The chemokine receptors ACKR2 and CCR2 reciprocally regulate lymphatic vessel density

Macrophages regulate lymphatic vasculature development; however, the molecular mechanisms regulating their recruitment to developing, and adult, lymphatic vascular sites are not known. Here, we report that resting mice deficient for the inflammatory chemokine-scavenging receptor, ACKR2, display increased lymphatic vessel density in a range of tissues under resting and regenerating conditions. This appears not to alter dendritic cell migration to draining lymph nodes but is associated with enhanced fluid drainage from peripheral tissues and thus with a hypotensive phenotype. Examination of embryonic skin revealed that this lymphatic vessel density phenotype is developmentally established. Further studies indicated that macrophages and the inflammatory CC-chemokine CCL2, which is scavenged by ACKR2, are associated with this phenotype. Accordingly, mice deficient for the CCL2 signalling receptor, CCR2, displayed a reciprocal phenotype of reduced lymphatic vessel density. Further examination revealed that proximity of pro-lymphangiogenic macrophages to developing lymphatic vessel surfaces is increased in ACKR2-deficient mice and reduced in CCR2-deficient mice. Therefore, these receptors regulate vessel density by reciprocally modulating pro-lymphangiogenic macrophage recruitment, and proximity, to developing, resting and regenerating lymphatic vessels.

Crystal structure of chemically synthesized [N33A] stromal cell-derived factor 1α, a potent ligand for the HIV-1 “fusin” coreceptor

Stromal cell-derived factor-1α (SDF-1α ) is a member of the chemokine superfamily and functions as a growth factor and chemoattractant through activation of CXCR4/LESTR/Fusin, a G protein-coupled receptor. This receptor also functions as a coreceptor for T-tropic syncytium-inducing strains of HIV-1. SDF-1α antagonizes infectivity of these strains by competing with gp120 for binding to the receptor. The crystal structure of a variant SDF-1α ([N33A]SDF-1α ) prepared by total chemical synthesis has been refined to 2.2-Å resolution. Although SDF-1α adopts a typical chemokine β-β-β-α topology, the packing of the α-helix against the β-sheet is strikingly different. Comparison of SDF-1α with other chemokine structures confirms the hypothesis that SDF-1α may be either an ancestral protein from which all other chemokines evolved or the chemokine that is the least divergent from a primordial chemokine. The structure of SDF-1α reveals a positively charged surface ideal for binding to the negatively charged extracellular loops of the CXCR4 HIV-1 coreceptor. This ionic complementarity is likely to promote the interaction of the mobile N-terminal segment of SDF-1α with interhelical sites of the receptor, resulting in a biological response.

CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood–brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell–cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood–brain barrier disruption and viral entry into the central nervous system.

A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5

HIV-1 entry into CD4+ cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.

Molecular mechanisms underlying differential odor responses of a mouse olfactory receptor

The prevailing paradigm for G protein-coupled receptors is that each receptor is narrowly tuned to its ligand and closely related agonists. An outstanding problem is whether this paradigm applies to olfactory receptor (ORs), which is the largest gene family in the genome, in which each of 1,000 different G protein-coupled receptors is believed to interact with a range of different odor molecules from the many thousands that comprise “odor space.” Insights into how these interactions occur are essential for understanding the sense of smell. Key questions are: (i) Is there a binding pocket? (ii) Which amino acid residues in the binding pocket contribute to peak affinities? (iii) How do affinities change with changes in agonist structure? To approach these questions, we have combined single-cell PCR results [Malnic, B., Hirono, J., Sato, T. & Buck, L. B. (1999) Cell 96, 713–723] and well-established molecular dynamics methods to model the structure of a specific OR (OR S25) and its interactions with 24 odor compounds. This receptor structure not only points to a likely odor-binding site but also independently predicts the two compounds that experimentally best activate OR S25. The results provide a mechanistic model for olfactory transduction at the molecular level and show how the basic G protein-coupled receptor template is adapted for encoding the enormous odor space. This combined approach can significantly enhance the identification of ligands for the many members of the OR family and also may shed light on other protein families that exhibit broad specificities, such as chemokine receptors and P450 oxidases.

