HIV-1 coreceptors CCR5 and CXCR4 both mediate neuronal cell death but CCR5 paradoxically can also contribute to protection

The chemokine receptors CCR5 and CXCR4 serve, in addition to CD4, as coreceptors for human immunodeficiency virus-1 (HIV-1), and infection with HIV-1 can cause dementia. In brain-derived cells, HIV-1 envelope glycoprotein gp120 initiates a signaling cascade that involves p38 mitogen-activated protein kinase and leads to neuronal cell death. Using mixed neuronal/glial cultures from rats and mice genetically deficient in one or both HIV coreceptors, we show here that CCR5, CXCR4 or both can mediate HIV/gp120 neurotoxicity depending on the viral strain. Paradoxically, we also found evidence for a CCR5-mediated neuroprotective pathway. We identify protein kinase Akt/PKB as an essential component of this pathway, which can be triggered by the CCR5 agonists macrophage inflammatory protein-1beta and regulated-and-normal-T-cell-expressed-and-secreted. Moreover, these CCR5 ligands prevent neuronal cell death induced by stromal cell-derived factor-1, a CXCR4 agonist. Both neurons and glia coexpress CXCR4 and CCR5. Ca2+ imaging experiments demonstrate that engagement of CCR5 prevents CXCR4-triggered increases in intracellular free Ca2+. This finding suggests that CCR5 ligands can protect neurons at least, in part, by modulating CXCR4-mediated toxicity through heterologous desensitization.     

Function of the follicular dendritic cell in the germinal center of lymphoid follicles

The authors made an immunocytochemical examination of the germinal centers (GCs) of (1) lymph follicles in physiological lymph nodes and (2) extra-nodal tissues of divergent diseases including thyroid disorders, rheumatoid arthritis, Warthin's tumor and Kimura's disease (Eosinophilic lymphfolliculoid granuloma). In these germinal centers, the presence of immunoglobulins (IgG and IgM), early acting complement components (C1q, C4, C3c, C3d), receptors for C3b and C3d and dendritic reticulum cell-1 was demonstrated in lace-like network patterns which were proven electron-microscopically to coincide with the surface of follicular dendritic cells. IgE was distributed in a lace-like pattern in the GCs of proliferating follicles of Kimura's disease, in which the lysis of follicles was frequently observed. This lysis appeared to be related to the presence of complement components. In the germinal centers of extra-nodal tissues, including the thyroid tissues accompanying the lymph follicles, rheumatoid arthritis synovial tissues as well as Warthin's tumors, thyroglobulin, rheumatoid factor and salivary amylase were detected as specific antigens, occurring in lace-like patterns. It is possible that follicular dendritic cells may play a role in the genesis of GCs and be responsible for the immune response through C3 receptors.     

FTY720-enhanced T cell homing is dependent on CCR2, CCR5, CCR7, and CXCR4: evidence for distinct chemokine compartments

FTY720 stimulates CCR7-driven T cell homing to peripheral lymph nodes (LN) by direct activation of sphingosine 1-phosphate receptors, along with the participation of multidrug transporters, 5-lipoxygenase, and G protein-coupled receptors for chemokines. In this study, we demonstrate that FTY720 also directly stimulates in vitro T cell chemotaxis to CCR2-CCL2, but not to a variety of other chemokines, including CCR5-CCL3/4/5 and CXCR4-CXCL12. FTY720 influences CCR2-CCL2-driven migration through activation of the multidrug transporters, Abcb1 and Abcc1, and through 5-lipoxygenase activity. In vivo administration of FTY720 induces chemokine-dependent migration of T cells in the thymus, peripheral blood, LN, and spleen. The CCR7 and CCR2 chemokine ligands are required for both T cell sequestration in LN and thymic T cell egress following FTY720 administration. Furthermore, FTY720 administration uncovers a requirement for CXCR4 ligands for LN homing, but not for thymic egress, and CCR5 for thymic egress, but not LN homing. FTY720-driven splenic and peripheral blood T cell egress are both independent of CCR2, CCR5, CCR7, or CXCR4. These results indicate that FTY720- and sphingosine 1-phosphate receptor-stimulated T cell migration are dependent on the restricted usage of chemokine receptor-ligand pairs within discrete anatomic compartments.     

