF) with the neurotrophin receptor p75. To analyse the mechanisms by which NGF promotes apoptosis, an in vitro culture system consisting of dissociated E5 retinal cells was established. In this system, NGF-induced apoptosis was only observed in the presence of insulin and neurotrophin-3, conditions that favour the birth of RGCs and other neurones expressing the glycoprotein G4. The pro-apoptotic effect of NGF on the G4-positive neurones was evident after 10 hours in vitro and was preceded by a significant upregulation of cyclin B2, but not cyclin D1, and the presence of mitotic nuclei in these cells. Brain-derived neurotrophic factor prevented both the increase of cyclin B2 expression in the G4-positive neurones and the NGF-induced cell death. Finally, pharmacologically blocking cell-cycle progression using the cyclin-dependent kinase inhibitor roscovitine prevented NGF-induced cell death in a dose-dependent manner. These results strongly suggest that the apoptotic signalling initiated by NGF requires a driving stimulus manifested by the neuronal birth and is preceded by the unscheduled re-entry of postmitotic neurones into the cell cycle.

HIV-1 infection through the CCR5 receptor is blocked by receptor dimerization.

The identification of the chemokine receptors as receptors for HIV-1 has boosted interest in these molecules, raising expectations for the development of new strategies to prevent HIV-1 infection. The discovery that chemokines block HIV-1 replication has focused attention on identifying their mechanism of action. Previous studies concluded that this inhibitory effect may be mediated by steric hindrance or by receptor down-regulation. We have identified a CCR5 receptor-specific mAb that neither competes with the chemokine for binding nor triggers signaling, as measured by Ca(2+) influx or chemotaxis. The antibody neither triggers receptor down-regulation nor interferes with the R5 JRFL viral strain gp120 binding to CCR5, but blocks HIV-1 replication in both in vitro assays using peripheral blood mononuclear cells as HIV-1 targets, as well as in vivo using human peripheral blood mononuclear cell-reconstituted SCID (severe combined immunodeficient) mice. Our evidence shows that the anti-CCR5 mAb efficiently prevents HIV-1 infection by inducing receptor dimerization. Chemokine receptor dimerization also is induced by chemokines and is required for their anti-HIV-1 activity. In addition to providing a molecular mechanism through which chemokines block HIV-1 infection, these results illustrate the prospects for developing new tools that possess HIV-1 suppressor activity, but lack the undesired inflammatory side effects of the chemokines.