CCR5 Signaling Research Articles

Induction of IL-13 production and upregulated expression of protease activated receptor-1 by RANTES in a mast cell line

RANTES is a potent chemoattractant for various important inflammatory cells such as eosinophils, memory T cells and mast cells. It has been long recognized as a crucial player in the pathogenesis of allergy. However, little is known of its effects on cytokine secretion and protease activated receptor (PAR) expression in mast cells. In the present study, we examined potential influence of RANTES on IL-13 and IL-12 release from P815 cells and PAR expression on P815 cells by using flow cytometry analysis, quantitative real-time PCR, ELISA and cellular activation of signaling ELISA (CASE) techniques. The results showed that RANTES induced up to 2.2-fold increase in IL-13, but not IL-12 release from P815 cells. Blocking antibodies against RANTES and CCR5 diminished RANTES induced IL-13 release. Furthermore, RANTES upregulated expression of PAR-1, PAR-2 and PAR-3 mRNAs, but enhanced only PAR-1 protein expression. At 1 ng/ml, RANTES can abolish tryptase induced IL-13 release, but enhance trypsin, tryptase and thrombin induced PAR-1, -2 and -4 expression. LY204002 abolished RANTES induced IL-13 release, indicating an Akt cell signaling pathway may be involved in the event. In conclusion, RANTES can stimulate IL-13 release from mast cells through a CCR5 and Akt cell signaling pathway dependent mechanism. It can also enhance trypsin, tryptase and thrombin-induced expression of PARs in mast cells. RANTES may contribute to modulation of IL-13 production and PAR expression in mast cells, through which participates in the mast cell related inflammation.

Na+/H+ Exchanger Regulatory Factor-1 Is Involved in Chemokine Receptor Homodimer CCR5 Internalization and Signal Transduction but Does Not Affect CXCR4 Homodimer or CXCR4-CCR5 Heterodimer

Chemokine receptors are members of the G protein-coupled receptor (GPCR) family. CCR5 is also the principal co-receptor for macrophage-tropic strains of human immunodeficiency virus, type 1 (HIV-1), and efforts have been made to develop ligands to inhibit HIV-1 infection by promoting CCR5 receptor endocytosis. Given the nature of GPCRs and their propensity to form oligomers, one can consider ligand-based therapies as unselective in terms of the oligomeric composition of complexes. For example, a ligand targeting a CCR5 homomer could likely induce signal transduction on a heteromeric CCR5-CXCR4. Other avenues could therefore be explored. We identified a receptor adaptor interacting specifically with one receptor complex but not others. NHERF1, an adaptor known for its role in desensitization, internalization, and regulation of the ERK signaling cascade for several GPCRs, interacts via its PDZ2 domain with the CCR5 homodimer but not with the CXCR4-CCR5 heterodimer or CXCR4 homodimer. To further characterize this interaction, we also show that NHERF1 increases the CCR5 recruitment of arrestin2 following stimulation. NHERF1 is also involved in CCR5 internalization, as we demonstrate that co-expression of constructs bearing the PDZ2 domain can block CCR5 internalization. We also show that NHERF1 potentiates RANTES (regulated on activation normal T cell expressed and secreted)-induced ERK1/2 phosphorylation via CCR5 activation and that this activation requires NHERF1 but not arrestin2. Taken together, our results suggest that oligomeric receptor complexes can associate specifically with partners and that in this case NHERF1 could represent an interesting new target for the regulation of CCR5 internalization and potentially HIV infection.

Restoration of dysregulated CC chemokine signaling for monocyte/macrophage chemotaxis in head and neck squamous cell carcinoma patients by neem leaf glycoprotein maximizes tumor cell cytotoxicity