Broad spectrum chemokine antagonistic activity of a human poxvirus chemokine homolog

A secreted CC chemokine homolog, encoded by the MC148 gene of molluscum contagiosum virus, potently interfered with the chemotaxis of human monocytes, lymphocytes, and neutrophils in response to a large number of CC and CXC chemokines with diverse receptor specificities. Evidence that the viral protein binds to human chemokine receptors was obtained by competition binding and calcium mobilization experiments. The broad spectrum chemokine antagonistic activity of MC148 can explain the prolonged absence of an inflammatory response in skin tumors that harbor replicating molluscum contagiosum virus.

Étude de la migration des populations de lymphocytes B du sang de patients infectés par le virus d’immunodéficience humaine (VIH)

La dérégulation du compartiment de cellules B est une conséquence importante de l’infection par le virus de l’immunodéficience humaine (VIH-1). On observe notamment une diminution des nombres de lymphocytes B sanguins ainsi qu’une variation des fréquences relatives des différentes populations de lymphocytes B chez les individus infectés par rapport aux contrôles sains. Notre laboratoire a précédemment démontré l’implication des cellules dendritiques dans la dérégulation des lymphocytes B via la roduction excessive de BLyS/BAFF, un stimulateur des cellules B. De plus, lors l’études menées chez la souris transgénique présentant une maladie semblable au SIDA, et chez la souris BLyS/BAFF transgénique, l’infection au VIH-1 fut associée à une expansion de la zone marginale (MZ) de la rate. De façon intéressante, nous observons chez les contrôleurs élites une diminution de la population B ‘mature’ de la MZ. Il s’agit du seul changement important chez les contrôleurs élites et reflète possiblement un recrutement de ces cellules vers la périphérie ainsi qu’une implication dans des mécanismes de contrôle de l’infection. Pour tenter d’expliquer et de mieux comprendre ces variations dans les fréquences des populations B, nous avons analysé les axes chimiotactiques CXCL13-CXCR5, CXCL12-CXCR4/CXCR7, CCL20-CCR6 et CCL25-CCR9. L’étude longitudinale de cohortes de patients avec différents types de progression clinique ou de contrôle de l’infection démontre une modulation des niveaux plasmatiques de la majorité des chimiokines analysées chez les progresseurs rapides et classiques. Au contraire, les contrôleurs élites conservent des niveaux normaux de chimiokines, démontrant leur capacité à maintenir l’homéostasie. La migration des populations de cellules B semble être modulée selon la progression ou le contrôle de l’infection. Les contrôleurs élites présentent une diminution de la population B ‘mature’ de la MZ et une augmentation de la fréquence d’expression du récepteur CXCR7 associé à la MZ chez la souris, suggérant un rôle important des cellules de la MZ dans le contrôle de l’infection au VIH-1. De façon générale, les résultats dans cette étude viennent enrichir nos connaissances du compartiment de cellules B dans le contexte de l’infection au VIH-1 et pourront contribuer à élaborer des stratégies préventives et thérapeutiques contre ce virus.

Transcription factor Tfec contributes to the IL-4-inducible expression of a small group of genes in mouse macrophages including the granulocyte colony-stimulating factor receptor