Constitutive plasmacytoid dendritic cell migration to the splenic white pulp is cooperatively regulated by CCR7- and CXCR4-mediated signaling

Although the spleen plays an important role in host defense against infection, the mechanism underlying the migration of the innate immune cells, plasmacytoid dendritic cells (pDCs), into the spleen remains ill defined. In this article, we report that pDCs constitutively migrate into the splenic white pulp (WP) in a manner dependent on the chemokine receptors CCR7 and CXCR4. In CCR7-deficient mice and CCR7 ligand-deficient mice, compared with wild-type (WT) mice, substantially fewer pDCs were found in the periarteriolar lymphoid sheath of the splenic WP under steady-state conditions. In addition, the migration of adoptively transferred CCR7-deficient pDCs into the WP was significantly worse than that of WT pDCs, supporting the idea that pDC trafficking to the splenic WP requires CCR7 signaling. WT pDCs responded to a CCR7 ligand with modest chemotaxis and ICAM-1 binding in vitro, and priming with the CCR7 ligand enabled the pDCs to migrate efficiently toward low concentrations of CXCL12 in a CXCR4-dependent manner, raising the possibility that CCR7 signaling enhances CXCR4-mediated pDC migration. In agreement with this hypothesis, CCL21 and CXCL12 were colocalized on fibroblastic reticular cells in the T cell zone and in the marginal zone bridging channels, through which pDCs appeared to enter the WP. Furthermore, functional blockage of CCR7 and CXCR4 abrogated pDC trafficking into the WP. Collectively, these results strongly suggest that pDCs employ both CCR7 and CXCR4 as critical chemokine receptors to migrate into the WP under steady-state conditions.     

Impaired apoptotic cell clearance in the germinal center by Mer-deficient tingible body macrophages leads to enhanced antibody-forming cell and germinal center responses

Germinal centers (GCs) are specialized microenvironments that generate high-affinity Ab-forming cells (AFCs) and memory B cells. Many B cells undergo apoptosis during B cell clonal selection in GCs. Although the factors that regulate the AFC and GC responses are not precisely understood, it is widely believed that dysregulated AFCs and GCs contribute to autoimmunity. The Mer receptor tyrosine kinase (Mer) facilitates macrophage clearance of apoptotic cells. The Tyro-3, Axl, and Mer receptors, including Mer, suppress TLRs and cytokine-mediated inflammatory responses. We report in this study that tingible body macrophages (TBMφs) in GCs express Mer. Compared to C57BL/6 (B6) controls, Mer-deficient (Mer(-/-)) mice had significantly higher AFC, GC, and Th1-skewed IgG2 Ab (especially IgG2c) responses against the T cell-dependent Ag (4-hydroxy-3-nitrophenyl) acetyl-chicken γ globulin. Mer(-/-) mice had a significantly higher percentage of GC B cells on days 9, 14, and 21 postimmunization compared with B6 controls. Significantly increased numbers of apoptotic cells accumulated in Mer(-/-) GCs than in B6 GCs, whereas the number of TBMφs remained similar in both strains. Our data are the first, to our knowledge, to demonstrate a critical role for Mer in GC apoptotic cell clearance by TBMφs and have interesting implications for Mer in the regulation of B cell tolerance operative in the AFC and GC pathways.     

Constitutive association of cell surface CCR5 and CXCR4 in the presence of CD4

Chemokine receptors CCR5 and CXCR4 are the major coreceptors of HIV-1 infection and also play fundamental roles in leukocyte trafficking, metastasis, angiogenesis, and embyogenesis. Here, we show that transfection of CCR5 into CXCR4 and CD4 expressing 3T3 cells enhances the cell surface level of CXCR4. In CCR5 high expressing cells, cell surface level of CXCR4 was incompletely modulated in the presence of the CXCR4 ligand CXCL12/SDF-1alpha. CCR5 was resistant to ligand-dependent modulation with the CCR5 ligand CCL5/RANTES. Confocal laser microscopy revealed that CCR5 was colocalized with CXCR4 on the cell surface. In CD4 expressing CCR5 and CXCR4 double positive NIH 3T3 cells, immunoprecipitation followed by Western blot analysis revealed that CCR5 was associated with CXCR4 and CD4. CXCR4 and CCR5 were not co-immunoprecipitated in cells expressing CCR5 and CXCR4 but without CD4 expression. Compared to NIH 3T3CD4 cells expressing CXCR4, the entry of an HIV-1 X4 isolate (HCF) into NIH 3T3CD4 expressing both CXCR4 and CCR5 was reduced. Our data indicate that chemokine receptors interact with each other, which may modulate chemokine-chemokine receptor interactions and HIV-1 coreceptor functions.     