Previous studies have shown that the CC chemokine receptor CCR5 is downregulated on monocyte/macrophage (MO/Mphi) surfaces in head and neck squamous cell carcinoma (HNSCC) patients (stage IIIB). Ligands (RANTES, MIP-1alpha and MIP-1beta) of this chemokine receptor were also secreted in lesser quantity from MO/Mphi of HNSCC patients in comparison with healthy individuals. In an aim to restore this dysregulated receptor-ligand signaling, we have used neem leaf glycoprotein (NLGP), a novel immunomodulator reported from our laboratory. NLGP upregulated CCR5 expression, as evidenced from studies on MO/Mphi of peripheral blood from HNSCC patients as well as healthy individuals. Expression of RANTES, MIP-1alpha and MIP-1beta was also upregulated following NLGP treatment of these cells in vitro. Interestingly, NLGP has little effect on the expression of CCR5 and the ligand RANTES in oral cancer cells. This restored CCR5 receptor-ligand signaling seen in MO/Mphi was reflected in improved CCR5-dependent, p38 mitogen-activated protein kinase (MAPK)-mediated migration of MO/Mphi after NLGP treatment to a standard chemoattractant. NLGP also induces better antigen presentation and simultaneous costimulation to effector T cells by MO/Mphi by upregulating human leucocyte antigen (HLA)-ABC, CD80 and CD86. In addition, NLGP-treated MO/Mphi-primed T cells can effectively lyse tumor cells in vitro. The effects of NLGP on monocyte migration and T cell-mediated oral tumor cell killing were further demonstrated in transwell assays with or without CCR5 neutralization. These results suggest a new approach in cancer immunotherapy by modulating dysregulated CCR5 signals from MO/Mphi.

Fat-Storing Multilocular Cells Expressing CCR5 Increase in the Thymus with Advancing Age: Potential Role for CCR5 Ligands on the Differentiation and Migration of Preadipocytes

Age-associated thymic involution is characterized by decreased thymopoiesis, adipocyte deposition and changes in the expression of various thymic microenvironmental factors. In this work, we characterized the distribution of fat-storing cells within the aging thymus. We found an increase of unilocular adipocytes, ERTR7(+) and CCR5(+ )fat-storing multilocular cells in the thymic septa and parenchymal regions, thus suggesting that mesenchymal cells could be immigrating and differentiating in the aging thymus. We verified that the expression of CCR5 and its ligands, CCL3, CCL4 and CCL5, were increased in the thymus with age. Hypothesizing that the increased expression of chemokines and the CCR5 receptor may play a role in adipocyte recruitment and/or differentiation within the aging thymus, we examined the potential role for CCR5 signaling on adipocyte physiology using 3T3-L1 pre-adipocyte cell line. Increased expression of the adipocyte differentiation markers, PPARgamma2 and aP2 in 3T3-L1 cells was observed under treatment with CCR5 ligands. Moreover, 3T3-L1 cells demonstrated an ability to migrate in vitro in response to CCR5 ligands. We believe that the increased presence of fat-storing cells expressing CCR5 within the aging thymus strongly suggests that these cells may be an active component of the thymic stromal cell compartment in the physiology of thymic aging. Moreover, we found that adipocyte differentiation appear to be influenced by the proinflammatory chemokines, CCL3, CCL4 and CCL5.

Global Gene Expression Signatures in CD34+ Hematopoietic Cells Following Exposure to 16,16-Dimethylprostaglandin E2.