Expression of the mouse transcription factor EC (Tfec) is restricted to the myeloid compartment, suggesting a function for Tfec in the development or function of these cells. However, mice lacking Tfec develop normally, indicating a redundant role for Tfec in myeloid cell development. We now report that Tfec is specifically induced in bone marrow-derived macrophages upon stimulation with the Th2 cytokines, IL-4 and IL-13, or LPS. LPS induced a rapid and transient up-regulation of Tfec mRNA expression and promoter activity, which was dependent on a functional NF-kappa B site. IL-4, however, induced a rapid, but long-lasting, increase in Tfec mRNA, which, in contrast to LPS stimulation, also resulted in detectable levels of Tfec protein. IL-4-induced transcription of Tfec was absent in macrophages lacking Stat6, and its promoter depended on two functional Stat6-binding sites. A global comparison of IL-4-induced genes in both wild-type and Tfec mutant macrophages revealed a surprisingly mild phenotype with only a few genes affected by Tfec deficiency. These included the G-CSFR (Csf3r) gene that was strongly up-regulated by IL-4 in wild-type macrophages and, to a lesser extent, in Tfec mutant macrophages. Our study also provides a general definition of the transcriptome in alternatively activated mouse macrophages and identifies a large number of novel genes characterizing this cell type.

A copper-hydrogen peroxide redox system induces dityrosine cross-links and chemokine oligomerisation

The activity of the chemoattractant cytokines, the chemokines, in vivo is enhanced by oligomerisation and aggregation on glycosaminoglycan (GAG), particularly heparan sulphate, side chains of proteoglycans. The chemokine RANTES (CCL5) is a T-lymphocyte and monocyte chemoattractant, which has a minimum tetrameric structure for in vivo activity and a propensity to form higher order oligomers. RANTES is unusual among the chemokines in having five tyrosine residues, an amino acid susceptible to oxidative cross-linking. Using fluorescence emission spectroscopy, Western blot analysis and LCMS-MS, we show that a copper/H2O2 redox system induces the formation of covalent dityrosine cross-links and RANTES oligomerisation with the formation of tetramers, as well as higher order oligomers. Amongst the transition metals tested, namely copper, nickel, mercury, iron and zinc, copper appeared unique in this respect. At high (400 µM) concentrations of H2O2, RANTES monomers, dimers and oligomers are destroyed, but heparan sulphate protects the chemokine from oxidative damage, promoting dityrosine cross-links and multimer formation under oxidative conditions. Low levels of dityrosine cross-links were detected in copper/H2O2-treated IL-8 (CXCL8), which has one tyrosine residue, and none were detected in ENA-78 (CXCL5), which has none. Redox-treated RANTES was fully functional in Boyden chamber assays of T-cell migration and receptor usage on activated T-cells following RANTES oligomerisation was not altered. Our results point to a protective, anti-oxidant, role for heparan sulphate and a previously unrecognised role for copper in chemokine oligomerisation that may offer an explanation for the known anti-inflammatory effect of copper-chelators such as penicillamine and tobramycin.

25-hydroxycholesterol, 7β-hydroxycholesterol and 7-ketocholesterol upregulate interleukin-8 expression independently of Toll-like receptor 1, 2, 4 or 6 signalling in human macrophages

Recent studies have shown that Toll-like receptor (TLR)- signalling contributes significantly to the inflammatory events of atherosclerosis. As products of cholesterol oxidation (oxysterols) accumulate within atherosclerotic plaque and have been proposed to contribute to inflammatory signalling in the diseased artery, we investigated the potential of 7-ketocholesterol (7-KC), 7β-hydroxycholesterol (7β-HC) and 25-hydroxycholesterol (25-HC) to stimulate inflammatory signalling via the lipid-recognising TLRs 1, 2, 4 and 6. Each oxysterol stimulated secretion of the inflammatory chemokine interleukin-8 (IL-8), but not I?B degradation or tumour necrosis factor- release from monocytic THP-1 cells. Transfection of TLR-deficient HEK-293 cells with TLRs 1, 2, 4 or 6 did not increase sensitivity to the tested oxysterols. Moreover, blockade of TLR2 or TLR4 with specific inhibitors did not reduce 25-hydroxycholesterol (25-HC) induced IL-8 release from THP-1 cells. We conclude that although the oxysterols examined in this study may contribute to increased expression of certain inflammatory genes, this occurs by mechanisms independent of TLR signalling.