Induction of a germinal center phenotype in B cells in vitro by a Th2 cell line

We have investigated the contribution of various stimuli for generating in vitro the changes in surface phenotype characteristic of B cells responding to a T-dependent antigen in a germinal center (GC). We show that, unlike many other stimuli such as B cell mitogens, cytokines, and surrogate antigen, alone or in combination, an alloreactive Th2 clonal line induces splenic B cells to become cell surface peanut agglutinin (PNA)(hi), Ig(lo), CD62L(lo), and CD44(hi) to produce mRNA for M17 and to express a GC-specific transgene even without B cell receptor ligation. Neither proliferation nor prior activation of responding B cells is needed, but B cells from CD45-null mice show reduced efficiency of this induction. These findings open up possibilities for separation and dissection of the various components of the GC response.     

Roles of mechanical force and CXCR1/CXCR2 in shear-stress-induced endothelial cell migration

We previously demonstrated that CXCR1 and CXCR2 are novel mechanosensors mediating laminar shear-stress-induced endothelial cell (EC) migration (Zeng et al. in Cytokine 53:42–51, 2011). In the present study, an analytical model was proposed to further analyze the underlying mechanisms, assuming the mechanical force (MF) and mechanosensor-mediated biochemical reactions induce cell migration together. Shear stress can regulate both mechanosensor-mediated migration in the flow direction (Ms–M_FD) and mechanosensor-mediated migration toward a wound (Ms–M_W). Next, the migration distance, the roles of MF-induced cell migration (MF–M), and the mobilization mechanisms of mechanosensors were analyzed. The results demonstrated that MF–M plays an important role in 15.27 dyn/cm² shear-stress-induced EC migration but is far weaker than Ms–M_W at 5.56 dyn/cm². Our findings also indicated that CXCR2 played a primary role, in synergy with CXCR1. The Ms–M_FD was primarily mediated by the synergistic effect of CXCR1 and CXCR2. In Ms–M_W, when shear stress was beyond a certain threshold, the synergistic effect of CXCR1 and CXCR2 was enhanced, and the effect of CXCR1 was inhibited. Therefore, the retarding of EC migration and wound closure capacity under low shear flow was related to the low magnitude of shear stress, which may contribute to atherogenesis and many other vascular diseases.     

Consequences of ChemR23 Heteromerization with the Chemokine Receptors CXCR4 and CCR7

Recent studies have shown that heteromerization of the chemokine receptors CCR2, CCR5 and CXCR4 is associated to negative binding cooperativity. In the present study, we build on these previous results, and investigate the consequences of chemokine receptor heteromerization with ChemR23, the receptor of chemerin, a leukocyte chemoattractant protein structurally unrelated to chemokines. We show, using BRET and HTRF assays, that ChemR23 forms homomers, and provide data suggesting that ChemR23 also forms heteromers with the chemokine receptors CCR7 and CXCR4. As previously described for other chemokine receptor heteromers, negative binding cooperativity was detected between ChemR23 and chemokine receptors, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other. We also showed, using mouse bone marrow-derived dendritic cells prepared from wild-type and ChemR23 knockout mice, that ChemR23-specific ligands cross-inhibited CXCL12 binding on CXCR4 in a ChemR23-dependent manner, supporting the relevance of the ChemR23/CXCR4 interaction in native leukocytes. Finally, and in contrast to the situation encountered for other previously characterized CXCR4 heteromers, we showed that the CXCR4-specific antagonist AMD3100 did not cross-inhibit chemerin binding in cells co-expressing ChemR23 and CXCR4, demonstrating that cross-regulation by AMD3100 depends on the nature of receptor partners with which CXCR4 is co-expressed.     