Abstract 2523Poster Board II-500Prostaglandin E2 (PGE2), a member of the eicosanoid family, is a local signaling molecule released from mammalian blood vessel walls and other hematopoietic niches. PGE2 produces tissue specific effects by interacting with G protein receptors in cell membranes. It has been found to be an important regulator of hematopoietic stem cell (HSC) number during embryogenesis and is required for the development of HSCs (North et al., Nature 447:1007–1011, 2007). Additionally, PGE2 enhances HSC engraftment in competitive transplantation assays (North et al., Nature 447:1007–1011, 2007) through alterations in the survival, proliferation and homing of transplanted cells (Hoggatt et al., Blood 113:5444–5455, 2009). Immunoselected CD34+ cells were isolated from the leukapheresis products of both human and non-human primates following cytokine mobilization with G-CSF. CD34+ cells were incubated in X-Vivo 10™ with either 50μM or no PGE2 for one hour at 37°C on RetroNectin™ coated plates. After one hour incubation, media was replaced with X-Vivo 10™ supplemented with 10ng/mL SCF and IL-6 and cells were kept at 37°C and collected either prior to or at 2, 6, 12 and 24 hours following PGE2 exposure. Total RNA was isolated from cells and amplified to cRNA. Control cRNA was labeled with Cy3 dye and experimental cRNA was labeled with Cy5 dye. Control and experimental labeled cRNA was co-hybridized to a custom-made 17.5K cDNA (UniGene cluster) microarray. The relationship between the cells was analyzed using an unsupervised Eisen's hierarchical clustering method and gene regulation was analyzed at each time point using an Array scatter plot comparison. Statistical analysis was done using Array Class Comparison analysis and pathway analysis was carried out using Ingenuity Pathway Analysis. Many of the significantly up regulated genes were associated with pathways activated by PGE2, including genes involved in the inflammatory response, signal transduction, and G proteins. Interestingly the human CD34+ cells showed at 2 hours a 21.8-fold increase of 3′,5′-cyclic AMP phosphodiesterase mRNA consistent with prior observations in zebrafish and murine cells (Goessling et al. Cell 136: 1136–1148). Pathways that increased at 2 hours include molecular mechanisms of cancer, and those that were down regulated include CCR5, CTLA4, and leukocyte extravasation signaling. At 6 hours the molecular mechanisms of cancer pathway predominated even more (diminishing by 12 hours) while oxidative phosphorylation was down regulated. Genes involved in cAMP and WNT signaling are observed to be up regulated at earlier time points but diminish with time. These results correlate with protein expression observations that have been made in the zebrafish and murine models following PGE2 treatment (Goessling et al. Cell 136: 1136–1148) and are consistent with the observation that PGE2 may influence the self renewal of HSCs. Disclosures:Goessling:Fate Therapeutics: Consultancy, Patents & Royalties.

Abstract 5055: CC Chemokine Receptor 5 Signaling Suppresses Inflammation and Attenuates Adverse Remodeling Following Myocardial Infarction by Recruiting Regulatory T Cells

Myocardial infarction triggers an inflammatory reaction that is critically involved in the pathogenesis of cardiac remodeling. Optimal cardiac repair is dependent on regulatory mechanisms that suppress inflammation preventing excessive matrix degradation. Chemokines are rapidly induced in the healing infarct mediating recruitment of leukocyte subsets with distinct properties. CCR5, the receptor for the CC chemokines MIP-1 α , MIP-1 β and RANTES plays an important role in immunoinflammatory responses. We hypothesized that CCR5 signaling may regulate the inflammatory response following myocardial infarction playing a role in the development of cardiac remodeling. CCR5 and its ligands MIP-1 α and MIP-1 β were markedly induced in reperfused mouse infarcts. Flow cytometry demonstrated that 38.3%+3.75 of the mononuclear cells infiltrating the infarcted myocardium after 24h of reperfusion expressed CCR5. CCR5 −/− mice and WT animals showed comparable macrophage and neutrophil infiltration in the infarct; however, CCR5 absence was associated with marked upregulation of pro-inflammatory cytokine (IL-1 β , TNF- α and IL-6) and chemokine (MIP-2, MIP-1 α , - β and IP-10) mRNA expression in the infarcted myocardium. CCR5+ mononuclear cells isolated from WT infarcts had anti-inflammatory properties exhibiting higher expression of the inhibitory cytokine IL-10 than CCR5- cells. In addition, mononuclear cells isolated from CCR5 −/− infarcts had reduced IL-10 expression. Enhanced inflammation in the absence of CCR5 was associated with impaired recruitment of foxp3+ regulatory T cells (Tregs), a CD4+ lymphocyte subpopulation with suppressive properties (Tregs as a percentage of T cells: WT 13.1%+ 1.1 vs. KO 7.2%+ 1.1; p<0.05). In WT mice the CCR5+ subset of Tregs exhibited enhanced expression of IL-10 reflecting potent anti-inflammatory activity. Enhanced inflammation in CCR5 −/− infarcts was associated with increased MMP and decreased TIMP expression, resulting in accentuated cardiac dilation (LVEDV: WT 52.99+2.43 mm3 vs. KO 65.13+4.53 mm3; p=0.02) without affecting the size of the infarct. CCR5-mediated recruitment of Tregs may restrain post-infarction inflammation preventing excessive matrix degradation and attenuating adverse remodeling.