The Influence of Age, Cardiorespiratory Fitness, Exercise and Sedentary Behaviours on Circulating Angiogenic Cells and Cell Surface Receptor Expression.

Cardiovascular disease (CVD) is the biggest killer of people in western civilisation. Age is a significant risk factor for the development for CVD, and treatments and therapies to address this increased risk are crucial to quality of life and longevity. Exercise is one such intervention which has been shown to reduce CVD risk. Age is also associated with endothelial dysfunction, reduced angiogenic capabilities, and reduced ability to repair the vessel wall. Circulating angiogenic cells (CACs) are a subset of circulating cells which assist in the repair and growth of the vasculature and in the maintenance of endothelial function. Reductions in these cells are observed in those with vascular disease compared to age-matched healthy controls. Exercise may reduce CVD risk by improvements in number and/or function of these CACs. Data was collected from human volunteers of various ages, cardiorespiratory fitness (CRF) levels and latent viral infection history status to investigate the effects of chronological age, CRF, viral serology and other lifestyle factors, such as sedentary behaviours and exercise on CACs. The levels of CACs in these volunteers were measured using four colour flow cytometry using various monoclonal antibodies specific to cell surface markers that are used to identify specific subsets of these CACs. In addition, the response to acute exercise of a specific subset of these CACs, termed ‘angiogenic T-cells’ (TANG) were investigated, in a group of well-trained males aged 20-40 years, using a strenuous submaximal exercise bout. Advancing age was associated with a decline in various subsets of CACs, including bone marrow-derived CD34+ progenitors, putative endothelial progenitor cells (EPCs) and also TANG cells. Individuals with a higher CRF were more likely to have higher circulating numbers of TANG cells, particularly in the CD4+ subset. CRF did not appear to modulate CD34+ progenitors or EPC subsets. Increasing sitting time was associated with reduction in TANG cells, but after correcting for the effects of fitness, sitting time no longer negatively affected the circulating number of these cells. Acute exercise was a powerful stimulus for increasing the number of TANG cells (140% increase), potentially through an SDF-1:CXCR4-dependent mechanism, but more studies are required to investigate this. Latent CMV infection was associated with higher number of TANG cells (CD8+), but only in 18-40 year old individuals, and not in an older age group (41-65 year old). The significance of this has yet to be understood. In conclusion, advancing age may contribute to increased CVD risk partly due to the observed reductions in angiogenic cells circulating in the peripheral compartment. Maintaining a high CRF may attenuate this CVD reduction by modulating TANG cell number, but potentially not CD34+ progenitor or EPC subsets. Acute exercise may offer a short window for vascular adaptation through the mobilisation of TANG cells into the circulation.

Impaired Compensatory Beta-cell Function And Growth In Response To High-fat Diet In Ldl Receptor Knockout Mice.

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr-/- mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr-/- mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr-/- mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr-/- mice showed no significant changes in beta-cell mass, but lower islet-duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr-/- mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion.

Fractalkine (cx3cl1) Is Involved In The Early Activation Of Hypothalamic Inflammation In Experimental Obesity.

Hypothalamic inflammation is a common feature of experimental obesity. Dietary fats are important triggers of this process, inducing the activation of toll-like receptor-4 (TLR4) signaling and endoplasmic reticulum stress. Microglia cells, which are the cellular components of the innate immune system in the brain, are expected to play a role in the early activation of diet-induced hypothalamic inflammation. Here, we use bone marrow transplants to generate mice chimeras that express a functional TLR4 in the entire body except in bone marrow-derived cells or only in bone marrow-derived cells. We show that a functional TLR4 in bone marrow-derived cells is required for the complete expression of the diet-induced obese phenotype and for the perpetuation of inflammation in the hypothalamus. In an obesity-prone mouse strain, the chemokine CX3CL1 (fractalkine) is rapidly induced in the neurons of the hypothalamus after the introduction of a high-fat diet. The inhibition of hypothalamic fractalkine reduces diet-induced hypothalamic inflammation and the recruitment of bone marrow-derived monocytic cells to the hypothalamus; in addition, this inhibition reduces obesity and protects against diet-induced glucose intolerance. Thus, fractalkine is an important player in the early induction of diet-induced hypothalamic inflammation, and its inhibition impairs the induction of the obese and glucose intolerance phenotypes.