Microglia Express CCR5, CXCR4, and CCR3, but of These, CCR5 Is the Principal Coreceptor for Human Immunodeficiency Virus Type 1 Dementia Isolates

Microglia are the main human immunodeficiency virus (HIV) reservoir in the central nervous system and most likely play a major role in the development of HIV dementia (HIVD). To characterize human adult microglial chemokine receptors, we analyzed the expression and calcium signaling of CCR5, CCR3, and CXCR4 and their roles in HIV entry. Microglia expressed higher levels of CCR5 than of either CCR3 or CXCR4. Of these three chemokine receptors, only CCR5 and CXCR4 were able to transduce a signal in microglia in response to their respective ligands, MIP-1β and SDF-1α, as recorded by single-cell calcium flux experiments. We also found that CCR5 is the predominant coreceptor used for infection of human adult microglia by the HIV type 1 dementia isolates HIV-1_DS-br, HIV-1_RC-br, and HIV-1_YU-2, since the anti-CCR5 antibody 2D7 was able to dramatically inhibit microglial infection by both wild-type and single-round luciferase pseudotype reporter viruses. Anti-CCR3 (7B11) and anti-CXCR4 (12G5) antibodies had little or no effect on infection. Last, we found that virus pseudotyped with the DS-br and RC-br envelopes can infect cells transfected with CD4 in conjunction with the G-protein-coupled receptors APJ, CCR8, and GPR15, which have been previously implicated in HIV entry.     

Gamma-ray irradiation impairs dendritic cell migration to CCL19 by down-regulation of CCR7 and induction of cell apoptosis

Dendritic cells (DCs) are the most potent antigen-presenting cells and play a crucial role in the regulation of immune response and migration of DCs into secondary lymphoid tissues also play an important role in the initiation of innate and adaptive immunity. Radiation therapy is now a routine treatment for certain types of cancer and over 20 percent of cancer patients will require radiation therapy during the treatment of their disease. However, the influence of ionizing irradiation on the migratory ability of DCs is largely unknown. In this article, we report that γ ray irradiation can significantly inhibit LPS-triggered up regulation of CCR7 expression and PGE2 production by DC, thus impairing DC migration towards CCL19 in vitro and in vivo. Moreover, γ ray exposed DC also displayed an increased apoptosis rate and decreased cell viability. Furthermore, we demonstrate that exogenous PGE2 can partly reduce the gamma-ray induced migratory impairment and restored CCR7 expression of DC. Our work suggests that γ irradiation affects DC function at multiple steps during the immune response including DC migration, and that PGE2, via control of CCR7 expression, is an important regulator of DC migration.     

Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in trans on the CCR2 chemokine receptor

The identification of chemokine receptors as HIV-1 coreceptors has focused research on developing strategies to prevent HIV-1 infection. We generated CCR2-01, a CCR2 receptor-specific monoclonal antibody that neither competes with the chemokine CCL2 for binding nor triggers signaling, but nonetheless blocks replication of monotropic (R5) and T-tropic (X4) HIV-1 strains. This effect is explained by the ability of CCR2-01 to induce oligomerization of CCR2 with the CCR5 or CXCR4 viral coreceptors. HIV-1 infection through CCR5 and CXCR4 receptors can thus be prevented in the absence of steric hindrance or receptor downregulation by acting in trans on a receptor that is rarely used by the virus to infect cells.     

Killing the messenger: The role of CXCR7 in regulating primordial germ cell migration