FROUNT Is a Common Regulator of CCR2 and CCR5 Signaling to Control Directional Migration

FROUNT is a known CCR2-binding protein that facilitates monocyte/macrophage infiltration. Here we report that FROUNT also binds to the C-terminal region of CCR5 and enhances CCR5-mediated cellular chemotaxis. We show that FROUNT overexpression enhances the directionality of chemotaxis, while FROUNT suppression results in impaired responsiveness. Furthermore, we found an increase in consolidated pseudopodium formation in FROUNT-overexpressing cells (FNT cells) on uniform stimulation with CCL4 (MIP1-beta), a specific ligand of CCR5. In most FNT cells, one to two pseudopodia directed toward higher chemokine concentration were found, whereas most FNT-suppressed cells had multiple pseudopodia. The data indicate that FROUNT is involved in sensing and amplifying a shallow extracellular chemokine gradient that leads to a limited number of accurate pseudopodia directed toward the chemokine concentration. In addition to its separate roles in CCR2- and CCR5-mediated chemotaxis, FROUNT, as a common regulator of these receptors, possibly plays a crucial role in the recruitment of immune cells expressing these receptors.

CCR5 signalling, but not DARC or D6 regulatory, chemokine receptors are targeted by herpesvirus U83A chemokine which delays receptor internalisation via diversion to a caveolin-linked pathway

BackgroundHerpesviruses have evolved chemokines and chemokine receptors, which modulate the recruitment of human leukocytes during the inflammatory response to infection. Early post-infection, human herpesvirus 6A (HHV-6A) infected cells express the chemokine receptor U51A and chemokine U83A which have complementary effects in subverting the CC-chemokine family thereby controlling anti-viral leukocyte recruitment. Here we show that, to potentiate this activity, the viral chemokine can also avoid clearance by scavenger chemokine receptors, DARC and D6, which normally regulate an inflammatory response. Conversely, U83A delays internalisation of its signalling target receptor CCR5 with diversion to caveolin rich membrane domains. This mechanism can redirect displaced human chemokines to DARC and D6 for clearance of the anti-viral inflammatory response, leaving the viral chemokine unchecked.MethodsCell models for competitive binding assays were established using radiolabeled human chemokines and cold U83A on CCR5, DARC or D6 expressing cells. Flow cytometry was used to assess specific chemotaxis of CCR5 bearing cells to U83A, and internalisation of CCR5 specific chemokine CCL4 after stimulation with U83A. Internalisation analyses were supported by confocal microscopy of internalisation and co-localisation of CCR5 with caveosome marker caveolin-1, after virus or human chemokine stimulation.ResultsU83A displaced efficiently human chemokines from CCR5, with a high affinity of 0.01nM, but not from DARC or D6. Signalling via CCR5 resulted in specific chemoattraction of primary human leukocytes bearing CCR5. However, U83A effective binding and signalling to CCR5 resulted in delayed internalisation and recycling up to 2 hours in the absence of continual re-stimulation. This resulted in diversion to a delayed caveolin-linked pathway rather than the rapid clathrin mediated endocytosis previously shown with human chemokines CCL3 or CCL4.ConclusionU83A diverts human chemokines from signalling, but not regulatory or scavenger, receptors facilitating their clearance, while occupying signalling receptors at the cell surface. This can enhance virus specific inflammation, facilitating dissemination to replication sensitive leukocytes while evading clearance; this has implications for linked neuro-inflammatory pathologies.