Peripheral P2x7 Receptor-induced Mechanical Hyperalgesia Is Mediated By Bradykinin.

P2X7 receptors play an important role in inflammatory hyperalgesia, but the mechanisms involved in their hyperalgesic role are not completely understood. In this study, we hypothesized that P2X7 receptor activation induces mechanical hyperalgesia via the inflammatory mediators bradykinin, sympathomimetic amines, prostaglandin E2 (PGE2), and pro-inflammatory cytokines and via neutrophil migration in rats. We found that 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (BzATP), the most potent P2X7 receptor agonist available, induced a dose-dependent mechanical hyperalgesia that was blocked by the P2X7 receptor-selective antagonist A-438079 but unaffected by the P2X1,3,2/3 receptor antagonist TNP-ATP. These findings confirm that, although BzATP also acts at both P2X1 and P2X3 receptors, BzATP-induced hyperalgesia was mediated only by P2X7 receptor activation. Co-administration of selective antagonists of bradykinin B1 (Des-Arg(8)-Leu(9)-BK (DALBK)) or B2 receptors (bradyzide), β1 (atenolol) or β2 adrenoceptors (ICI 118,551), or local pre-treatment with the cyclooxygenase inhibitor indomethacin or the nonspecific selectin inhibitor fucoidan each significantly reduced BzATP-induced mechanical hyperalgesia in the rat hind paw. BzATP also induced the release of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1), an effect that was significantly reduced by A-438079. Co-administration of DALBK or bradyzide with BzATP significantly reduced BzATP-induced IL-1β and CINC-1 release. These results indicate that peripheral P2X7 receptor activation induces mechanical hyperalgesia via inflammatory mediators, especially bradykinin, which may contribute to pro-inflammatory cytokine release. These pro-inflammatory cytokines in turn may mediate the contributions of PGE2, sympathomimetic amines and neutrophil migration to the mechanical hyperalgesia induced by local P2X7 receptor activation.

Icatibant, An Inhibitor Of Bradykinin Receptor 2, For Hereditary Angioedema Attacks: Prospective Experimental Single-cohort Study.

Hereditary angioedema (HAE) with C1 inhibitor deficiency manifests as recurrent episodes of edema involving the skin, upper respiratory tract and gastrointestinal tract. It can be lethal due to asphyxia. The aim here was to evaluate the response to therapy for these attacks using icatibant, an inhibitor of the bradykinin receptor, which was recently introduced into Brazil. Prospective experimental single-cohort study on the efficacy and safety of icatibant for HAE patients. Patients with a confirmed HAE diagnosis were enrolled according to symptoms and regardless of the time since onset of the attack. Icatibant was administered in accordance with the protocol that has been approved in Brazil. Symptom severity was assessed continuously and adverse events were monitored. 24 attacks in 20 HAE patients were treated (female/male 19:1; 19-55 years; median 29 years of age). The symptoms were: subcutaneous edema (22/24); abdominal pain (15/24) and upper airway obstruction (10/24). The time taken until onset of relief was: 5-10 minutes (5/24; 20.8%); 10-20 (5/24; 20.8%); 20-30 (8/24; 33.4%); 30-60 (5/24; 20.8%); and 2 hours (1/24; 4.3%). The time taken for complete resolution of symptoms ranged from 4.3 to 33.4 hours. Adverse effects were only reported at injection sites. Mild to moderate erythema and/or feelings of burning were reported by 15/24 patients, itching by 3 and no adverse effects in 6. HAE type I patients who received icatibant responded promptly; most achieved improved symptom severity within 30 minutes. Local adverse events occurred in 75% of the patients.