Primordial Germ Cell (PGC) migration in zebrafish is guided by SDF-1a. Binding of this chemokine to its receptor CXCR4b activates downstream signalling cascades leading to cell polarization and directed migration towards the attractant source. Despite the detailed information available concerning the role of SDF-1 in guiding the PGCs to their targets, little was known regarding the molecular mechanisms controlling the distribution of SDF-1a within the tissue. We have recently shown that the activity of a second SDF-1/CXCL12 receptor, CXCR7 is crucial for proper migration of PGCs. Although CXCR4 and CXCR7 are structurally related and serve as receptors for the same ligand, they appear to serve very different functions during PGC migration. Here we discuss a model according to which CXCR4b translates the polarized distribution of SDF-1 into directed PGC migration, while CXCR7 acts as a high-affinity decoy receptor and facilitates the migration of PGCs by shaping the distribution of the chemokine in the environment.Key words: cell migration, CXCR4, CXCR7, SDF-1, chemokine, chemotaxisChemokine-guided cell migration is central for many processes in normal development and homeostasis (e.g., embryogenesis) as well as in pathological conditions (e.g., inflammation). Zebrafish primordial germ cells (PGCs) serve as a useful model for studying chemokine-controlled cell migration in vivo as the migrating PGCs sense and respond to the dynamic distribution of the chemokine SDF-1a through its receptor CXCR4b.^1,2Recent reports identified CXCR7 as a receptor for SDF-1^3,4 that controls processes such as cell adhesion, survival and tumor progression. A role for this receptor in regulating cell migration during development was demonstrated in the zebrafish lateral line.^5,6 The zebrafish lateral line primordium migrates directionally on a stripe of uniform sdf-1a expression to deposit a set of sensory organs along the fish tail. While the authors raised the hypothesis that antagonistic interactions between CXCR4b and CXCR7 polarize the developing organ to allow its migration, the precise function of CXCR7 in this process remained unclear.To address this question in an in vivo context, we examined the role CXCR7 plays in zebrafish PGC migration.⁷ Our experiments revealed that knockdown of cxcr7 translation using morpholino antisense oligo nucleotides results in impaired polarity and aberrant migration of PGCs. Unlike cxcr4b, cxcr7 is not specifically expressed in the PGCs but is initially uniformly distributed throughout the embryo. Furthermore, in contrast to activity of CXCR4, CXCR7 function was found to be required in tissues surrounding the migrating cells rather than in the PGCs themselves.To examine the function of CXCR7 in somatic cells we determined the subcellular localization of the protein as compared with that of CXCR4b and SDF-1a. Interestingly, while CXCR4b is predominantly localized to the plasma membrane, CXCR7 is found primarily in intracellular structures. The fact that SDF-1α and CXCR7 colocalized in the cell and that SDF-1α was found in vesicles that contained the lysosomal marker LAMP-1 suggested that the prime role of CXCR7 is to bind and internalize SDF-1a thereby controlling the level of the diffusible chemokine in the extracellular space. Indeed, observing PGCs expressing CXCR4b on their membrane we detected strong receptor internalization when CXCR7 function was knocked down. The enhanced internalization, a typical response to high levels of SDF-1a⁸ could be reversed by concomitant removal of SDF-1.These findings provided an explanation for the CXCR7 knock-down phenotype as abnormally high levels of SDF-1a in the environment have been shown before to interfere with cell motility.^1,2 Indeed, PGCs in CXCR7 knocked-down embryos displayed strong inhibition of motility manifested in short migration tracks—a phenotype that could be reversed by simultaneous removal of CXCR7 and SDF-1.The implication of the results presented above is that the sole function of CXCR7 in the context of PGC migration is ligand sequestration. Consistent with this idea, two typical signalling responses acting downstream of chemokine receptors namely, elevation of intracellular calcium levels and PI3K activation^9–13 were not altered in cells knocked down for CXCR7. Thus, consistent with other reports,^4,14 our results imply that CXCR7 signalling is not required for PGC migration.An important outstanding question concerns the molecular basis for the dramatic difference between the activity of CXCR4 and that of CXCR7. Defining domains and amino acids responsible for this difference would provide extensive information regarding chemokine receptor signalling and trafficking within the cell. Whereas random mutagenesis and generation of various CXCR4-CXCR7 chimeric molecules might provide an answer to this question, it is tempting to speculate that known protein motifs are responsible for the differences between the two receptors. For example, an obvious candidate region is that around its DRY motif,¹⁴ a motif within the second intracellular loop that is important for Gprotein coupling and signalling.¹⁵ Whereas uncoupling downstream signalling in the case of CXCR7 is an interesting research avenue, other non-mutually exclusive options should be examined (Fig. 1). For example, CXCR7 could possess domains that facilitate interaction with components that enhance internalization. Such an interaction could remove the receptor from the location where it normally interacts with the signalling machinery, while effectively internalizing SDF-1a.Open in a separate windowFigure 1Proposed model for differential functions of CXCR4b and CXCR7. (A) CXCR4b signalling in PGCs controls cell polarization and directional migration in response to SDF-1a binding (squares), through interaction with G-proteins and elevation of calcium levels. (B) Binding of SDF-1a by CXCR7 does not elicit signalling. Endocytosis of the lignad-bound CXCR7 leads to sequestration and degradation of SDF-1a in the somatic environment.Taken together, we show that proper PGC migration requires a mechanism to remove the guidance cue thereby allowing the formation of an informative chemotactic gradient. It would be very interesting to examine whether the paradigm demonstrated for the PGC migration model applies for other chemokine-guided events in development and disease.     