Distinct modes of molecular regulation of CCL3 induced calcium flux in monocytic cells

The chemokine receptor CCR5 has been shown to be targeted to cholesterol- and sphingolipid-rich membrane microdomains. Here we elucidate the effects of membrane fluidity on CCR5 signalling and expression using the monocytic THP-1 cells. MCD treatment of THP-1 cells, which removes nearly all cholesterol from the plasma membrane, leads to an increase in the signalling properties of CCR5. In contrast, the prevention of cholesterol production with lovastatin and simvastatin decreases the release of intracellular calcium and also decreases receptor cell surface expression. The loss of response in lovastatin treated cells can be rescued by MCD addition, which shows that the cholesterol content in the membrane is only one factor in determining the amount of receptor response. We show that CCR5 signalling is dependent on thapsigargin-sensitive Ca 2+ stores and on activation of ryanodine receptors as well as InsP3 receptors or store-operated channels. Cholesterol depletion with MCD changes the thapsigargin sensitivity in THP-1 cells and also alters receptor-G-protein coupling towards pertussis toxin (PTX) independent G-proteins. Cholesterol removal by MCD in THP-1 cells has far reaching consequences for receptor activation and signalling and emphasises the need for a clearer understanding of how membrane fluidity affects receptor signalling events.

CCR5 signaling suppresses inflammation and attenuates adverse remodeling in healing myocardial infarcts.

Chemokine‐mediated interactions may regulate chemotactic recruitment of leukocyte subsets and modulate cellular responses in healing infarcts. We hypothesized that CCR5, a receptor for the CC chemokines MIP‐1α, MIP‐1β and RANTES may play a crucial role in the post‐infarction inflammatory response. Morphometric, molecular and echocardiographic endpoints were compared in WT and CCR5 ‐/‐ mice undergoing reperfused infarction protocols. MIP‐1α, MIP‐1β and CCR5 were markedly induced in WT infarcts; however, RANTES mRNA was not upregulated. Neutrophil and macrophage density was comparable in CCR5 ‐/‐ and WT infarcts. However, in the absence of CCR5, expression of the pro‐inflammatory cytokines TNF‐α, IL‐1β and IL‐6 and the chemokines MIP‐1α and MIP‐1β was significantly increased after 6‐24h of reperfusion. CCR5 ‐/‐ hearts exhibited enhanced ventricular dilation following infarction associated with increased levels of MMP‐8 and MMP‐9 and reduced TIMP‐1 expression. The surprising accentuation of the inflammatory response in CCR5 ‐/‐ infarcts was associated with reduced foxp3 mRNA levels suggesting impaired recruitment of regulatory T cells (Tregs). The anti‐inflammatory effects of CCR5 in healing infarcts may be mediated through selective recruitment of leukocyte subsets with suppressive properties (R01 HL‐76246 and HL‐85440).

Highly potent, fully recombinant anti-HIV chemokines: Reengineering a low-cost microbicide

New prevention strategies for use in developing countries are urgently needed to curb the worldwide HIV/AIDS epidemic. The N-terminally modified chemokine PSC-RANTES is a highly potent entry inhibitor against R5-tropic HIV-1 strains, with an inhibitory mechanism involving long-term intracellular sequestration of the HIV coreceptor, CCR5. PSC-RANTES is fully protective when applied topically in a macaque model of vaginal HIV transmission, but it has 2 potential disadvantages related to further development: the requirement for chemical synthesis adds to production costs, and its strong CCR5 agonist activity might induce local inflammation. It would thus be preferable to find a recombinant analogue that retained the high potency of PSC-RANTES but lacked its agonist activity. Using a strategy based on phage display, we set out to discover PSC-RANTES analogs that contain only natural amino acids. We sought molecules that retain the potency and inhibitory mechanism of PSC-RANTES, while trying to reduce CCR5 signaling to as low a level as possible. We identified 3 analogues, all of which exhibit in vitro potency against HIV-1 comparable to that of PSC-RANTES. The first, 6P4-RANTES, resembles PSC-RANTES in that it is a strong agonist that induces prolonged intracellular sequestration of CCR5. The second, 5P12-RANTES, has no detectable G protein-linked signaling activity and does not bring about receptor sequestration. The third, 5P14-RANTES, induces significant levels of CCR5 internalization without detectable G protein-linked signaling activity. These 3 molecules represent promising candidates for further development as topical HIV prevention strategies.

CCR5 internalisation and signalling have different dependence on membrane lipid raft integrity

The chemokine receptor, CCR5, acts as a co-receptor for human immunodeficiency virus entry into cells. CCR5 has been shown to be targeted to cholesterol- and sphingolipid-rich membrane microdomains termed lipid rafts or caveolae. Cholesterol is essential for CCL4 binding to CCR5 and for keeping the conformational integrity of the receptor. Filipin treatment leads to loss of caveolin-1 from the membrane and therefore to a collapse of the caveolae. We have found here that sequestration of membrane cholesterol with filipin did not affect receptor signalling, however a loss of ligand-induced internalisation of CCR5 was observed. Cholesterol extraction with methyl-β-cyclodextrin (MCD) reduced signalling through CCR5 as measured by release of intracellular Ca 2+ and completely abolished the inhibition of forskolin-stimulated cAMP accumulation with no effect on internalisation. Pertussis toxin (PTX) treatment inhibited the intracellular release of calcium that is transduced via Gαi G-proteins. Depletion of cholesterol destroyed microdomains in the membrane and switched CCR5/G-protein coupling to a PTX-independent G-protein. We conclude that cholesterol in the membrane is essential for CCR5 signalling via the Gαi G-protein subunit, and that integrity of lipid rafts is not essential for effective CCR5 internalisation however it is crucial for proper CCR5 signal transduction via Gαi G-proteins.

The Duffy Antigen/Receptor for Chemokines Exists in an Oligomeric Form in Living Cells and Functionally Antagonizes CCR5 Signaling through Hetero-Oligomerization

The Duffy antigen/receptor for chemokines (DARC) is an unusual chemokine receptor that binds a large number of inflammatory chemokines of both the CC and CXC families with nanomolar affinity, yet it lacks the ability to signal upon ligand binding. Using bioluminescent resonant energy transfer, we have demonstrated for the first time that DARC exists as a constitutive homo-oligomer in living cells and furthermore that DARC hetero-oligomerizes with the CC chemokine receptor CCR5. DARC-CCR5 interaction impairs chemotaxis and calcium flux through CCR5, whereas internalization of CCR5 in response to ligand binding remains unchanged. These results suggest a novel mechanism by which DARC could modulate inflammatory responses to chemokines in vivo.

Controls for Lung Dendritic Cell Maturation and Migration during Respiratory Viral Infection

Dendritic cells are ideally suited to orchestrate the innate and adaptive immune responses to infection, but we know little about how these cells respond to infection with common respiratory viruses. Paramyxoviral infections are the most frequent cause of serious respiratory illness in childhood and are associated with an increased risk of asthma. We therefore used a high-fidelity mouse model of paramyxoviral respiratory infection triggered by Sendai virus to examine the response of conventional and plasmacytoid dendritic cells (cDCs and pDCs, respectively) in the lung. We found that pDCs are scarce at baseline but become the predominant population of lung dendritic cells during infection. This recruitment allows for a source of IFN-alpha locally at the site of infection. In contrast, cDCs rapidly differentiate into myeloid cDCs and begin to migrate from the lung to draining lymph nodes within 2 h after viral inoculation. These events cause the number of lung cDCs to decrease rapidly and remain decreased at the site of viral infection. Maturation and migration of lung cDCs depends on Ccl5 and Ccr5 signals because these events are significantly impaired in Ccl5(-/-) and Ccr5(-/-) mice. cDCs failure to migrate to draining lymph nodes in Ccl5(-/-) or Ccr5(-/-) mice is associated with impaired up-regulation of CCR7 that would normally direct this process. Our results indicate that pDCs and cDCs respond distinctly to respiratory paramyxoviral infection with patterns of movement that should serve to coordinate the innate and adaptive immune responses, respectively.