gp120 induces cell death in human neuroblastoma cells through the CXCR4 and CCR5 chemokine receptors

To infect target cells, the human immunodeficiency virus (HIV) type I (HIV-1) must engage not only the well-known CD4 molecule, but it also requires one of several recently described coreceptors. In particular, the CXCR4 (LESTR/fusin) receptor allows fusion and entry of T-tropic strains of HIV, whereas CCR5 is the major coreceptor used by primary HIV-1 strains that infect macrophages and CD4(+) T-helper cells (M-tropic viruses). In addition, the alpha chemokine SDF1alpha and the beta chemokines MIP1alpha, MIP1beta, and RANTES, natural ligands of CXCR4 and CCR5, respectively, are potent soluble inhibitors of HIV infection by blocking the binding between the viral envelope glycoprotein gp120 and the coreceptors. Approximately two-thirds of individuals with acquired immunodeficiency syndrome (AIDS) show neurologic complications, which are referred to a syndrome called AIDS dementia complex or HIV-1-associated cognitive/motor complex. The HIV-1 coat glycoprotein gp120 has been proposed as the major etiologic agent for neuronal damage, mediating both direct and indirect effects on the CNS. Furthermore, recent findings showing the presence of chemokine receptors on the surface of different cell types resident in the CNS raise the possibility that the association of gp120 with these receptors may contribute to the pathogenesis of neurological dysfunction. Here, we address the possible role of alpha and beta chemokines in inhibiting gp120-mediated neurotoxicity using the human neuroblastoma CHP100 cell line as an experimental model. We have previously shown that, in CHP100 cells, picomolar concentrations of gp120 produce a significant increase in cell death, which seems to proceed through a Ca(2+) - and NMDA receptor-dependent cascade. In this study, we gained insight into the mechanism(s) of neurotoxicity elicited by the viral glycoprotein. We found that CHP100 cells constitutively express both CXCR4 and CCR5 receptors and that stimulation with phorbol 12-myristate 13-acetate down-regulates their expression, thus preventing gp120-induced cell death. Furthermore, all the natural ligands of these receptors exerted protective effects against gp120-mediated neuronal damage, although with different efficiencies. These findings, together with our previous reports, suggest that the neuronal injury observed in HIV-1 infection could be due to direct (or indirect) interactions between the viral protein gp120 and chemokine and/or NMDA receptors.     

Quantitatively reduced participation of anti-nuclear antigen B cells that down-regulate B cell receptor during primary development in the germinal center/memory B cell response to foreign antigen

The peripheral B cell compartment contains high levels of "polyreactivity" including autospecificities. We have described a pathway that certain autoreactive B cells may take in gaining stable access to the foreign Ag-responsive peripheral compartment. This pathway was revealed in mice expressing a targeted Ig H chain transgene encoding BCRs with "multireactivity" for the hapten arsonate and DNA-based autoantigens. B cells expressing such BCRs develop to mature follicular phenotype and locale, and are not short-lived. These B cells express very low levels of BCR, indicating that they are not "ignorant" of self Ag, but do not display features of anergy in in vitro assays. Nonetheless, a variety of states of lymphocyte anergy has been described, and some may only be manifested in vivo. As such, we analyzed the ability of these B cells to participate in a T cell-dependent immune response to arsonate in vivo. These B cells mount an early primary response similar to control B cells, including homing to follicles, migration to the T-B interface, and induction of costimulatory molecules, proliferation, differentiation to AFCs, class switching, and entry into GCs and somatic hypermutation. Nonetheless, these B cells display reduced participation in the latter stages of the GC response and in the anamnestic AFC response. In total, these data suggest that while the autoreactivity of this type of B cell does not result in anergy, the ability of such B cells to participate in a cross-reactive immune response to foreign Ag is compromised.