CCR5 signaling through phospholipase D involves p44/42 MAP‐kinases and promotes HIV‐1 LTR‐directed gene expression

The chemokine receptor CCR5 plays an important role as an entry gate for the human immunodeficiency virus-1 (HIV-1) and for viral postentry events. Among signal transducers used by chemoattractant receptors, the phosphatidylcholine-specific phospholipase D (PLD) produces large amounts of second messengers in most cell types. However, the relevance of PLD isoforms to CCR5 signaling and HIV-1 infection process remains unexplored. We show here that CCR5 activation by MIP-1beta in HeLa-MAGI cells triggered a rapid and substantial PLD activity, as assessed by mass choline production. This activity required the activation of ERK1/2-MAP kinases and involved both PLD1 and PLD2. MIP-1beta also promoted the activation of an HIV-1 long terminal repeat (LTR) by the transactivator Tat in HeLa P4.2 cells through a process involving ERK1/2. Expression of wild-type and catalytically inactive PLDs dramatically boosted and inhibited the LTR activation, respectively, without altering Tat expression. Wild-type and inactive PLDs also respectively potentiated and inhibited HIV-1(BAL) replication in MAGI cells. Finally, in monocytic THP-1 cells, antisense oligonucleotides to both PLDs dramatically inhibited the HIV-1 replication. Thus, PLD is activated downstream of ERK1/2 upon CCR5 activation and plays a major role in promoting HIV-1 LTR transactivation and virus replication, which may open novel perspectives to anti-HIV-1 strategies.

Study of the role of CCR5 in a mouse model of intranasal challenge with Yersinia pestis

CCR5 is a chemokine receptor used by HIV-1 to enter cells and has recently been found to act as a pathogen associated molecule pattern receptor. Current positive selection for the high frequency of a CCR5-Δ32 allele in humans has been attributed to resistance to HIV, smallpox, and plague infections. Using an intranasal mouse model of Y. pestis infection, we have found that lack of CCR5 does not enhance host resistance to Y. pestis infection and that CCR5-mediated responses might have a protective role. CCR5−/− mice exhibited higher levels of circulating RANTES and MIP-1α than those exhibited by wild-type mice at the baseline and throughout the course of Y. pestis infection. High levels of RANTES and MIP-1α, which are CCR5 ligands that mediate Natural Killer cell migration, may reflect compensation for the absence of CCR5 signaling.

Neuronal Apoptotic Signaling Pathways Probed and Intervened by Synthetically and Modularly Modified (SMM) Chemokines

As the main coreceptors for human immunodeficiency virus type 1 (HIV-1) entry, CXCR4 and CCR5 play important roles in HIV-associated dementia (HAD). HIV-1 glycoprotein gp120 contributes to HAD by causing neuronal damage and death, either directly by triggering apoptotic pathways or indirectly by stimulating glial cells to release neurotoxins. Here, to understand the mechanism of CXCR4 or CCR5 signaling in neuronal apoptosis associated with HAD, we have applied synthetically and modularly modified (SMM)-chemokine analogs derived from natural stromal cell-derived factor-1alpha or viral macrophage inflammatory protein-II as chemical probes of the mechanism(s) whereby these SMM-chemokines prevent or promote neuronal apoptosis. We show that inherently neurotoxic natural ligands of CXCR4, such as stromal cell-derived factor-1alpha or viral macrophage inflammatory protein-II, can be modified to protect neurons from apoptosis induced by CXCR4-preferring gp120(IIIB), and that the inhibition of CCR5 by antagonist SMM-chemokines, unlike neuroprotective CCR5 natural ligands, leads to neurotoxicity by activating a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Furthermore, we discover distinct signaling pathways activated by different chemokine ligands that are either natural agonists or synthetic antagonists, thus demonstrating a chemical biology strategy of using chemically engineered inhibitors of chemokine receptors to study the signaling mechanism of neuronal apoptosis and survival.

Persistent resistance to HIV-1 infection in CD4 T cells from exposed uninfected Vietnamese individuals is mediated by entry and post-entry blocks

BackgroundWe have previously reported that CD4 T cells from some exposed uninfected (EU) Vietnamese intravenous drug users are relatively resistant to HIV infection in vitro. Here, we further characterized the restriction of viral replication in CD4 T cells from five EUs and assessed its persistence in serial samples.ResultsCD4 T cells and/or PBMC sampled during a period of between 2 and 6 years were challenged with replication-competent HIV-1 and other retroviral particles pseudotyped with envelope proteins of various tropisms. CCR5 expression and function in resistant CD4 T cells was evaluated. The step at which HIV-1 replication is restricted was investigated by real-time PCR quantification of HIV-1 reverse transcripts.We identified three patterns of durable HIV-1 restriction in EU CD4 T cells. CD4 T cells from four of the five EU subjects were resistant to HIV-1 R5 infection. In two cases this resistance was associated with low CCR5 surface expression, which was itself associated with heterozygous CCR5 mutations. In the other two cases, CD4 T cells were resistant to HIV-1 R5 infection despite normal CCR5 expression and signaling function, and normal β-chemokine secretion upon CD4 T cell activation. Instead, restriction appeared to be due to enhanced CD4 T cell sensitivity to β-chemokines in these two subjects. In the fifth EU subject the restriction involved post-entry steps of viral replication and affected not only HIV-1 but also other lentiviruses. The restriction was not overcome by a high viral inoculum, suggesting that it was not mediated by a saturable inhibitory factor.ConclusionVarious constitutive mechanisms of CD4 T cell resistance to HIV-1 infection, affecting entry or post-entry steps of viral replication, are associated with resistance to HIV-1 in subjects who remain uninfected despite long-term high-risk behavior.

The Src kinase Lyn is required for CCR5 signaling in response to MIP-1β and R5 HIV-1 gp120 in human macrophages

AbstractCCR5 is a receptor for several β chemokines and the entry coreceptor used by macrophage-tropic (R5) strains of HIV-1. In addition to supporting viral entry, CCR5 ligation by the HIV-1 envelope glycoprotein 120 (gp120) can activate intracellular signals in macrophages and trigger inflammatory mediator release. Using a combination of in vitro kinase assay, Western blotting for phospho-specific proteins, pharmacologic inhibition, CCR5 knockout (CCR5Δ32) cells, and kinase-specific blocking peptide, we show for the first time that signaling through CCR5 in primary human macrophages is linked to the Src kinase Lyn. Stimulation of human monocyte-derived macrophages with either HIV-1 gp120 or MIP-1β results in the CCR5-mediated activation of Lyn and the concomitant Lyn-dependent activation of the mitogen-activated protein (MAP) kinase ERK-1/2. Furthermore, activation of the CCR5/Lyn/ERK-1/2 pathway is responsible for gp120-triggered production of TNF-α by macrophages, which is believed to contribute to HIV-1 pathogenesis. Thus, Lyn kinase may play an important role both in normal CCR5 function in macrophages and in AIDS pathogenesis in syndromes such as AIDS dementia where HIV-1 gp120 contributes to inappropriate macrophage activation, mediator production, and secondary injury.

Diverse signalling by different chemokines through the chemokine receptor CCR5

We have investigated the signalling properties of the chemokine receptor, CCR5, using several assays for agonism: stimulation of changes in intracellular Ca 2+ or CCR5 internalisation in CHO cells expressing CCR5 or stimulation of [ 35S]GTPγS binding in membranes of CHO cells expressing CCR5. Four isoforms of the chemokine CCL3 with different amino termini (CCL3, CCL3(2–70), CCL3(5–70), CCL3L1) were tested in these assays in order to probe structure/activity relationships. Each isoform exhibited agonism. The pattern of agonism (potency, maximal effect) was different in the three assays, although the rank order was the same with CCL3L1 being the most potent and efficacious. The data show that the amino terminus of the chemokine is important for signalling. A proline at position 2 (CCL3L1) provides for high potency and efficacy but the isoform with a serine at position 2 (CCL3(2–70)) is as efficacious in some assays showing that the proline is not the only determinant of high efficacy. We also increased the sensitivity of CCR5 signalling by treating cells with sodium butyrate, thus increasing the receptor/G protein ratio. This allowed the detection of a change in intracellular Ca 2+ after treatment with CCL7 and Met-RANTES showing that these ligands possess measurable but low efficacy. This study therefore shows that sodium butyrate treatment increases the sensitivity of signalling assays and enables the detection of efficacy in ligands previously considered as antagonists. The use of different assay systems, therefore, provides different estimates of efficacy for some ligands at this receptor.